US00888.9371B2

(12) United States Patent (10) Patent No.: US 8,889,371 B2 Miasnikov et al. (45) Date of Patent: Nov. 18, 2014

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Cerial Foods World, p. 561. Nagao et al. J. Biochem 124, 1124-1129, 1998. Ohta, Yoshifumi, et al., “Inhibition and Inactivation of Lipase by Fat Nagao et al. J. of Bioscience and Bioengineering vol. 89, No. 5, Peroxide in the Course of Batch and Continuous Glycerolyses of Fat 446-450, 2000. by Lipase”, Agric. Biol. Chem., vol.53, No. 7, pp. 1885-1890, 1989. Nagao et al. J. of Molecular Catalysis B. Enzymatic 17 (2002) 125 Okiy D.A. (1977) Partial glycerides and palm oil Crystallisation, in 132. Journal of Science and Food Agriculture 28:955. Nagao et al. JAOCS vol. 78, No. 2, 2001. Okiy D.A. (1978) Interaction of triglycerides and diglycerides of Nagao, Toshihiro et al., “Cloning and Nucleotide Sequence of CDNA palm oil, in Oleagineux 33:625-628. Encoding a Lipase from Fusarium heterosporum'. J. Biochem... vol. Okiy D.A., Wright, W.B., Berger, K.G. & Morton I.D. (1978), The 116, pp. 535-540, 1994. physical properties of modified palm oil, in Journal of Science of Nagao, Toshihiro et al., “Expression of Lipase cDNA from Fusarium Food and Agriculture 29:1061-1068. heterosporum by Saccharomyces cerevisiae: High-Level Production Oluwatosin, Yemisi E. et al., “Phenotype: A Possible Role for the and Purification'. Journal of Fermentation and Bioengineering, Kex2 Endoprotease in Vacuolar Acidification'. Molecular and Cel 1996, vol. 81, No. 6, pp. 488-492. lular Biology, 1998, pp. 1534-1543. Nagodawlthana et al., “Enzymes in Food Processing. Third Edition, Oluwatosin, Yemisi E., et al., “Mutations in the Yeast KEX2 Gene 1993, Academic Press, Inc. Cause a Vma-Like Phenotype: a Possible Role for the Kex2 US 8,889,371 B2 Page 16

(56) References Cited Peters, G.H., et al., “Active Serine Involved in the Stabilization of the Active Site Loop in the Humicola Ianuginosa Lipase'. Biochemistry, OTHER PUBLICATIONS 1998 vol. 17 pp. 12375-12383. Peters, Günther H., et al., “Theoretical Investigation of the Dynamics Endoprotease in Vacuolar Acidification'. Molecular and Cellular of the Active Site Lid in Rhizomucor miehei Lipase'. Biophysical Biology, vol. 18, No. 3, pp. 1534-1543, Mar. 1998. Journal, vol. 71, 1996, pp. 119-129. Pliter J and JHGM Mutsaers, The surface rheological properties of Oberg, Marie-Louise, “Self-assembly Structures Formed by Wheat dough and the influence of lipase on it, Gist-brocades, Bakery Ingre Polar Lipids and their Interaction with Lipases', Master of Scient dients Division, Oct. 1994. Thesis, Apr. 2005. Plouetal, J. Biotechnology 92 (2002) 55-66. Orskov, Janne, et al., “Solubilisation of poorly water-soluble drugs Ponte J. G. Cereal Chemistry (1969), vol. 46(3), p. 325-329. during in vitro lipolysis of medium- and long-chain triacylglycerols'. Punt and van den Hondel, Meth. Enzym., 1992, 216:447-457. European Journal of Pharmaceutical Sciences, vol. 23, 2004, pp. Pyler, E.J., “Baking Science and Technology Third Edition', vol. 1, 287-296. 1988. Osman, Mohamed, et al., “Lipolytic activity of Alternaria alternata Pyler, E.J., “Baking Science and Technology Third Edition', vol. II, and Fusarium oxysporum and certain properties of their lipids'. 1988. Queener et al. (1994) Ann NY Acad Sci. 721, 178-93. Microbios Letters, vol.39, pp. 131-135, 1988. Rambosek and Leach, CRC Crit. Rev. Biotechnol., 1987, 6:357-393. O'Sullivan et al. J Plant Physiol, vol. 313, (1987) p. 393-404. Rapp, Peter, et al., “Formation of extracellular lipases by filamentous Palomo, Jose M. et al., “Enzymatic production of (3S, 4R)-(-)-4- fungi, yeasts, and bacteria'. Enzyme Microb. Technol., 1992, vol. 14. (4-fluorophenyl)-6-oxo-piperidin-3-carboxylic acid using a com Nov. merical preparation of lipase a from Candida antarctica: the role of Rapp, Peter; “Production, regulation, and some properties of lipase a contaminant esterase” Tetrahedron: Asymmetry, vol. 13, 2002, pp. activity from Fusarium Oxysporum f.sp. vasinfectum', Enzyme and 2653-2659. Microbial Technology(1995); vol. 17: pp. 832-838. Palomo, Jose M., et al., “Enzymatic resolution of (+)-glycidyl Reetz M.T., Jaeger K.E. ChemPhys Lipids. Jun. 1998; 93(1-2): 3-14. butyrate in aquenous media. Strong modulation of the properties of Reetz Manfred T. Current Opinion in Chemical Biology, Apr. 2002, the lipase from Rhizopus oryzae via immobilization techniques'. vol. 6, No. 2, pp. 145-150. Tetrahedron: Asymmetry, vol. 15, 2004, pp. 1157-1161. Reiser J et al. (1990) Adv Biochem Eng Biotechnol. 43, 75-102. Palomo, Jose M., et al., “Modulation of the enantioselectivity of Richardson & Hyslop, pp. 371-476 in Food Chemistry, 1985, second Candida antarctica B lipase via conformational engineering: kinetic edition, Owen R. Fennema (ed) Manel Dekker, Inc, New York and resolution of (+)-O-hydroxy-phenylacetic acid derivatives'. Tetrahe Basel. dron: Asymmetry, vol. 13, 2002, pp. 1337-1345. Richardson and Hyslop, “Enzymes: XI Enzymes Added to Foods Patent Abstracts of Japan vol. 016, No. 528 (C-1001), Oct. 29, 1992 During Processing” in Food Chemistry, Marcel Dekker, Inc., New & JP 04 200339 A See abstract. York, NY 1985. Patent Abstracts of Japan vol. 095, No. 001, Feb. 28, 1995 & JP 06 Arskog and Joergensen, "Baking performance of prior art lipases 296467 A see abstract. from Candida cylindraceaand Aspergillus foeditus and their activity Peelman F, et al. Protein Science Mar. 1998; 7(3): 587-99. on galactolipids in dough', Novozymes Report 2005. Penninga et al. Biochemistry (1995), 3368-3376. Arskog and Joergensen, "Baking performance of prior art lipases Persson, Mattias, et al., “Enzymatic fatty acid exchange in from Humicola lanuginosa, Aspergillus tubigensis, Rhizopus digalactosyldiacylglycerol'. Chemistry and Physics of Lipids, vol. deleimar and Rhizomucor miehei, and their activity on galactolipids in 104, 2000, pp. 13-21. dough', Novozymes Report 2005.

U.S. Patent Nov. 18, 2014 Sheet 3 of 15 US 8,889,371 B2

FIGURE 5

pGTK(131) 4960 bp

pET (13-51)

Cole pBR322 origin Psi U.S. Patent Nov. 18, 2014 Sheet 4 of 15 US 8,889,371 B2

FIGURE 6

Dough slurry treated with Streptomyces sp. L. 130. Sample id.182

0,15 0,13 0,11 s O,09 ais . 9 0,07 O. g.S o 0.05 s o 0.03 s 0.01 0,4 0,6 Units/gram flour U.S. Patent Nov. 18, 2014 Sheet 5 Of 15 US 8,889,371 B2

FIGURE 7

Dough slurry treated with Streptomyces sp. 131. Sample id.185 0,16

0,4 0.6 O,8 Units gran flour U.S. Patent Nov. 18, 2014 Sheet 6 of 15 US 8,889,371 B2

FIGURE 8

Triglyce de in U.S. Patent Nov. 18, 2014 Sheet 7 Of 15 US 8,889,371 B2

FIGURE 9

pH profile L131

2 2.5 3 3.5 4 4.5 5 5.5 6 65 7 7.5 8 8.5 9 95 10 pH U.S. Patent Nov. 18, 2014 Sheet 8 of 15 US 8,889,371 B2

FIGURE 10

U.S. Patent Nov. 18, 2014 Sheet 9 Of 15 US 8,889,371 B2

Figure 11

Kuni (10238) Ban.H.I (10225). Psil (10211) HindIII (101.95), P(LAC) Psil (10193A EcoRI (1) Prio's all r-or neoR Psi (9050). APr tST Y Clai (8121) P(BLA) NdeI (7891). Ndel (2421) 10245 bp HinamHindi (263(2636) PstI (2652) Clai (2968) '-.lipase ORF

rep ("EcoRI (4031) visis) - 7 F fd terminator Koni (5285 Psi (4932) U.S. Patent Nov. 18, 2014 Sheet 10 of 15 US 8,889,371 B2

FIGURE 12

-- Stability -H activity 120

100

80

60

40

20

22 30 40 50 60 70 8O 90 Temp (degrees C) U.S. Patent Nov. 18, 2014 Sheet 11 of 15 US 8,889,371 B2

(Z,·)vae

9 9

TEO

9IERIO!!OIH U.S. Patent Nov. 18, 2014 Sheet 12 of 15 US 8,889,371 B2

FIGURE 14

eIninator? SmaI (6759) Ban.H.I.6393) Xiba I () Mature BamHI (7) Apa I (6001) f tufterminator area GDSY g Hairpin Sig pep cleavage N HindIII (275)

Signal P Bell (401) CDS

Bell (3557) f SENSEPRN Bigl (3552) U.S. Patent Nov. 18, 2014 Sheet 13 of 15 US 8,889,371 B2

FIGURE 15 l. 31 2. S. aver Initilis 3. T. fusca 4. Consensus

1. 50 1 () ------MRLTRSSAASWIVFALLLALLGISPAOAAG------2 (1) ------MRRSRITAYWTSLLIAVGCALTGAATAOASPA------3 (1) WGSGPRAATRRRLFLGIPALVLVTALTLVLAVPTGREFLWRMWCEATQDW 4 (...) MRRSRELA ALILITA AL GAA ARAAP

51 OO 1 (32) ------PaAYWA 2 (33) ------AAAG 3 (51) CLGVPWDSRGQPAEDGEELLLSPVQAATWGNYY 4 5)

10 150 1 (53) SSGD-CHRSNNAYPARWAAANAP---SSETEAACSGAVTTDWIN---- 2 (57) SSGD---CKRSSKAYPYLWOAAHSP--SSFSFMACSGARTGDVLA---- 3 (101) GTAVKGGCWRSANAYPELVAEAYDFA-GHLSFLACSGQRGYAMEDAIDE 4 (101) SSGD C RSTKAYPALWAAAHA SSFSF ACSGARTYDVLA

5. 200 1 (93) --NOLGALNAST--GLVSITIGGNDAGFADAMTTCVTS------SDSTC, 2 (97) --NQLGTLNSST--GLVSLIGGNDAGFSDVMTTCVLQ------SSAC, 3 (149) WGSQLDWNSPHT--SLVFIGIGGNDGFSTVLKTCMVR------WPDS 4 (151) OL, LNS T LVSITIGGNDAGEAD MTTCWL, SOSAC,

2O 250 1 (133) NRLATATNYINTELLA------RLDAVYSOIKARAPNARVWWLGYPRMY 2 (137). SRINTAKAYVDSTLPG------OLDSVYTAISTKAPSAHVAVIGYPREY 3 (191) KACTDOEDAIRKRMAKF----ETTFEELISEVRTRAPDARILVWGYPRIF 4 (201) RIA AK YI TLPA RLDSWYSA TRAP ARVWWLGYPRY

25. 300 1 (176) LASNPWYCLGLSNTKRAAINTTADTLNSWISSRATAH------GF 2 (1.80) KLGG-SCLAGLSETKRSAINDAADYLNSAIAKRAADH------GE 3 (237) PEEPTGAYYTLTASNQRWLNETIQEFNQQLAEAVAVHDEELAASGGWGSW 4 (251) SG LGLS TKRAAINDAAD LNSVIAKRAADH GF

3O O 1 (215) RFGDVRPTFNNHELFFGNDWLHSLTLP------WWES 2 (218) TFGDVKSTFTGHEICSSSTWLHSLDLLN------IGOS 3 (287) EFVDVYHALDGHEIGSDEPWVNGVQLRDLATG------WTWDRST 4 (301) TFGDW TF GHELCSA PWLHSLTLP W

35. 395 1 (248) PTSTGHQSGYLPVLNANSST------2 (252) PTAAGQSGGYLPVMNSVA------3 (328) PNAAGHRAVGERVIEQIETGPGRPLYATEAVVAGATVDTLAGEVG 4 (351) PTA GHAAGYLPVLNSI T U.S. Patent Nov. 18, 2014 Sheet 14 of 15 US 8,889,371 B2

U.S. Patent Nov. 18, 2014 Sheet 15 Of 15 US 8,889,371 B2

Oles Jued

IOIONOW

Je)SeOJeSeOUO US 8,889,371 B2 1. 2 LIPOLYTIC ENZYME USES THEREOF IN WO02/00852 discloses five lipase enzymes and their THE FOOD INDUSTRY encoding polynucleotides, isolated from Fusarium venena tum, F sulphureum, Aspergillus berkeleyanum, F, culmorum REFERENCE TO RELATED APPLICATIONS and F. Solani. All five enzymes are described as having tria cylglycerol hydrolysing activity, phospholipase and galacto This application is a divisional of U.S. patent application lipase activity. Ser. No. 1 1/623,607 filed Jan. 16, 2007 now U.S. Pat. No. Lipolytic enzyme variants, with specific amino acid Sub 7,666,618, which is a continuation-in-part of International stitutions and fusions, have been produced; some of which Patent Application PCT/IB2005/002602 filed Jul 18, 2005 have an enhanced activity on the polar lipids compared to the and published as WO 2006/008653 on Jan. 26, 2006, which 10 wildtype parent enzymes. WOO1/396.02 describes such a claims priority from Great Britain Patent Application Nos. variant, referred to as SP979, which is a fusion of the Ther 0513859.9 filed Jul. 7, 2005 and 0416035.4 filed Jul. 16, momyces lanuginosus lipase, and the Fusarium oxysporum 2004, and from U.S. Patent Application No. 60/591,185 filed lipase described in EP 0869 167. This variant has been found Jul. 26, 2004. to have a significantly high ratio of activity on phospholipids Each of the above referenced applications, and each docu 15 and glycolipids compared to triglycerides. ment cited in this text (“application cited documents') and each document cited or referenced in each of the application In WO02/094123 it was discovered that by selecting cited documents, and any manufacturer's specifications or lipolytic enzymes which were active on the polar lipids (gly instructions for any products mentioned in this text and in any colipids and phospholipids) in a dough, but Substantially not document incorporated into this text, are hereby incorporated active on triglycerides or 1-mono-glycerides an improved herein by reference; and, technology in each of the documents functionality could be achieved. incorporated herein by reference can be used in the practice of In co-pending PCT application number PCT/IB2005/ this invention. 000875, wild-type lipolytic enzymes having a higher ratio of It is noted that in this disclosure, terms such as "com activity on polar lipids as compared with triglycerides are prises”, “comprised”, “comprising”, “contains”, “contain 25 taught. However, this document does not teach lipolytic ing” and the like can have the meaning attributed to them in enzymes from Streptomyces, Thermobifida or Corynebacte U.S. Patent law; e.g., they can mean “includes”, “included'. rium species. “including and the like. Terms such as "consisting essen Prior to the present invention no lipolytic enzymes having tially of and “consists essentially of have the meaning activity or significant activity on glycolipids had been pub attributed to them in U.S. Patent law, e.g., they allow for the 30 lished from Streptomyces species. Likewise, no lipolytic inclusion of additional ingredients or steps that do not detract enzymes having activity or significant activity on glycolipids from the novel or basic characteristics of the invention, i.e., had been published from Thermobifida species or Coryne they exclude additional unrecited ingredients or steps that bacterium species. Although lipases, i.e. triacylglycerol detract from novel or basic characteristics of the invention, hydrolysing enzymes, have been isolated from Streptomyces and they exclude ingredients or steps of the prior art, such as 35 species (see Vujaklija et al Arch Microbiol (2002) 178: 124 documents in the art that are cited herein or are incorporated 130 for example), these enzymes have never been identified by reference herein, especially as it is a goal of this document as having glycolipid hydrolysing activity. to define embodiments that are patentable, e.g., novel, non obvious, inventive, over the prior art, e.g., over documents ASPECTS OF THE INVENTION cited herein or incorporated by reference herein. And, the 40 terms “consists of and "consisting of have the meaning The present invention in predicated upon the seminal find ascribed to them in U.S. Patent law; namely, that these terms ing of a lipolytic enzyme having significant galactolipid are closed ended. activity from the genus Streptomyces. In particular the lipoly tic enzyme from the genus Streptomyces has significant FIELD OF INVENTION 45 galactolipid hydrolysing activity and/or significant galacto lipid acyltransferase activity, particularly when used in the The present invention relates to a novel lipolytic enzyme, methods and uses according to the present invention. in particular a novel lipolytic enzyme, and nucleotide In addition, the present invention in predicated upon the sequences encoding same. The present invention also relates seminal finding that lipolytic enzymes from the genera Ther to methods of production of the novellipolytic enzyme and to 50 mobifida or Corynebacterium have significant galactolipid uses thereof. The present invention also relates to methods activity. In particular the lipolytic enzymes from the genera and uses of a lipolytic enzyme. Thermobifida or Corynebacterium have significant galacto lipid hydrolysing activity and/or significant galactolipid acyl TECHNICAL BACKGROUND transferase activity, particularly when used in the methods 55 and uses of the present invention. The beneficial use of lipolytic enzymes active on glycolip In a broad aspect the present invention relates to a lipolytic ids in bread making was taught in EP 1 193314. It was taught enzyme capable of hydrolysing at least glycolipids and/or that the partial hydrolysis products the lyso-glycolipids were capable of transferring an acyl group from at least a glycolipid found to have very high emulsifier functionality. However, to one or more acyl acceptor Substrates, wherein the enzyme the enzymes taught in EP 1 193314 were also found to have 60 is obtainable, preferably obtained, from Streptomyces spe significant non-selective activity on triglycerides which C1GS. resulted in unnecessarily high free fatty acid. Inafurther aspect the present invention relates to a lipolytic A lipolytic enzyme from Fusarium oxysporum having enzyme capable of hydrolysing at least galactolipids and/or phospholipase activity has been taught in EP 0869 167. This capable of transferring an acyl group from at least a galacto lipolytic enzyme has high triacylglyceride hydrolysing (li 65 lipid to one or more acyl acceptor Substrates, wherein the pase) activity. This enzyme is now sold by Novozymes A/S enzyme is encoded by a nucleic acid selected from the group (Denmark) as Lipopan FTM. consisting of: US 8,889,371 B2 3 4 a) a nucleic acid comprising a nucleotide sequence shown in In another aspect of the present invention there is provided SEQID No. 3; the use of a lipolytic enzyme according to the present inven b) a nucleic acid which is related to the nucleotide sequence of tion in a process of treating egg or egg-based products to SEQID No. 3 by the degeneration of the genetic code; and produce lysophospholipids. c) a nucleic acid comprising a nucleotide sequence which has 5 In another aspect of the present invention there is provided at least 70% identity with the nucleotide sequence shown in the use of a lipolytic enzyme according to the present inven SEQID No. 3. tion in a process of treating egg or egg-based products to The present invention yet further provides a lipolytic produce lysoglycolipids. enzyme comprising an amino acid sequence as shown in SEQ A further aspect of the present invention provides a process ID No. 4 or an amino acid sequence which has at least 60%. 10 identity thereto. of enzymatic degumming of vegetable or edible oils, com In another aspect the present invention provides a lipolytic prising treating the edible or vegetable oil with a lipolytic enzyme capable of hydrolysing at least a galactolipid and/or enzyme according to the present invention so as to hydrolyse capable of transferring an acyl group from at least a galacto a major part of the polar lipids (e.g. phospholipid and/or lipid to one or more acyl acceptor Substrates, wherein the 15 glycolipid). enzyme comprises an amino acid sequence as shown in SEQ In another aspect the present invention provides the use of ID No. 4 or an amino acid sequence which has at least 60% a lipolytic enzyme according to the present invention in a identity thereto. process comprising treatment of a phospholipid so as to In a further aspect the present invention provides a nucleic hydrolyse fatty acyl groups. acid encoding a lipolytic enzyme comprising an amino acid 20 In another aspect the present invention provides the use of sequence as shown in SEQ ID No. 4 or an amino acid a lipolytic enzyme according to the present invention in a sequence which has at least 60% identity therewith. process for reducing the content of a phospholipid in an edible SEQ ID No. 3 is shown in FIG. 3 and SEQ ID No. 4 is oil, comprising treating the oil with the lipolytic enzyme shown in FIG. 4. according to the present invention so as to hydrolyse a major The present invention yet further provides a nucleic acid 25 part of the phospholipid, and separating an aqueous phase encoding a lipolytic enzyme, which nucleic acid is selected containing the hydrolysed phospholipid from the oil. from the group consisting of There is also provided a method of preparing a lipolytic a) a nucleic acid comprising a nucleotide sequence shown in enzyme according to the present invention, the method com SEQID No. 3; prising transforming a host cell with a recombinant nucleic b) a nucleic acid which is related to the nucleotide sequence of 30 acid comprising a nucleotide sequence coding for the lipoly SEQID No. 3 by the degeneration of the genetic code; and tic enzyme, the host cell being capable of expressing the c) a nucleic acid comprising a nucleotide sequence which has nucleotide sequence coding for the polypeptide of the lipoly at least 70% identity with the nucleotide sequence shown in tic enzyme, cultivating the transformed host cell under con SEQID No. 3. ditions where the nucleic acid is expressed and harvesting the The present invention yet further provides the use of a 35 lipolytic enzyme. lipolytic enzyme according to the present invention in a Sub Inafurther aspect the present invention relates to the use of strate (preferably a foodstuff) for preparing a lyso-glycolipid, a lipolytic enzyme in accordance with the present invention in for example digalactosyl monoglyceride (DGMG) or the bioconversion of polar lipids (preferably glycolipids) to monogalactosyl monoglyceride (MGMG) by treatment of a make high value products, such as carbohydrate esters and/or glycolipid (e.g. digalactosyl diglyceride (DGDG) or monoga- 40 protein esters and/or protein subunit esters and/or a hydroxy lactosyl diglyceride (MGDG)) with the lipolytic enzyme acid ester. according to the present invention to produce the partial Another aspect of the present invention relates to the use of hydrolysis product, i.e. the lyso-glycolipid. a lipolytic enzyme in accordance with the present invention in In a further aspect, the present invention provides the use of a process of enzymatic degumming of vegetable or edible oil, a lipolytic enzyme according to the present invention in a 45 comprising treating said edible or vegetable oil with said Substrate (preferably a foodstuff) for preparing a lyso-phos lipolytic enzyme so as to hydrolyse a major part of the polar pholipid, for example lysolecithin, by treatment of a phos lipids. pholipid (e.g. lecithin) with the enzyme according to the A further aspect of the present invention relates to the use present invention to produce a partial hydrolysis product, i.e. of a lipolytic enzyme in accordance with the present invention a lyso-phospholipid. 50 in a process comprising treatment of a phospholipid so as to In one broad aspect the present invention relates to a hydrolyse fatty acyl groups. method of preparing a foodstuff the method comprising The present invention yet further relates to an immobilised admixing a lipolytic enzyme of the present invention with one lipolytic enzyme in accordance with the present invention. or more ingredients of the foodstuff. Another aspect of the present invention relates to a method Another broad aspect of the present invention relates to a 55 of preparing a lysoglycolipid comprising treating a substrate method of preparing a baked product from a dough, the comprising a glycolipid with at least one lipolytic enzyme to method comprising admixing a lipolytic enzyme of the produce said lysoglycolipid, wherein said lipolytic enzyme present invention with the dough. has glycolipase activity and wherein said lipolytic enzyme is In a further aspect the present invention relates to a method obtainable from one of the following genera: Streptomyces, of preparing a dairy product, the method comprising admix 60 Corynebacterium and Thermobifida. ing a lipolytic enzyme of the present invention with one or A further aspect of the present invention relates to a method more ingredients of the dairy product. of preparing a lysophospholipid comprising treating a Sub In another aspect the present invention relates to the use of strate comprising a phospholipid with at least one lipolytic a lipolytic enzyme of the present invention in the manufacture enzyme to produce said lysophospholipid, wherein said of a dairy product to reduce one or more of the following 65 lipolytic enzyme has phospholipase activity and wherein said detrimental effects: off-odours and/or off-flavours and/or lipolytic enzyme is obtainable from one of the following Soapy taste. genera: Streptomyces, Corynebacterium and Thermobifida. US 8,889,371 B2 5 6 Another aspect of the present invention relates to a method The present invention further encompasses methods of of enzymatic degumming of vegetable or edible oil, compris isolating the nucleotide sequence, Such as isolating from a ing treating said edible or vegetable oil with a lipolytic host cell. enzyme obtainable from one of the following genera: Strep Other aspects concerning the amino acid sequence for use tomyces, Corynebacterium and Thermobifida capable of 5 in the present invention include: a construct encoding the hydrolysing a major part of the polar lipids. amino acid sequences for use in the present invention; a The present invention further relates to a method of bio vector encoding the amino acid sequences for use in the conversion of polar lipids to make high value products com present invention; a plasmid encoding the amino acid prising treating said polar lipids with a lipolytic enzyme sequences for use in the present invention; a transformed cell obtainable from one of the following genera: Streptomyces, 10 expressing the amino acid sequences for use in the present Corynebacterium and Thermobifida to produce said high invention; a transformed tissue expressing the amino acid sequences for use in the present invention; a transformed value products, wherein said lipolytic enzyme is capable of organ expressing the amino acid sequences for use in the hydrolysing said polar lipids. present invention; a transformed host expressing the amino Another aspect of the present invention relates to a method 15 acid sequences for use in the present invention; a transformed of preparing a foodstuff comprising admixing at least one organism expressing the amino acid sequences for use in the lipolytic enzyme with one or more ingredients of a foodstuff present invention. The present invention also encompasses wherein said lipolytic enzyme is capable of hydrolysing a methods of purifying the amino acid sequence for use in the glycolipid and/or a phospholipid present in or as at least one present invention using the same. Such as expression in a host of said ingredients and wherein said lipolytic enzyme is cell; including methods of transferring same, and then puri obtainable from one of the following genera: Streptomyces, fying said sequence. Corynebacterium and Thermobifida. For the ease of reference, these and further aspects of the A further aspect of the present invention relates the use of present invention are now discussed under appropriate sec a lipolytic enzyme in a Substrate for preparing a lysophos tion headings. However, the teachings under each section are pholipid wherein said lipolytic enzyme has phospholipase 25 not necessarily limited to each particular section. activity and wherein said lipolytic enzyme is obtainable from one of the following: Streptomyces, Corynebacterium and DETAILED DISCLOSURE OF THE INVENTION Thermobifida. The present invention additionally relates to the use of a Suitably, the lipolytic enzyme for use in the methods and lipolytic enzyme obtainable from one of the following gen 30 uses according to the present invention may be a lipolytic era: Streptomyces, Corynebacterium and Thermobifida for enzyme comprising any one of the amino acid sequences enzymatic degumming of vegetable or edible oil so as to shown as SEQID No. 4, 5, 7, 8, 12, 14 or 16 or an amino acid hydrolyse a major part of the polar lipids. sequence which has at least 70%, 75%,80%,85%, 90%,95%, Another aspect of the present invention relates to the use of 96%, 97% or 98% identity therewith, or encoded by any one a lipolytic enzyme obtainable from one of the following gen 35 of the nucleotide sequences shown as SEQID No. 3, 6, 9, 13, era: Streptomyces, Corynebacterium and Thermobifida in a 15 or 17 or a nucleotide sequence which has at least 70%, process comprising treatment of a phospholipid so as to 75%, 80%, 85%, 90%. 95%, 96%, 97% or 98% identity hydrolyse fatty acyl groups. therewith. A further aspect of the present invention relates to use of a Preferably, the lipolytic enzyme for use in the methods and lipolytic enzyme in the bioconversion of polar lipids to make 40 uses according to the present invention is a lipolytic enzyme high value products, wherein said lipolytic enzyme is capable capable of hydrolysing at least galactolipids and/or capable of of hydrolysing said polar lipids and wherein said lipolytic transferring an acyl group from at least agalactolipid to one or enzymes is obtainable from one of the following genera: more acyl acceptor Substrates, wherein the enzyme is obtain Streptomyces, Corynebacterium and Thermobifida. able, preferably obtained, from Streptomyces species. A further aspect of the present invention relates to the use 45 In one embodiment the lipolytic enzyme for use in the of a lipolytic enzyme obtainable from one of the following methods and uses according to the present invention is pref genera: Streptomyces, Corynebacterium and Thermobifida in erably a lipolytic enzyme capable of hydrolysing at least the preparation of a foodstuff, wherein said lipolytic enzyme galactolipids and/or capable of transferring an acyl group is capable of hydrolysing a glycolipid and/or a phospholipid. from at least a galactolipid to one or more acyl acceptor Aspects of the present invention are presented in the claims 50 Substrates, wherein the enzyme is encoded by a nucleic acid and in the following commentary. selected from the group consisting of: Other aspects concerning the nucleotide sequences which a) a nucleic acid comprising a nucleotide sequence shown can be used in the present invention include: a construct in SEQID No. 3; comprising the sequences of the present invention; a vector b) a nucleic acid which is related to the nucleotide sequence comprising the sequences for use in the present invention; a 55 of SEQID No. 3 by the degeneration of the genetic code: plasmid comprising the sequences for use in the present and invention; a transformed cell comprising the sequences for c) a nucleic acid comprising a nucleotide sequence which use in the present invention; a transformed tissue comprising has at least 70% identity with the nucleotide sequence the sequences for use in the present invention; a transformed shown in SEQID No. 3. organ comprising the sequences for use in the present inven 60 In one embodiment, the lipolytic enzyme for use in the tion; a transformed host comprising the sequences for use in methods and uses according to the present invention is pref the present invention; a transformed organism comprising the erably a lipolytic enzyme comprising an amino acid sequence sequences for use in the present invention. The present inven as shown in SEQID No. 4 or an amino acid sequence which tion also encompasses methods of expressing the nucleotide has at least 60% identity thereto. sequence for use in the present invention using the same. Such 65 In another embodiment the lipolytic enzyme for use in the as expression in a host cell; including methods for transfer methods and uses according to the present invention is pref ring same. erably a lipolytic enzyme capable of hydrolysing at least a US 8,889,371 B2 7 8 galactolipid and/or capable of transferring an acyl group from a) a nucleic acid comprising a nucleotide sequence shown at least a galactolipid to one or more acyl acceptor Substrates, in SEQID No. 3; wherein the enzyme comprises an amino acid sequence as b) a nucleic acid which is related to the nucleotide sequence shown in SEQID No. 4 or an amino acid sequence which has of SEQID No. 3 by the degeneration of the genetic code: at least 60% identity thereto. 5 and Preferably, the lipolytic enzyme for use in the methods and c) a nucleic acid comprising a nucleotide sequence which uses according to the present invention is a lipolytic enzyme has at least 70% identity with the nucleotide sequence capable of hydrolysing at least galactolipids and/or capable of shown in SEQID No. 3. transferring an acyl group from at least agalactolipid to one or In one embodiment the lipolytic enzyme according to the more acyl acceptor Substrates, wherein the enzyme is obtain 10 present invention may be a lipolytic enzyme obtainable, pref able, preferably obtained, from Thermobifida species, pref erably obtained, from the Streptomyces strains L130 or L131 erably Thermobifida fisca. deposited by Danisco A/S of Langebrogade 1, DK-1001 Preferably, the lipolytic enzyme for use in the methods and Copenhagen K, Denmark under the Budapest Treaty on the uses according to the present invention is a lipolytic enzyme International Recognition of the Deposit of Microorganisms capable of hydrolysing at least galactolipids and/or capable of 15 for the purposes of Patent Procedure at the National Collec transferring an acyl group from at least agalactolipid to one or tion of Industrial, Marine and Food Bacteria (NCIMB) 23 St. more acyl acceptor Substrates, wherein the enzyme is obtain Machar Street, Aberdeen Scotland, GB on 25 Jun. 2004 under able, preferably obtained, from Corynebacterium species, accession numbers NCIMB 41226 and NCIMB 41227, preferably Corynebacterium efficiens. respectively. In a further embodiment the lipolytic enzyme for use in the Preferably, the lipolytic enzyme according to the present methods and uses according to the present invention may be invention acts on at least a glycolipid, Such as digalactosyl a lipolytic enzyme comprising any one of the amino acid diglyceride (DGDG) for example. Suitably, the lipolytic sequences shown as SEQID No. 4, 5, 7, 8, 12, 14 or 16 or an enzyme according to the present invention may also act on amino acid sequence which has at least 70%, 75%, 80%, 85%, one or more other polar lipid substrates, such as a phospho 90%. 95%, 96%, 97% or 98% identity therewith, or encoded 25 lipid, for example a lecithin, e.g. phosphatidylcholine. by any one of the nucleotide sequences shown as SEQID No. An alternative way of expressing the term “capable of 3, 6, 9, 13, 15 or 17 or a nucleotide sequence which has at least hydrolysing glycolipids' as used herein would be to say that 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity the lipolytic enzyme has glycolipid hydrolysing activity. therewith. Preferably, the lipolytic enzyme according to the present In a further embodiment the lipolytic enzyme for use in the 30 invention hydrolyses a glycolipid, such as digalactosyldig methods and uses according to the present invention may be lyceride (DGDG) for example, and also a phospholipid, such a lipolytic enzyme comprising any one of amino sequences as a lecithin, e.g. phosphatidylcholine. shown as SEQ ID No. 5, 7, 8, 14 or 16 or an amino acid Preferably the lipolytic enzyme according to the present sequence which has at least 70%, 75%, 80%,85%.90%,95%, invention acts on glycolipids such as DGDG or MGDG. 96%, 97% or 98% identity therewith for the uses described 35 In one aspect the lipolytic enzyme according to the present herein. invention hydrolyses DGDG to DGMG and/or MGDG to In a further embodiment the lipolytic enzyme for use in the MGMG. methods and uses according to the present invention may be In one aspect the lipolytic enzyme according to the present a lipolytic enzyme comprising any one of amino sequences invention hydrolyses lecithin to lysolecithin. shown as SEQID No. 5, 7 or 16 or an amino acid sequence 40 When it is the case that the lipolytic enzyme is capable of which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, transferring an acyl group from at least a glycolipid to a donor 97% or 98% identity therewith for the uses described herein. substrate, the polar lipid substrate may be referred to hereinas More preferably in one embodiment the lipolytic enzyme the “lipid acyl donor. for use in the methods and uses according to the present In one embodiment, the enzyme according to the present invention may be a lipolytic enzyme comprising the amino 45 invention which as well as having phospholipase and/or gly acid sequence shown as SEQ ID No. 16 or an amino acid colipase activity (generally classified as E.C. 3.1.1.26; E.C. sequence which has at least 70%, 75%, 80%,85%.90%,95%, 3.1.1.4 or E.C. 3.1.1.32 in accordance with the Enzyme 96%, 97% or 98% identity therewith. Nomenclature Recommendations (1992) of the Nomencla In another embodiment the lipolytic enzyme for use in the ture Committee of the International Union of Biochemistry methods and uses according to the present invention may be 50 and Molecular Biology) also has acyltransferase activity a lipolytic enzyme comprising the amino acid sequence (generally classified as E.C. 2.3.1.x), whereby the enzyme is shown as SEQID Nos. 12 or 14 or an amino acid sequence capable of transferring an acyl group from a lipid acyl donor which has at least 80%, 85%, 90%, 95%, 96%, 97% or 98% to one or more acceptor Substrates, such as one or more of the identity therewith. following: a sterol; a stanol; a carbohydrate; a protein; a In another embodiment the lipolytic enzyme for use in the 55 protein subunit; glycerol. methods and uses according to the present invention may be Lipid acyltransferases and their uses are taught in co-pend a lipolytic enzyme comprising the amino acid sequence ing International Patent Application number PCT/IB2004/ shown as SEQID No. 8 or an amino acid sequence which has 000655. This document is incorporated herein by reference. at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% However, the lipolytic enzymes from the genera Streptomy identity therewith. 60 ces according to the present invention are not taught in PCT/ In one embodiment the lipolytic enzyme for use in the IB2004/OOO655. methods and uses according to the present invention may be In some aspects, the lipolytic enzyme for use in the meth a lipolytic enzyme capable of hydrolysing at least galactolip ods and/or uses of the present invention may be capable of ids and/or capable of transferring an acyl group from at least transferring an acyl group from a polar lipid (as defined a galactolipid to one or more acyl acceptor Substrates, 65 herein) to one or more of the following acyl acceptor Sub wherein the enzyme is encoded by a nucleic acid selected strates: a sterol, a stanol, a carbohydrate, a protein or subunits from the group consisting of thereof, or a glycerol. US 8,889,371 B2 9 10 For some aspects the “acyl acceptor according to the corn oil, olive oil, peanut oil, coconut oil, and rapeseed oil. present invention may be any compound comprising a Lecithin from soya, rapeseed or egg yolk is also a suitable hydroxy group (-OH). Such as for example, polyvalentalco lipid substrate. The lipid substrate may be an oatlipid or other hols, including glycerol; sterol; stanols; carbohydrates; plant based material containing galactolipids. hydroxy acids including fruit acids, citric acid, tartaric acid, 5 In one aspect the lipid substrate or lipid acyl donor is lactic acid and ascorbic acid; proteins or a Sub-unit thereof, preferably lecithin (such as phosphatidylcholine) in egg yolk. Such as amino acids, protein hydrolysates and peptides (partly For some aspects of the present invention, the lipid may be hydrolysed protein) for example; and mixtures and deriva selected from lipids having a fatty acid chain length of from 8 tives thereof. to 22 carbons. In some aspects, the “acyl acceptor according to the 10 present invention may be preferably not water. For some aspects of the present invention, the lipid may be In one embodiment, the acyl acceptor is preferably not a selected from lipids having a fatty acid chain length of from monoglyceride and/or a diglyceride. 16 to 22 carbons, more preferably of from 16 to 20 carbons. In one aspect, preferably the enzyme is capable of trans For some aspects of the present invention, the lipid may be ferring an acyl group from a lipid to a sterol and/or a stanol. 15 selected from lipids having a fatty acid chain length of no In one aspect, preferably the enzyme is capable of trans greater than 14 carbons, Suitably from lipids having a fatty ferring an acyl group from a lipid to a carbohydrate. acid chain length of from 4 to 14 carbons, suitably 4 to 10 In one aspect, preferably the enzyme is capable of trans carbons, suitably 4 to 8 carbons. ferring an acyl group from a lipid to a protein or a subunit Suitably, the lipolytic enzyme according to the present thereof. Suitably the protein subunit may be one or more of invention exhibits at least glycolipase activity (E.C.3.1.1.26). the following: anamino acid, a protein hydrolysate, a peptide, Suitably, the lipolytic enzyme according to the present inven a dipeptide, an oligopeptide, a polypeptide. tion may also exhibit phospholipase A2 activity (E.C.3.1.1.4) Suitably in the protein or protein subunit the acyl acceptor and/or phospholipase A1 activity (E.C.3.1.1.32). may be one or more of the following constituents of the For Some aspects, the lipolytic enzyme according to the protein or protein subunit: a serine, a threonine, a tyrosine, or 25 present invention may solely have glycolipase activity (E.C. a cysteine. 3.1.1.26). When the protein subunit is an amino acid, suitably the For Some aspects, the lipolytic enzyme according to the amino acid may be any suitable amino acid. Suitably the present invention is a galactolipase (E.C. 3.1.1.26). The fact amino acid may be one or more of a serine, a threonine, a that the enzyme is designated at a galactolipase does not, tyrosine, or a cysteine for example. 30 In one aspect, preferably the enzyme is capable of trans however, prevent it from having other side-activities, such as ferring an acyl group from a lipid to glycerol. activity towards other polar lipids for example. In one aspect, preferably the enzyme is capable of trans The terms 'glycolipase activity” and "galactolipase activ ferring an acyl group from a lipid to a hydroxy acid. ity as used herein are used interchangeably. In one aspect, preferably the enzyme is capable of trans 35 Suitably, for some aspects the lipolytic enzyme according ferring an acyl group from a lipid to a polyvalent alcohol. to the present invention may be capable of transferring an acyl In one aspect, the lipolytic enzyme may, as well as being group from a glycolipid and/or a phospholipid to one or more able to transfer an acyl group from a lipid to a sterol and/or a acceptor Substrates. stanol, additionally be able to transfer the acyl group from a Suitably the acceptor substrate may be one or more of the lipid to one or more of the following: a carbohydrate, a pro 40 following Substrates: a sterol, a stanol, a carbohydrate, a tein, a protein subunit, glycerol. protein, glycerol. The term lecithin as used herein encompasses phosphati The term “polar lipids” as used herein means phospholip dylcholine, phosphatidylethanolamine, phosphatidylinositol, ids and/or glycolipids. In some aspects, the term polar lipids phosphatidylserine and phosphatidylglycerol. preferably means at least glycolipids. For some aspects, preferably the lipid substrate is at least a 45 The glycolipase activity; phospholipase activity and/or glycolipid, such as DGDG for example. triacylglycerol lipase activity of an enzyme can be deter For some aspects, preferably the lipid substrate may be mined using the assays presented hereinbelow. additionally a phospholipid, such as lecithin, for example Determination of Galactolipase Activity (Glycolipase Activ phosphatidylcholine. Other phospholipid substrates in accor ity Assay (GLU-7)): dance with the present invention may be one or more of Nacyl 50 Substrate phosphatidyl ethanolamine (APE) or N acyl lyso-phosphati 0.6% digalactosyldiglyceride (Sigma D 4651), 0.4% Tri dyl ethanolamine (ALPE). Preferably the lipid substrate is a food lipid, that is to say a ton-X 100 (Sigma X-100) and 5 mM CaCl was dissolved in lipid component of a foodstuff. 0.05M HEPES buffer pH 7. For some aspects, preferably the lipolytic enzyme accord 55 Assay Procedure: ing to the present invention is incapable, or Substantially 400 uL substrate was added to an 1.5 mL Eppendorf tube incapable, of acting on a triglyceride and/or a 1-monoglycer and placed in an Eppendorf Thermomixer at 37° C. for 5 ide and/or 2-monoglyceride. minutes. At time t—0 min, 50 uL enzyme solution was added. In one embodiment the lipolytic enzyme according to the Also a blank with water instead of enzyme was analyzed. The present invention has no activity or no significant activity on 60 sample was mixed at 10x100 rpm in an Eppendorf Thermo triglyceride and/or 1-monoglycerides and/or 2-monoglycer mixer at 37°C. for 10 minutes. At time t—10 min the Eppen ides. dorf tube was placed in another thermomixer at 99°C. for 10 Suitably, the lipid substrate or lipid acyl donor may be one minutes to stop the reaction. or more lipids present in one or more of the following Sub Free fatty acid in the samples was analyzed by using the strates: fats, including lard, tallow and butter fat; oils includ 65 NEFA C kit from WAKO GmbH. ing oils extracted from or derived from palm oil, sunflower Enzyme activity GLU at pH 7 was calculated as micromole oil, Soya bean oil, safflower oil, cotton seed oil, groundnut oil, fatty acid produced per minute under assay conditions US 8,889,371 B2 11 12 Determination of Phospholipase Activity (Phospholipase preferably at least 85%, preferably at least 90%, preferably at Activity Assay (PLU-7)): least 95%, preferably at least 98%, preferably at least 99% Substrate identity with the nucleotide sequence shown in SEQID No. 3. 0.6% L-C. Phosphatidylcholine 95% Plant (Avanti In one embodiment suitably the pH optimum of the #441601), 0.4% Triton-X 100 (Sigma X-100) and 5 mM enzyme on a galactolipid substrate is about 6-8, preferably CaCl was dispersed in 0.05M HEPES buffer pH 7. about 6.5 to 7.5, more preferably about 7. Assay Procedure: Suitably, the lipolytic enzyme according to the present 400 uL substrate was added to a 1.5 mL Eppendorf tube and invention may not be inhibited or not significantly be inhib placed in an Eppendorf Thermomixer at 37°C. for 5 minutes. ited by lipases inhibitors present in wheat flour. The term “not At time t—0 min, 50 uL enzyme solution was added. Also a 10 significantly inhibited as used herein means that the enzyme blank with water instead of enzyme was analyzed. The is less sensitive to lipase inhibitors present in the wheat flour sample was mixed at 10x100 rpm in an Eppendorf Thermo when compared to an equivalent dosage (PLU) of Lipo mixer at 37°C. for 10 minutes. At time t—10 min the Eppen panFTM (Novozymes A/S, Denmark), as based on the stan dorf tube was placed in another thermomixer at 99 C. for 10 dard phospholipase (PLU-7) assay defined herein. minutes to stop the reaction. 15 Suitably, the lipolytic enzyme according to the present Free fatty acid in the samples was analyzed by using the invention is capable of hydrolysing at least 10% of the galac NEFA C kit from WAKO GmbH. tolipid diester in the Substrate (i.e. in the foodstuff, e.g. dough, Enzyme activity PLU-7 at pH 7 was calculated as micro for instance) to the monoester. Preferably, the enzyme is mole fatty acid produced per minute under assay conditions capable of hydrolysing at least 20%, more preferably at least Determination of Triacylglyceride Lipase Activity: Assay 30%, at least 40%, at least 50%, at least 60%, at least 70%, at Based on Triglyceride (Tributyrin) as Substrate (LIPU): least 80% or at least 90% of the galactolipid diester to the Lipase activity based on tributyrin is measured according mono ester. Suitably, the galactolipid diester may be one or to Food Chemical Codex, Forth Edition, National Academy more of MGDG or DGDG and the monoester may be one or Press, 1996, p. 803. With the modification that the sample is more of MGMG or DGMG, respectively. dissolved in deionized water in stead of glycine buffer, and 25 Suitably, the lipolytic enzyme according to the present the pH stat set point is 5.5 instead of 7. invention may be isolated from a fermentation broth of Strep 1 LIPU is defined as the quantity of enzyme which can tomyces strain L131 or Streptomyces strain L130. liberate 1 micromole butyric acid per min. under assay con Suitably, the enzyme may be purified by liquid chromatog ditions. raphy. In one embodiment, preferably the lipolytic enzyme 30 The amino acid sequence of the purified lipolytic enzyme according to the present invention is a wild-type lipolytic may be determined by Edman degradation, LC-MS and enzyme. MALDI-TOF analysis. The terms “natural and “wild type' as used herein mean a Suitably, the enzyme as defined herein may catalyse one or naturally-occurring enzyme. That is to say an enzyme more of the following reactions: interesterification, transes expressed from the endogenous genetic code and isolated 35 terification, alcoholysis, hydrolysis. from its endogenous host organism and/or a heterologously The term “interesterification” refers to the enzymatic produced enzyme which has not been mutated (i.e. does not catalysed transfer of acyl groups between a lipid donor and contain amino acid deletions, additions or Substitutions) lipid acceptor, wherein the lipid donor is not a free acyl group. when compared with the mature protein sequence (after co The term “transesterification' as used herein means the and post-translational cleavage events) endogenously pro 40 enzymatic catalysed transfer of an acyl group from a lipid duced. Natural and wild-type proteins of the present invention donor (other than a free fatty acid) to an acyl acceptor (other may be encoded by codon optimised polynucleotides for than water). heterologous expression, and may also comprise a non-en As used herein, the term “alcoholysis” refers to the enzy dogenous signal peptide selected for expression in that host. matic cleavage of a covalent bond of an acid derivative by The term “variant as used herein means a protein 45 reaction with an alcohol ROH so that one of the products expressed from a non-endogenous genetic code resulting in combines with the H of the alcohol and the other product one or more amino acid alterations (i.e. amino acid deletions, combines with the OR group of the alcohol. additions or substitutions) when compared with the natural or As used herein, the term “alcohol refers to an alkyl com wild-type sequence within the mature protein sequence. pound containing a hydroxyl group. Preferably, the lipolytic enzyme according to the present 50 As used herein, the term “hydrolysis” refers to the enzy invention is obtainable (suitably may be obtained) from a matic catalysed transfer of an acyl group from a lipid to the bacterium. OH group of a water molecule. Acyl transfer which results Preferably, the lipolytic enzyme according to the present from hydrolysis requires the separation of the water mol invention may be obtainable (preferably obtained) from ecule. Streptomyces spp. Preferably, the lipolytic enzyme according 55 The term “foodstuff as used herein means a substance to the present invention may be obtainable (preferably which is suitable for human and/or animal consumption. obtained) from Streptomyces strain L131 or Streptomyces Suitably, the term “foodstuff as used herein may mean a strain L130. foodstuff in a form which is ready for consumption. Alterna Preferably, the lipolytic enzyme according to the present tively or in addition, however, the term foodstuff as used invention comprises an amino acid sequence which has at 60 herein may mean one or more food materials which are used least 70%, preferably at least 75%, preferably at least 80%, in the preparation of a foodstuff. By way of example only, the preferably at least 90%, preferably at least 95%, preferably at term foodstuffencompasses both baked goods produced from least 98%, preferably at least 99% identity with the amino dough as well as the dough used in the preparation of said acid sequence shown as SEQID No. 4. baked goods. Preferably, the nucleic acid encoding the lipolytic enzyme 65 In a preferred aspect the present invention provides a food according to the present invention comprises a nucleotide stuff as defined above wherein the foodstuff is selected from sequence which has at least 75%, preferably at least 80%, one or more of the following: eggs, egg-based products, US 8,889,371 B2 13 14 including but not limited to mayonnaise, Salad dressings, mayonnaise, Salad dressings, sauces, ice cream, egg powder, sauces, ice creams, egg powder, modified egg yolk and prod modified egg yolk and products made therefrom. ucts made therefrom: baked goods, including breads, cakes, Preferably the foodstuff according to the present invention Sweet dough products, laminated doughs, liquid batters, muf is a water containing foodstuff. Suitably the foodstuffmay be fins, doughnuts, biscuits, crackers and cookies; confection comprised of 10-98% water, suitably 14-98%, suitably of ery, including chocolate, candies, caramels, halawa, gums, 18-98% water, suitably of 20-98%, suitably of 40-98%, suit including Sugar free and Sugar Sweetened gums, bubblegum, ably of 50-98%, suitably of 70-98%, suitably of 75-98%. Softbubblegum, chewing gum and puddings; frozen products For some aspects, the foodstuff in accordance with the including sorbets, preferably frozen dairy products, including present invention may not be a pure plant derived oil. Such as 10 olive oil, Sunflower oil, peanut oil, rapeseed oil for instance. ice cream and ice milk; dairy products, including cheese, For the avoidance of doubt, in some aspects of the present butter, milk, coffee cream, whipped cream, custard cream, invention the foodstuff according to the present invention milk drinks and yoghurts; mousses, whipped vegetable may comprise an oil, but the foodstuff is not primarily com creams, meat products, including processed meat products; posed of oil or mixtures of oil. For some aspects, preferably edible oils and fats, aerated and non-aerated whipped prod 15 the foodstuff comprises less than 95% lipids, preferably less ucts, oil-in-water emulsions, water-in-oil emulsions, marga than 90% lipids, preferably less than 85%, preferably less rine, shortening and spreads including low fat and very low fat than 80% lipids. Thus, for some aspects of the present inven spreads; dressings, mayonnaise, dips, cream based sauces, tion oil may be a component of the foodstuff, but preferably cream based soups, beverages, spice emulsions and sauces. the foodstuff is not an oil perse. Suitably the foodstuff in accordance with the present The advantages of using a lipolytic enzyme capable of invention may be a “fine foods', including cakes, pastry, transferring an acyl group in food applications is taught in confectionery, chocolates, fudge and the like. patent applications WO2004/064987, WO2004/064537, In one aspect the foodstuff in accordance with the present PCT/IB2004/004374 and GB0513859.9 which are incorpo invention may be a dough productor a baked product, Such as rated herein by reference. a bread, a fried product, a Snack, cakes, pies, brownies, cook 25 The production of free fatty acids can be detrimental to ies, noodles, Snack items such as crackers, graham crackers, foodstuffs. Free fatty acids have been linked with off-odours pretzels, and potato chips, and pasta. and/or off-flavours in foodstuffs, as well other detrimental In a further aspect, the foodstuff in accordance with the effects, including a Soapy taste in dairy products such as present invention may be a plant derived food product such as cheese for instance. Suitably in some embodiments of the flours, pre-mixes, oils, fats, cocoa butter, coffee whitener, 30 present invention the lipolytic enzyme is capable of transfer salad dressings, margarine, spreads, peanut butter, shorten ring the fatty acid from the lipid to an acyl acceptor, for ings, ice cream, cooking oils. example a sterol and/or a stanol. Hence, the overall level of In another aspect, the foodstuff in accordance with the free fatty acids in the foodstuff does not increase or increases present invention may be a dairy product, including butter, only to an insignificant degree. Thus, a lipolytic enzyme milk, cream, cheese such as natural, processed, and imitation 35 capable of transferring an acyl group according to the present cheeses in a variety of forms (including shredded, block, invention may provide one or more of the following unex slices or grated), cream cheese, ice cream, frozen desserts, pected technical effects in the production of cheese: a yoghurt, yoghurt drinks, butter fat, anhydrous milk fat, other decrease in the oiling-off effect in cheese; an increase in dairy products. The enzyme according to the present inven cheese yield; an improvement in flavour; a reduced mal tion may improve fat stability in dairy products. 40 odour, a reduced 'soapy' taste. It is particularly advantageous to utilise the enzyme The utilisation of a lipolytic enzyme taught herein which according to the present invention in cheese. Thus, a lipolytic can transfer the acyl group to a carbohydrate as well as to a enzyme in accordance with the present invention can advan sterol and/or a stanol is particularly advantageous for food tageously be used to produce cheese. The lipolytic enzyme stuffs comprising eggs. In particular, the presence of Sugars, catalyses the hydrolysis of phospholipids in the milk which 45 in particular glucose, in eggs and egg products is often seen as contributes to increased cheese yield. Preferably the lipolytic disadvantageous. Egg yolk may comprise up to 1% glucose. enzyme according to the present invention may be added to In accordance with the present invention this unwanted Sugar milk (referred to as cheese milk) prior to or during the cheese can be readily removed by “esterifying the sugar to form a making process. Sugar ester. In another aspect, the foodstuff in accordance with the 50 The presence of diglycerides in edible oils is disadvanta present invention may be a food product containing animal geous. In particular, diglycerides in edible oils (in particular derived ingredients, such as processed meat products, cook palm oil) can lead to a low quality oil. Suitably in some ing oils, shortenings. embodiments of the present invention a lipolytic enzyme In a further aspect, the foodstuff in accordance with the taught herein is capable of transferring the fatty acid from the present invention may be a beverage, a fruit, mixed fruit, a 55 lipid to an acyl acceptor which reduces the level of diglycer Vegetable or wine. In some cases the beverage may contain up ides in the oil without increasing or significantly increasing to 20 g/l of added phytosterols. the level of free fatty acids. In another aspect, the foodstuff in accordance with the A lipolytic enzyme taught herein is able to hydrolyse a present invention may be an animal feed. The animal feed major part of the phospholipids in an edible or vegetable oil. may be enriched with phytosterol and/or phytostanols, pref 60 This is highly advantageous in the enzymatic degumming of erably with beta-sitosterol/stanol. Suitably, the animal feed vegetable or edible oils. Suitably in some embodiments of the may be a poultry feed. When the foodstuff is poultry feed, the present invention the lipolytic enzyme may be capable of present invention may be used to lower the cholesterol con transferring the fatty acid from the lipid to an acyl acceptor. tent of eggs produced by poultry fed on the foodstuff accord Hence, advantageously the overall level of free fatty acids in ing to the present invention. 65 the oil does not increase or increases only to an insignificant In one aspect preferably the foodstuff is selected from one degree. The production of free fatty acids can be detrimental or more of the following: eggs, egg-based products, including in the edible oil. Preferably, the method according to the US 8,889,371 B2 15 16 present invention results in the degumming of an edible oil (whether of white, light or dark type) which may advanta wherein the accumulation of free fatty acids is reduced and/or geously produced by the present invention include one or eliminated. more of the following: bread (including white, whole-meal The claims of the present invention are to be construed to and rye bread), typically in the form of loaves or rolls, steam include each of the foodstuffs listed above. buns, French baguette-type bread, pita bread, tacos, corn In some of the applications mentioned herein, particularly tortilla, wheat tortilla, cakes, pancakes, biscuits, crisp bread, the food applications, such as the bakery applications, the pasta, noodles and the like. lipolytic enzyme according to the present invention may be The dough in accordance with the present invention may be used with one or more conventional emulsifiers, including for a leavened dough or a dough to be subjected to leavening. The example monoglycerides, diacetyl tartaric acid esters of 10 dough may be leavened in various ways such as by adding mono- and diglycerides of fatty acids, Sugar esters, sodium Sodium bicarbonate or the like, or by adding a Suitable yeast stearoyl lactylate (SSL) and lecithins. culture Such as a culture of Saccharomyces cerevisiae (bak In addition or alternatively, the enzyme according to the er's yeast). present invention may be used with one or more other suitable The present invention further relates to the use of the food grade enzymes. Thus, it is within the scope of the present 15 lipolytic enzyme in accordance with the present invention to invention that, in addition to the lipolytic enzyme of the produce a pasta dough, preferably prepared from durum flour present invention, at least one further enzyme may be added or a flour of comparable quality. to the baked product and/or the dough. Such further enzymes The lipolytic enzyme according to the present invention is include starch degrading enzymes Such as endo- or exoamy Suitable for use in the enzymatic degumming of vegetable or lases, pullulanases, debranching enzymes, hemicellulases edible oils. In processing of vegetable or edible oil the edible including Xylanases, cellulases, oxidoreductases, e.g. glucose or vegetable oil is treated with lipolytic enzyme according to oxidase, pyranose oxidase, Sulfhydryl oxidase or a carbohy the present invention so as to hydrolyse a major part of the drate oxidase Such as one which oxidises maltose, for polar lipids (e.g. phospholipid). Preferably, the fatty acyl example hexose oxidase (HOX), lipases, and groups are hydrolysed from the polar lipids. The degumming hexose oxidase, proteases, and acyltransferases (such as 25 process typically results in the reduction of the content of the those described in PCT/IB2004/000575 for instance). polar lipids, particularly of phospholipids, in an edible oil due The present invention encompasses food enzyme compo to hydrolyse of a major part (i.e. more than 50%) of the polar sitions, including bread and/or dough improving composi lipid, e.g. phospholipid. Typically, the aqueous phase con tions comprising the enzyme according to the present inven taining the hydrolysed polar lipid (e.g. phospholipid) is sepa tion, and optionally further comprising another enzyme. Such 30 rated from the oil. Suitably, the edible or vegetable oil may as one or more other Suitable food grade enzymes, including initially (pre-treatment with the enzyme according to the Starch degrading enzymes such as endo- or exoamylases, present invention) have a phosphorus content of 50-250 ppm. pullulanases, debranching enzymes, hemicellulases includ In one embodiment, the present invention relates to the use ing Xylanases, cellulases, oxidoreductases, e.g. glucose oxi of the lipolytic enzyme in accordance with the present inven dase, pyranose oxidase, Sulfhydryl oxidase or a carbohydrate 35 tion in the bioconversion of polar lipids (preferably glycolip oxidase Such as one which oxidises maltose, for example ids) to make high value products, such as carbohydrate esters hexose oxidase (HOX), lipases, phospholipases and hexose and/or protein esters and/or protein subunit esters and/or a oxidase, proteases and acyltransferases (such as those hydroxy acid ester. The use of a lipolytic enzyme, particularly described in PCT/IB2004/000575 for instance). a lipolytic enzyme capable of transferring acyl groups from a In some applications mentioned herein, particularly in 40 polar lipid substrate (preferably a glycolipid) to a acyl accep food applications, such as the bakery applications, the lipoly tor, in the bioconversion of polar lipids and the advantages tic enzyme according to the present invention may be added in thereof is detailed in PCT/IB2004/004374 incorporated combination or sequentially with one or more enzyme Sub herein by reference. strates. By way of example only, the lipolytic enzyme accord In one embodiment the lipolytic enzyme for use in the ing to the present invention may be added together with one or 45 methods of the present invention may be immobilised. When more polar lipid Substrates and/or one or more acyl acceptor it is the case that the enzyme is immobilised the admixture Substrates. comprising an acyl donor, optionally an acyl acceptor, and In some applications mentioned herein, particularly in optionally water may be passed through a column for food applications, such as the bakery applications, the lipoly example comprising the immobilised enzyme. By immobil tic enzyme according to the present invention may be used 50 ising the enzyme it is possible to easily reuse it. with one or more hydroxy acids, including for example tar Suitably, the immobilised enzyme may be used in a flow taric acid, citric acid, lactic acid, Succinic acid or ascorbic reactor or in a batch reactor containing a reaction mixture acid for example. which comprises a lipid acyl donor and optionally an acyl The term “improved properties” as used herein means any acceptor dissolved in water. When the acyl acceptor is present property which may be improved by the action of the lipolytic 55 the donor and acceptor are in a two-phase system or an emul enzyme of the present invention. In particular, the use of the Sion. The reaction mixture may be optionally stirred or Soni lipolytic enzyme according to the present invention results in cated. Once the reaction has reached equilibrium for one or more of the following characteristics: increased Vol example, the reaction mixture and the immobilised enzyme ume of the baked product; improved crumb structure of the may be separated. Suitably, the reaction product may be frac baked product; anti-Staling properties in the baked product; 60 tionated for example by hydrophobic interaction chromatog increased strength, increased stability, reduced Stickiness raphy, crystallisation or high vacuum distillation. and/or improved machinability of the dough. Immobilised lipid acyl transferase can be prepared using The improved properties are evaluated by comparison with immobilisation techniques known in the art. There are numer a dough and/or a baked product prepared without addition of ous methods of preparing immobilised enzymes, which will the lipolytic enzyme according to the present invention. 65 be apparent to a person skilled in the art (for example the The term “baked product as used herein includes a prod techniques referred to in EP 0746 608; or Balcao V. M. etal uct prepared from a dough. Examples of baked products Enzyme Microb Technol. 1996 May 1; 18 (6):392-416; or

US 8,889,371 B2 23 24 - Continued 1141 ccgt.cggc cc atgtggcc.gt gctgggctac ccc.cgcttct acaaactggg cggct Cotgc 12 O1 Ctcgcgggcc t ct cq9agac Caagcggit Co gcc at Caacg acgcggc.cga citatctgaac 1261 agcgc.cat cq cCaagcgc.gc cgc.cgaccac ggctt cacct tcggcgacgt. Caagagc acc 1321 ttcaccggcc atgagatctg Ctc.ca.gcagc acctggctgc acagt ct cqa cctgctgaac 1381 atcggc.cagt CCtaCCaCCC gaccgcggcc ggc.cagt cc.g gcggctat ct gcc.ggtcatg 1441 alacagcgtgg cctgagct co cacggcctga atttitta agg cctgaattitt taaggcgaag 15 O1. gtgaaccgga agcggaggcc cc.gtc.cgt.cg gggtctic.cgt. cgcacaggto accgagaacg 15 61 gcacggagtt ggacgt.cgtg cgcaccgggit cqc.gcacctic gacggcgatc tcgttcgaga 1621 tcqttic cqct cgtgtcgtac gtggtgacga acacctgctt Ctgctgggtc titt cogcc.gc 1681 tcgc.cgggaa gga cagcgt.c titc.ca.gc.ccg gatcc.gggac citc.gc.ccttc ttggit caccc 1741 agcgg tactic cacct cqacc ggc acccggc ccaccgtgaa ggtc.gc.cgtg aacgtgggcg 18O1 CCtggg.cggit ggg.cgg.cggg Caggcaccgg agtag toggt gtgcacgc.cg gtgaccgt.ca 1861 cctt cacgga Ctgggc.cggc ggggtcgt.cg taccgcc.gc.c gcc accgc.cg Cct cocggag 1921 tggagc.ccga gctgttggtc.g CCC cc.gc.cgt. C9gcgttgtc. gtCct C9ggg gttitt cqaac A / Thermobifidavifuscaw - GDSix (SEQ ID NO: 31) SEQ ID No. 16 1 mgsgpraatr rirlflgipal willwtaltl will avptgretlw rmwceat qdw clgvpvdisrg 61 qipaedgefill lspvgaatwg nyyalgdsys Sgdgardyyip gtavkggcWr sanaypelva 121 eaydfaghills flac Sggrgy amlidaidevg sqldwnspht silvtigiggin dlgfstvlkt 181 cmvrvpillds kactdgedai rkrmakfett feeliSevrt rapidarilvv gyprifpeep 241 togayytiltas ngrwilnetic efnqqlaeav avhdeeiaas ggvgsvefvd vyhaldghei 3 O1 gsdepwVngv qlrdlatgvt vdrstfhpna aghravgerv ieqietgpgr ply at favva 3 61 gatvdtlage A / Thermobifidavifuscaw - GDSix (SEQ ID NO: 31) SEQ ID No. 17 Ctgcagacac cc.gc.ccc.gc.c ttct c ccgga tcqt catgtt cggcgacticc Ctcagcgaca 61 ccggcaagat gtactic caag atgcgcggct acctg.ccgt.c ctic ccc.gc.cg tactacgagg 121 gocgcttct c gaacggc.ccg gtctggctgg agcagctgac gaagcagttc cc.cggcctga 181 cqatcqccaa cgaggcc.gag ggggg.cgcga cc.gcagt cqc ctacaacaag atctoctdga 241 accc.gaagta c caggit catt aacaacct cq actacgaggit cacccagttc ttgcagaagg 301 actic gttcaa gcc.cgacgac ctggit catcc tgttgggtggg cgc.caacgac tacctggcct 361 acggttggaa cacggagcag gacgc.caa.gc gggtgcgcga cgc catctog gacgcggcaa. 421 accgcatggit Cctgaacggc gcgaa.gcaga tcc tigctgtt caacctg.ccc gacctgggcc 481 agaaccc.gtc cgc.ccgct Co Cagalaggtog tdgaggcc.gt Ctc.gcacgtg to cqcct acc 541 acaacaagct gct cotcaac Ctcgc.ccggc agct cqc ccc gacgggCatg gtcaa.gctgt 6O1 to gagat.cga caa.gcagttc gcggagatgc tgcgcgaccc ccagaacttic ggCCtgagcg 661 acgtggagaa cc.cgtgctac gacgg.cggct acgtgtggaa gcc.gttcgc.c acccggit cog 721 totcgaccga ccggcagctg tcqgccttct cgc.cccagga gcgc.ctggcg atcgctggca 781 accogctcct ggCacagg.cg gtagctt.cgc cgatggc.ccg cc.gct cqgcc tcqc.ccct ca 841 actg.cgaggg caagatgttc tgggaccagg to Cacco CaC Caccgtggtc cacgcc.gc.cc 901 totcggagcg cgc.cgccacc ttcatcgaga CCC agtacga gttcc togcc cactagt cta 961 gaggat.cc

Thus, in a further aspect, the present invention provides the 40 In another aspect, the present invention yet further provides use of a lipolytic enzyme comprising any one of the amino the use of a lipolytic enzyme comprising any one of the amino acid sequences shown as SEQID No. 4, 5, 7, 8, 12, 14, or 16 acid sequences shown as SEQID No. 4, 5, 7, 8, 12, 14 or 16 oran amino acid sequence which has at least 70%, 75%, 80%, oran amino acid sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith, or encoded by any one of the nucleotide sequences shown as 45 85%, 90%, 95%, 96%, 97% or 98% identity therewith, or SEQ ID No. 3, 6, 9, 13, 15 or 17 or a nucleotide sequence encoded by any one of the nucleotide sequences shown as which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, SEQ ID No. 3, 6, 9, 13, 15 or 17 or a nucleotide sequence 97% or 98% identity therewith, in a foodstuff for the prepa which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, ration of a lyso-glycolipid, for example digalactosyl 97% or 98% identity therewith, in an egg or an egg-based monoglyceride (DGMG) or monogalactosyl monoglyceride 50 product for the hydrolysis of phospholipids and/or glycolip (MGMG) by treatment of a glycolipid (e.g. digalactosyl dig ids. lyceride (DGDG) or monogalactosyl diglyceride (MGDG)) In another aspect the present invention provides the use of with the lipolytic enzyme according to the present invention a lipolytic enzyme comprising any one of the amino acid to produce the partial hydrolysis product, i.e. the lyso-gly colipid. sequences shown as SEQID No. 4, 5, 7, 8, 12, 14 or 16 or an In a further aspect, the present invention yet further pro 55 amino acid sequence which has at least 70%, 75%, 80%, 85%, vides the use of a lipolytic enzyme comprising any one of the 90%. 95%, 96%, 97% or 98% identity therewith, or encoded amino acid sequences shown as SEQID No. 4, 5, 7, 8, 12, 14 by any one of the nucleotide sequences shown as SEQID No. or 16 oran amino acid sequence which has at least 70%, 75%, 3, 6, 9, 13, 15 or 17 or a nucleotide sequence which has at least 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith, 70%, 75%, 80%, 85%, 90%, 95%,96%.97% or 98% identity or encoded by any one of the nucleotide sequences shown as 60 therewith, in a substrate (preferably a foodstuff) for hydrol SEQ ID No. 3, 6, 9, 13, 15 or 17 or a nucleotide sequence ysing fatty acyl groups. which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, In another aspect the present invention provides the use of 97% or 98% identity therewith, in a foodstuff for the prepa a lipolytic enzyme comprising any one of the amino acid ration of a lyso-phospholipid, for example lysolecithin, by sequences shown as SEQID No. 4, 5, 7, 8, 12, 14 or 16 or an treatment of a phospholipid (e.g. lecithin) with the enzyme to 65 amino acid sequence which has at least 70%, 75%, 80%, 85%, produce the partial hydrolysis product, i.e. a lyso-phospho 90%. 95%, 96%, 97% or 98% identity therewith, or encoded lipid. by any one of the nucleotide sequences shown as SEQID No. US 8,889,371 B2 25 26 3, 6, 9, 13, 15 or 17 or a nucleotide sequence which has at least In a further aspect the present invention may provide a 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity lipolytic enzyme capable of hydrolysing at least a glycolipid therewith, in an edible oil for reducing the content of a phos and/or capable of transferring an acyl group from at least a pholipid. galactolipid to one or more acyl acceptors, wherein the In a further aspect the present invention relates to the use of 5 enzyme comprises SEQID No. 16 oran amino acid sequence the lipolytic enzyme comprising any one of the amino acid which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, sequences shown as SEQID No. 4, 5, 7, 8, 12, 14 or 16 or an 97% or 98% identity therewith, or the enzyme is encoded by amino acid sequence which has at least 70%, 75%, 80%, 85%, any one of the nucleotide sequences shown as SEQID No. 17 90%. 95%, 96%, 97% or 98% identity therewith, or encoded or a nucleotide sequence which has at least 70%, 75%, 80%, 10 85%, 90%. 95%, 96%, 97% or 98% identity therewith. by any one of the nucleotide sequences shown as SEQID No. In one embodiment of the present invention preferably the 3, 6, 9, 13, 15 or 17 or a nucleotide sequence which has at least Streptomyces species from which the lipolytic enzyme is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity obtainable (or obtained) is not Streptomyces rimosus. therewith, in a substrate (preferably a bioconversion mixture In one embodiment of the present invention preferably the comprising polar lipids (preferably glycolipids)) for the pro 15 Streptomyces species from which the lipolytic enzyme is duction of make high value products, such as carbohydrate obtainable (or obtained) is not Streptomyces coelicolor: esters and/or protein esters and/or protein Subunit esters and/ Advantages or a hydroxy acid ester. One advantage of the present invention is that the lipolytic In a preferable aspect, the present invention relates to a enzyme has significant glycolipid hydrolysing activity. This lipolytic enzyme comprising any one of amino sequences was Surprising for a lipolytic enzyme from Streptomyces spp. shown as SEQID No. 8, 14 or 16 or an amino acid sequence In addition, this was surprising for a lipolytic enzyme from which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, Thermobifida and Corynebacterium spp. 97% or 98% identity therewith for the uses described herein. A further advantage of the present invention is that the More preferably the present invention relates to the use of lipolytic enzyme has no or no significant triacylglycerol a lipolytic enzyme comprising the amino acid sequence 25 hydrolysing activity. shown as SEQID No. 16 or an amino acid sequence which Isolated has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or In one aspect, preferably the sequence is in an isolated 98% identity therewith. form. The term "isolated' means that the sequence is at least In a broad aspect the present invention may provide a substantially free from at least one other component with lipolytic enzyme capable of hydrolysing at least a glycolipid 30 which the sequence is naturally associated in nature and as and/or capable of transferring an acyl group from at least a found in nature. galactolipid to one or more acyl acceptors, wherein the Purified enzyme is obtainable, preferably obtained, from Thermobi In one aspect, preferably the sequence is in a purified form. fida spp., preferably T. fusca. The term “purified” means that the sequence is in a relatively In another broad aspect the present invention may provide 35 pure state—e.g. at least about 90% pure, or at least about 95% a lipolytic enzyme capable of hydrolysing at least a glycolipid pure or at least about 98% pure. and/or capable of transferring an acyl group from at least a Nucleotide Sequence galactolipid to one or more acyl acceptors, wherein the The scope of the present invention encompasses nucleotide enzyme is obtainable, preferably obtained, from Corynebac sequences encoding enzymes having the specific properties terium spp., preferably C. efficiens. 40 as defined herein. In another broad aspect the present invention may provide The term “nucleotide sequence” as used herein refers to an a lipolytic enzyme capable of hydrolysing at least a glycolipid oligonucleotide sequence or polynucleotide sequence, and and/or capable of transferring an acyl group from at least a variants, homologues, fragments and derivatives thereof galactolipid to one or more acyl acceptors, wherein the (such as portions thereof). The nucleotide sequence may be of enzyme is obtainable, preferably obtained, from Streptomy 45 genomic or synthetic or recombinant origin, which may be ces avermitilis. double-stranded or single-stranded whether representing the In a further aspect the present invention may provide a sense or anti-sense Strand. lipolytic enzyme capable of hydrolysing at least a glycolipid The term “nucleotide sequence” in relation to the present and/or capable of transferring an acyl group from at least a invention includes genomic DNA, cDNA, synthetic DNA, galactolipid to one or more acyl acceptors, wherein the 50 and RNA. Preferably it means DNA, more preferably cDNA enzyme comprises SEQID No. 5, 7, 8, 12, or 16 or an amino sequence coding for the present invention. acid sequence which has at least 70%, 75%, 80%, 85%, 90%, In a preferred embodiment, the nucleotide sequence when 95%, 96%, 97% or 98% identity therewith, or the enzyme is relating to and when encompassed by the perse scope of the encoded by any one of the nucleotide sequences shown as present invention does not include the native nucleotide SEQID No. 6, 9, 13, or 17 or a nucleotide sequence which has 55 sequence according to the present invention when in its natu at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% ral environment and when it is linked to its naturally associ identity therewith. ated sequence(s) that is/are also in its/their natural environ In a further aspect the present invention may provide a ment. For ease of reference, we shall call this preferred lipolytic enzyme capable of hydrolysing at least a glycolipid embodiment the “non-native nucleotide sequence'. In this and/or capable of transferring an acyl group from at least a 60 regard, the term “native nucleotide sequence” means an entire galactolipid to one or more acyl acceptors, wherein the nucleotide sequence that is in its native environment and enzyme comprises SEQID No. 14 or an amino acid sequence when operatively linked to an entire promoter with which it is which has at least 80%, 85%, 90%, 95%, 96%, 97% or 98% naturally associated, which promoter is also in its native identity therewith, or the enzyme is encoded by any one of the environment. However, the amino acid sequence encom nucleotide sequences shown as SEQID No. 15 or a nucleotide 65 passed by Scope the present invention can be isolated and/or sequence which has at least 80%, 85%, 90%. 95%,96%.97% purified post expression of a nucleotide sequence in its native or 98% identity therewith. organism. Preferably, however, the amino acid sequence US 8,889,371 B2 27 28 encompassed by scope of the present invention may be nal nucleotide sequence but which encodes the same, or a expressed by a nucleotide sequence in its native organism but variant, amino acid sequence as encoded by the original wherein the nucleotide sequence is not under the control of nucleotide sequence. For example, for most amino acids the the promoter with which it is naturally associated within that degeneracy of the genetic code is at the third position in the organism. 5 triplet codon (wobble position) (for reference see Stryer, Preparation of the Nucleotide Sequence Lubert, Biochemistry, Third Edition, Freeman Press, ISBN Typically, the nucleotide sequence encompassed by scope 0-7167-1920-7) therefore, a nucleotide sequence in which all of the present invention is prepared using recombinant DNA triplet codons have been “wobbled in the third position techniques (i.e. recombinant DNA). However, in an alterna would be about 66% identical to the original nucleotide tive embodiment of the invention, the nucleotide sequence 10 sequence however, the amended nucleotide sequence would could be synthesised, in whole or in part, using chemical encode for the same, or a variant, primary amino acid methods well known in the art (see Caruthers M H et al., sequence as the original nucleotide sequence. (1980) Nuc Acids Res Symp Ser 215-23 and Horn Tet al., Therefore, the present invention further relates to any (1980) Nuc Acids Res Symp Ser 225-232). nucleotide sequence that has alternative triplet codon usage A nucleotide sequence encoding an enzyme which has the 15 for at least one amino acid encoding triplet codon, but which specific properties as defined herein may be identified and/or encodes the same, or a variant, polypeptide sequence as the isolated and/or purified from any cell or organism producing polypeptide sequence encoded by the original nucleotide said enzyme. Various methods are well known within the art Sequence. for the identification and/or isolation and/or purification of Furthermore, specific organisms typically have a bias as to nucleotide sequences. By way of example, PCR amplification which triplet codons are used to encode amino acids. Pre techniques to prepare more of a sequence may be used once a ferred codon usage tables are widely available, and can be suitable sequence has been identified and/or isolated and/or used to prepare codon optimised genes. Such codon optimi purified. sation techniques are routinely used to optimise expression of By way of further example, a genomic DNA and/or cDNA transgenes in a heterologous host. library may be constructed using chromosomal DNA or mes 25 Molecular Evolution senger RNA from the organism producing the enzyme. If the Once an enzyme-encoding nucleotide sequence has been amino acid sequence of the enzyme or a part of the amino acid isolated, or a putative enzyme-encoding nucleotide sequence sequence of the enzyme is known, labelled oligonucleotide has been identified, it may be desirable to modify the selected probes may be synthesised and used to identify enzyme nucleotide sequence, for example it may be desirable to encoding clones from the genomic library prepared from the 30 mutate the sequence in order to prepare an enzyme in accor organism. Alternatively, a labelled oligonucleotide probe dance with the present invention. containing sequences homologous to another known enzyme Mutations may be introduced using synthetic oligonucle gene could be used to identify enzyme-encoding clones. In otides. These oligonucleotides contain nucleotide sequences the latter case, hybridisation and washing conditions of lower flanking the desired mutation sites. stringency are used. 35 A suitable method is disclosed in Morinaga et al (Biotech Alternatively, enzyme-encoding clones could be identified nology (1984) 2. p 646-649). Another method of introducing by inserting fragments of genomic DNA into an expression mutations into enzyme-encoding nucleotide sequences is vector, Such as a plasmid, transforming enzyme-negative bac described in Nelson and Long (Analytical Biochemistry teria with the resulting genomic DNA library, and then plating (1989), 180, p 147-151). the transformed bacteria onto agar plates containing a Sub 40 Instead of site directed mutagenesis, such as described strate for the enzyme (e.g. maltose for a glucosidase (maltase) above, one can introduce mutations randomly for instance producing enzyme), thereby allowing clones expressing the using a commercial kit such as the GeneMorph PCR enzyme to be identified. mutagenesis kit from Stratagene, or the Diversify PCR ran In a yet further alternative, the nucleotide sequence encod dom mutagenesis kit from Clontech. EPO 583 265 refers to ing the enzyme may be prepared synthetically by established 45 methods of optimising PCR based mutagenesis, which can standard methods, e.g. the phosphoroamidite method also be combined with the use of mutagenic DNA analogues described by Beucage S. L. et al., (1981) Tetrahedron Letters such as those described in EP 0 866 796. Error prone PCR 22, p 1859-1869, or the method described by Matthes et al., technologies are suitable for the production of variants of (1984) EMBO J. 3, p. 801-805. In the phosphoroamidite lipolytic enzymes with preferred characteristics. method, oligonucleotides are synthesised, e.g. in an auto 50 WO0206457 refers to molecular evolution of lipases. matic DNA synthesiser, purified, annealed, ligated and A third method to obtain novel sequences is to fragment cloned in appropriate vectors. non-identical nucleotide sequences, either by using any num The nucleotide sequence may be of mixed genomic and ber of restriction enzymes or an enzyme such as Dnase I, and synthetic origin, mixed synthetic and cDNA origin, or mixed reassembling full nucleotide sequences coding for functional genomic and cDNA origin, prepared by ligating fragments of 55 proteins. Alternatively one can use one or multiple non-iden synthetic, genomic or cDNA origin (as appropriate) in accor tical nucleotide sequences and introduce mutations during the dance with standard techniques. Each ligated fragment cor reassembly of the full nucleotide sequence. DNA shuffling responds to various parts of the entire nucleotide sequence. and family shuffling technologies are suitable for the produc The DNA sequence may also be prepared by polymerase tion of variants of lipid acyl with preferred char chain reaction (PCR) using specific primers, for instance as 60 acteristics. Suitable methods for performing shuffling can described in U.S. Pat. No. 4,683.202 or in Saiki R K et al., be found in EPO 752 008, EP1 138763, EP1 103 606. Shuf (Science (1988) 239, pp. 487-491). fling can also be combined with other forms of DNA Due to degeneracy in the genetic code, nucleotide mutagenesis as described in U.S. Pat. No. 6, 180,406 and WO sequences may be readily produced in which the triplet codon O1/34835. usage, for Some or all of the amino acids encoded by the 65 Thus, it is possible to produce numerous site directed or original nucleotide sequence, has been changed thereby pro random mutations into a nucleotide sequence, either in vivo ducing a nucleotide sequence with low homology to the origi or in vitro, and to Subsequently screen for improved function US 8,889,371 B2 29 30 ality of the encoded polypeptide by various means. Using in onymous with the term "peptide'. In some instances, the term silico and exo mediated recombination methods (see WO “amino acid sequence' is synonymous with the term 00/58517, U.S. Pat. No. 6,344,328, U.S. Pat. No. 6,361974), “enzyme'. for example, molecular evolution can be performed where the The amino acid sequence may be prepared/isolated from a variant produced retains very low homology to known Suitable source, or it may be made synthetically or it may be enzymes or proteins. Such variants thereby obtained may prepared by use of recombinant DNA techniques. have significant structural analogy to known lipolytic The enzyme encompassed in the present invention may be enzymes, but have very low amino acid sequence homology. used in conjunction with other enzymes. Thus the present As a non-limiting example. In addition, mutations or natu invention also covers a combination of enzymes wherein the ral variants of a polynucleotide sequence can be recombined 10 combination comprises the enzyme of the present invention with either the wildtype or other mutations or natural variants and another enzyme, which may be another enzyme accord to produce new variants. Such new variants can also be ing to the present invention. This aspect is discussed in a later screened for improved functionality of the encoded polypep section. tide. Preferably the amino acid sequence when relating to and The application of the above-mentioned and similar 15 when encompassed by the perse scope of the present inven molecular evolution methods allows the identification and tion is not a native enzyme. In this regard, the term “native selection of variants of the enzymes of the present invention enzyme” means an entire enzyme that is in its native environ which have preferred characteristics without any prior knowl ment and when it has been expressed by its native nucleotide edge of protein structure or function, and allows the produc Sequence. tion of non-predictable but beneficial mutations or variants. Identity/Homology There are numerous examples of the application of molecular The present invention also encompasses the use of homo evolution in the art for the optimisation or alteration of logues of any amino acid sequence of an enzyme or of any enzyme activity. Such examples include, but are not limited to nucleotide sequence encoding such an enzyme. one or more of the following: optimised expression and/or Here, the term "homologue' means an entity having a activity in a host cellor in vitro, increased enzymatic activity, 25 certain homology with the amino acid sequences and the altered substrate and/or product specificity, increased or nucleotide sequences. Here, the term "homology” can be decreased enzymatic or structural stability, altered enzymatic equated with “identity”. These terms will be used inter activity/specificity in preferred environmental conditions, changeably herein. e.g. temperature, pH, Substrate In the present context, a homologous amino acid sequence As will be apparent to a person skilled in the art, using 30 is taken to include an amino acid sequence which may be at molecular evolution tools an enzyme may be altered to least 87 or 90% identical, preferably at least 95, 96.97, 98 or improve the functionality of the enzyme. 99% identical to the sequence. Typically, the homologues will Suitably, the lipolytic enzyme used in the invention may be comprise the same active sites etc.—e.g. as the Subject amino a variant, i.e. may contain at least one amino acid substitution, acid sequence. Although homology can also be considered in deletion or addition, when compared to a parental enzyme. 35 terms of similarity (i.e. amino acid residues having similar Variant enzymes retain at least 20%, 30%, 40%, 50%, 60%, chemical properties/functions), in the context of the present 70%, 80%, 90%, 95%, 97%, 99% homology with the parent invention it is preferred to express homology in terms of enzyme. Suitable parent enzymes may include any enzyme sequence identity. with esterase or lipase activity. Preferably, the parent enzyme In the present context, an homologous nucleotide sequence aligns to the pfamO0657 consensus sequence. 40 is taken to include a nucleotide sequence which may be at In a preferable embodiment a variant lipolytic enzyme least 85 or 90% identical, preferably at least 95, 96.97, 98 or retains or incorporates at least one or more of the pfamO0657 99% identical to a nucleotide sequence encoding an enzyme consensus sequence amino acid residues found in the GDSX of the present invention (the Subject sequence). Typically, the (SEQ ID NO: 31), GANDY (SEQ ID NO: 35) and HPT homologues will comprise the same sequences that code for blocks. 45 the active sites etc. as the Subject sequence. Although homol Enzymes, such as lipases with no or low galactolipase ogy can also be considered in terms of similarity (i.e. amino and/or phospholipase activity in an aqueous environment acid residues having similar chemical properties/functions), may be mutated using molecular evolution tools to introduce in the context of the present invention it is preferred to express or enhance the galactolipase and/or phospholipase activity, homology in terms of sequence identity. thereby producing a lipolytic enzyme with significant galac 50 For the amino acid sequences and the nucleotide tolipase and/or phospholipase activity Suitable for use in the sequences, homology comparisons can be conducted by eye, compositions and methods of the present invention. or more usually, with the aid of readily available sequence Suitably the variant enzyme may have no activity on trig comparison programs. These commercially available com lycerides and/or monoglycerides and/or diglycerides. puter programs can calculate % homology between two or Alternatively, the variant enzyme for use in the invention 55 more Sequences. may have increased activity on triglycerides, and/or may also % homology may be calculated over contiguous have increased activity on one or more of the following, polar sequences, i.e. one sequence is aligned with the other lipids, phospholipids, lecithin, phosphatidylcholine, gly sequence and each amino acid in one sequence is directly colipids, digalactosyl monoglyceride, monogalactosyl compared with the corresponding amino acid in the other monoglyceride. 60 sequence, one residue at a time. This is called an “ungapped' Amino Acid Sequences alignment. Typically, such ungapped alignments are per The scope of the present invention also encompasses formed only over a relatively short number of residues. amino acid sequences of enzymes having the specific prop Although this is a very simple and consistent method, it erties as defined herein. fails to take into consideration that, for example, in an other As used herein, the term "amino acid sequence' is synony 65 wise identical pair of sequences, one insertion ordeletion will mous with the term “polypeptide' and/or the term “protein'. cause the following amino acid residues to be put out of In some instances, the term "amino acid sequence' is syn alignment, thus potentially resulting in a large reduction in 96 US 8,889,371 B2 31 32 homology when a global alignment is performed. Conse sible pair of amino acid sequences. Settings are default quently, most sequence comparison methods are designed to parameters (Gap opening penalty -10, Gap extension penalty produce optimal alignments that take into consideration pos 0.1). sible insertions and deletions without penalising unduly the For nucleic acid sequences percentage of identities (ho overall homology score. This is achieved by inserting 'gaps' mology) or “positives” are calculated by the AlignX Vec in the sequence alignment to try to maximise local homology. torNTI programme from Informax Inc. (USA) for each pos However, these more complex methods assign 'gap pen sible pair of nucleic acid sequences. Settings are default alties' to each gap that occurs in the alignment so that, for the settings which for DNA is: Gap opening penalty: 15 and Gap same number of identical amino acids, a sequence alignment extension penalty: 6.66. (same settings for multiple align with as few gaps as possible—reflecting higher relatedness 10 between the two compared sequences—will achieve a higher ments). score than one with many gaps. "Affine gap costs are typi Preferably the amino acid identity (homology) is calcu cally used that charge a relatively high cost for the existence lated across the full-length amino acid sequence (e.g. SEQ of a gap and a smaller penalty for each Subsequent residue in IDs 4, 5, 7, 8, 10, 12 and 14), or for nucleic acid to a corre the gap. This is the most commonly used gap scoring system. 15 sponding polynucleotide which encodes the respective the High gap penalties will of course produce optimised align full-length amino acid sequence. Amino acid or nucleic acid ments with fewer gaps. Most alignment programs allow the identity (homology) may be, preferably, calculated by com gap penalties to be modified. However, it is preferred to use paring the homology/identity over the mature polypeptide the default values when using Such software for sequence sequence, i.e. a polypeptide sequence which has been co- or comparisons. For example when using the GCG Wisconsin post-translationally processed, for example by cleavage of an Bestfit package the default gap penalty for amino acid N-terminal signal peptide, or a C-terminal cleavage event. sequences is -12 for a gap and -4 for each extension. The sequences may also have deletions, insertions or Sub Calculation of maximum '% homology therefore firstly stitutions of amino acid residues which produce a silent requires the production of an optimal alignment, taking into change and result in a functionally equivalent Substance. consideration gap penalties. A Suitable computer program for 25 Deliberate amino acid substitutions may be made on the basis carrying out such an alignment is the GCG Wisconsin Bestfit of similarity in amino acid properties (such as polarity, package (Devereux et al 1984 Nuc. Acids Research 12 p 387). charge, solubility, hydrophobicity, hydrophilicity, and/or the Examples of other software than can perform sequence com amphipathic nature of the residues) and it is therefore useful parisons include, but are not limited to, the BLAST package to group amino acids together in functional groups. Amino (see Ausubel et al., 1999 Short Protocols in Molecular Biol 30 acids can be grouped together based on the properties of their ogy, 4' Ed Chapter 18), FASTA (Altschul et al., 1990.J. side chain alone. However it is more useful to include muta Mol. Biol. 403-410) and the GENEWORKS suite of compari tion data as well. The sets of amino acids thus derived are son tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Proto likely to be conserved for structural reasons. These sets can be cols in Molecular Biology, pages 7-58 to 7-60). 35 described in the form of a Venn diagram (Livingstone C. D. However, for some applications, it is preferred to use the and Barton G. J. (1993) “Protein sequence alignments: a GCG Bestfit program. A new tool, called BLAST 2 strategy for the hierarchical analysis of residue conservation' Sequences is also available for comparing protein and nucle Comput. Appl Biosci. 9; 745-756) (Taylor W. R. (1986) “The otide sequence (see FEMS Microbiol Lett 1999 174 (2): 247 classification of amino acid conservation' J. Theor: Biol. 119, 50; and FEMS Microbiol Lett 1999 177(1): 187-8). 40 205-218). Conservative substitutions may be made, for Although the final % homology can be measured in terms example according to the table below which describes agen of identity, the alignment process itself is typically not based erally accepted Venn diagram grouping of amino acids. on an all-or-nothing pair comparison. Instead, a scaled simi larity Score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or 45 SET SUB-SET evolutionary distance. An example of Such a matrix com Hydro- F W Y H K M I L. W. A. G. Aromatic F. W. Y H monly used is the BLOSUM62 matrix the default matrix phobic C Aliphatic I L for the BLAST suite of programs. GCG Wisconsin programs Polar W Y H K R E D C S T N Charged H. K. R. E. D generally use either the public default values or a custom Q Positively H K R symbol comparison table if Supplied (see user manual for 50 charged further details). For some applications, it is preferred to use Negatively E D the public default values for the GCG package, or in the case charged of other software, the default matrix, such as BLOSUM62. Alternatively, percentage homologies may be calculated Small W. C. A. G. S P T N ID Tiny A G S using the multiple alignment feature in DNASISTM (Hitachi 55 Software), based on an algorithm, analogous to CLUSTAL The present invention also encompasses homologous Sub (Higgins D G & Sharp PM (1988), Gene 73 (1), 237-244). stitution (substitution and replacement are both used hereinto Once the Software has produced an optimal alignment, it is mean the interchange of an existing amino acid residue, with possible to calculate % homology, preferably '% sequence an alternative residue) that may occur i.e. like-for-like Sub identity. The software typically does this as part of the 60 stitution Such as basic for basic, acidic for acidic, polar for sequence comparison and generates a numerical result. polar etc. Non-homologous Substitution may also occur i.e. In a preferable aspect of the present invention the following from one class of residue to another or alternatively involving Software and settings for calculating percentage sequence the inclusion of unnatural amino acids such as ornithine homology/identity are used. For amino acid sequences per (hereinafter referred to as Z), diaminobutyric acid ornithine centage of identities (homology) or “positives are calculated 65 (hereinafter referred to as B), norleucine ornithine (hereinaf by the AlignX Vector NTI (Vector NTI Advance 9.1 from ter referred to as O), pyriylalanine, thienylalanine, naphthy Invitrogen Corporation, Carlsbad, Calif., USA.) for each pos lalanine and phenylglycine. US 8,889,371 B2 33 34 Replacements may also be made by unnatural amino acids. changes are required to optimise codon preferences for a Variant amino acid sequences may include Suitable spacer particular host cell in which the polynucleotide sequences are groups that may be inserted between any two amino acid being expressed. Other sequence changes may be desired in residues of the sequence including alkyl groups such as order to introduce recognition sites, or to methyl, ethyl or propyl groups in addition to amino acid 5 alter the property or function of the polypeptides encoded by spacers such as glycine or B-alanine residues. A further form the polynucleotides. of variation involves the presence of one or more amino acid Polynucleotides (nucleotide sequences) of the invention residues in peptoid form, and will be well understood by those may be used to produce a primer, e.g. a PCR primer, a primer skilled in the art. For the avoidance of doubt, “the peptoid for an alternative amplification reaction, a probe e.g. labelled form' is used to refer to variant amino acid residues wherein 10 the C-carbon Substituent group is on the residue's nitrogen with a revealing label by conventional means using radioac atom rather than the C-carbon. Processes for preparing pep tive or non-radioactive labels, or the polynucleotides may be tides in the peptoid form are known in the art, for example cloned into vectors. Such primers, probes and other frag Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Hor ments will be at least 15, preferably at least 20, for example at well DC, Trends Biotechnol. (1995) 13 (4), 132-134. 15 least 25, 30 or 40 nucleotides in length, and are also encom The nucleotide sequences for use in the present invention passed by the term polynucleotides of the invention as used may include within them synthetic or modified nucleotides. A herein. number of different types of modification to oligonucleotides Polynucleotides such as DNA polynucleotides and probes are known in the art. These include methylphosphonate and according to the invention may be produced recombinantly, phosphorothioate backbones and/or the addition of acridine synthetically, or by any means available to those of skill in the or polylysine chains at the 3' and/or 5' ends of the molecule. art. They may also be cloned by standard techniques. For the purposes of the present invention, it is to be under In general, primers will be produced by synthetic means, stood that the nucleotide sequences described herein may be involving a stepwise manufacture of the desired nucleic acid modified by any method available in the art. Such modifica sequence one nucleotide at a time. Techniques for accom tions may be carried out in order to enhance the in vivo 25 plishing this using automated techniques are readily available activity or life span of nucleotide sequences of the present in the art. invention. Longer polynucleotides will generally be produced using The present invention also encompasses the use of nucle recombinant means, for example using a PCR (polymerase otide sequences that are complementary to the sequences chain reaction) cloning techniques. The primers may be presented herein, or any derivative, fragment or derivative 30 designed to contain Suitable restriction enzyme recognition thereof. If the sequence is complementary to a fragment sites so that the amplified DNA can be cloned into a suitable thereof then that sequence can be used as a probe to identify cloning vector. similar coding sequences in other organisms etc. Biologically Active Polynucleotides which are not 100% homologous to the Preferably, the variant sequences etc. are at least as bio sequences of the present invention but fall within the scope of 35 logically active as the sequences presented herein. the invention can be obtained in a number of ways. Other As used herein “biologically active' refers to a sequence variants of the sequences described herein may be obtained having a similar structural function (but not necessarily to the for example by probing DNA libraries made from a range of same degree), and/or similar regulatory function (but not individuals, for example individuals from different popula necessarily to the same degree), and/or similar biochemical tions. In addition, other homologues may be obtained and 40 function (but not necessarily to the same degree) of the natu Such homologues and fragments thereof in general will be rally occurring sequence. capable of selectively hybridising to the sequences shown in Hybridisation the sequence listing herein. Such sequences may be obtained The present invention also encompasses sequences that are by probing cDNA libraries made from or genomic DNA complementary to the nucleic acid sequences of the present libraries from other species, and probing such libraries with 45 invention or sequences that are capable of hybridising either probes comprising all or part of any one of the sequences in to the sequences of the present invention or to sequences that the attached sequence listings under conditions of medium to are complementary thereto. high Stringency. Similar considerations apply to obtaining The term “hybridisation' as used herein shall include “the species homologues and allelic variants of the polypeptide or process by which a strand of nucleic acid joins with a comple nucleotide sequences of the invention. 50 mentary Strand through base pairing as well as the process of Variants and strain/species homologues may also be amplification as carried out in polymerase chain reaction obtained using degenerate PCR which will use primers (PCR) technologies. designed to target sequences within the variants and homo The present invention also encompasses the use of nucle logues encoding conserved amino acid sequences within the otide sequences that are capable of hybridising to the sequences of the present invention. Conserved sequences can 55 sequences that are complementary to the sequences presented be predicted, for example, by aligning the amino acid herein, or any derivative, fragment or derivative thereof. sequences from several variants/homologues. Sequence The term “variant also encompasses sequences that are alignments can be performed using computer Software complementary to sequences that are capable of hybridising known in the art. For example the GCG Wisconsin PileUp to the nucleotide sequences presented herein. program is widely used. 60 Preferably, the term “variant' encompasses sequences that The primers used in degenerate PCR will contain one or are complementary to sequences that are capable of hybrid more degenerate positions and will be used at Stringency ising under stringent conditions (e.g. 50° C. and 0.2xSSC conditions lower than those used for cloning sequences with {1xSSC=0.15 M NaCl, 0.015 M Nacitrate pH 7.0}) to the single sequence primers against known sequences. nucleotide sequences presented herein. Alternatively, such polynucleotides may be obtained by 65 More preferably, the term “variant' encompasses site directed mutagenesis of characterised sequences. This sequences that are complementary to sequences that are may be useful where for example silent codon sequence capable of hybridising under high Stringent conditions (e.g. US 8,889,371 B2 35 36 65° C. and 0.1XSSC (1xSSC=0.15 M NaCl, 0.015 M The vectors for use in the present invention may be trans Nacitrate pH 7.0) to the nucleotide sequences presented formed into a suitable host cell as described below to provide herein. for expression of a polypeptide of the present invention. The present invention also relates to nucleotide sequences The choice of vector e.g. a plasmid, cosmid, or phage that can hybridise to the nucleotide sequences of the present vector will often depend on the host cell into which it is to be invention (including complementary sequences of those pre introduced. sented herein). The vectors for use in the present invention may contain The present invention also relates to nucleotide sequences one or more selectable marker genes-such as a gene, which that are complementary to sequences that can hybridise to the confers antibiotic resistance e.g. amplicillin, kanamycin, nucleotide sequences of the present invention (including 10 chloramphenicol or tetracyclin resistance. Alternatively, the complementary sequences of those presented herein). selection may be accomplished by co-transformation (as described in WO91/17243). Also included within the scope of the present invention are Vectors may be used in vitro, for example for the produc polynucleotide sequences that are capable of hybridising to tion of RNA or used to transfect, transform, transduce or the nucleotide sequences presented herein under conditions 15 infect a host cell. of intermediate to maximal stringency. Thus, in a further embodiment, the invention provides a In a preferred aspect, the present invention covers nucle method of making nucleotide sequences of the present inven otide sequences that can hybridise to the nucleotide sequence tion by introducing a nucleotide sequence of the present of the present invention, or the complement thereof, under invention into a replicable vector, introducing the vector into stringent conditions (e.g. 50° C. and 0.2xSSC). a compatible host cell, and growing the host cell under con In a more preferred aspect, the present invention covers ditions which bring about replication of the vector. nucleotide sequences that can hybridise to the nucleotide The vector may further comprise a nucleotide sequence sequence of the present invention, or the complement thereof, enabling the vector to replicate in the host cell in question. under high stringent conditions (e.g. 65° C. and 0.1xSSC). Examples of Such sequences are the origins of replication of Recombinant 25 plasmids puC19, p.ACYC177, puB110, pE194, p.AMB1 and In one aspect the sequence for use in the present invention pIJ702. is a recombinant sequence—i.e. a sequence that has been Regulatory Sequences prepared using recombinant DNA techniques. In some applications, the nucleotide sequence for use in the These recombinant DNA techniques are within the capa present invention is operably linked to a regulatory sequence bilities of a person of ordinary skill in the art. Such techniques 30 which is capable of providing for the expression of the nucle are explained in the literature, for example, J. Sambrook, E. F. otide sequence, such as by the chosen host cell. By way of Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Labo example, the present invention covers a vector comprising the ratory Manual, Second Edition, Books 1-3, Cold Spring Har nucleotide sequence of the present invention operably linked bor Laboratory Press. to such a regulatory sequence, i.e. the vector is an expression Synthetic 35 Vector. In one aspect the sequence for use in the present invention The term “operably linked’ refers to a juxtaposition is a synthetic sequence—i.e. a sequence that has been pre wherein the components described are in a relationship per pared by in vitro chemical or enzymatic synthesis. It includes, mitting them to function in their intended manner. A regula but is not limited to, sequences made with optimal codon tory sequence “operably linked to a coding sequence is usage for host organisms—such as the methylotrophic yeasts 40 ligated in Such a way that expression of the coding sequence Pichia and Hansenula. is achieved under condition compatible with the control Expression of Enzymes Sequences. The nucleotide sequence for use in the present invention The term “regulatory sequences' includes promoters and may be incorporated into a recombinant replicable vector. enhancers and other expression regulation signals. The vector may be used to replicate and express the nucle 45 The term “promoter' is used in the normal sense of the art, otide sequence, in enzyme form, in and/or from a compatible e.g. an RNA polymerase binding site. host cell. Enhanced expression of the nucleotide sequence encoding Expression may be controlled using control sequences e.g. the enzyme of the present invention may also be achieved by regulatory sequences. the selection of heterologous regulatory regions, e.g. pro The enzyme produced by a host recombinant cell by 50 moter, secretion leader and terminator regions. expression of the nucleotide sequence may be secreted or may Preferably, the nucleotide sequence according to the be contained intracellularly depending on the sequence and/ present invention is operably linked to at least a promoter. or the vector used. The coding sequences may be designed Examples of Suitable promoters for directing the transcrip with signal sequences which direct secretion of the Substance tion of the nucleotide sequence in a bacterial, fungal or yeast coding sequences through a particular prokaryotic or eukary 55 host are well known in the art. otic cell membrane. Constructs Expression Vector The term “construct” which is synonymous with terms The term “expression vector” means a construct capable of such as “conjugate”, “cassette' and “hybrid' includes a in vivo or in vitro expression. nucleotide sequence for use according to the present inven Preferably, the expression vector is incorporated into the 60 tion directly or indirectly attached to a promoter. genome of a Suitable host organism. The term “incorporated An example of an indirect attachment is the provision of a preferably covers stable incorporation into the genome. Suitable spacer group Such as an intron sequence. Such as the The nucleotide sequence of the present invention may be Sh1-intron or the ADH intron, intermediate the promoter and present in a vector in which the nucleotide sequence is oper the nucleotide sequence of the present invention. The same is ably linked to regulatory sequences capable of providing for 65 true for the term “fused in relation to the present invention the expression of the nucleotide sequence by a suitable host which includes direct or indirect attachment. In some cases, organism. the terms do not cover the natural combination of the nucle US 8,889,371 B2 37 38 otide sequence coding for the protein ordinarily associated yeast. Preferred are Eubacteria, for example, Firmicutes (low with the wild type gene promoter and when they are both in GC-Gram positive bacteria), such as Bacillus subtilis and their natural environment. other bacillus species, lactic acid bacteria Such as species of The construct may even contain or express a marker, which genera Lactobacillus and Lactococcus. allows for the selection of the genetic construct. Also preferred are Gram-negative Proteobacteria, in par For some applications, preferably the construct of the ticular Gammaproteobacteria, Such as host species belonging present invention comprises at least the nucleotide sequence to the genera Pseudomonas, Xanthomonas, Citrobacter and of the present invention operably linked to a promoter. Escherichia, especially Escherichia coli. Host Cells In another embodiment the host cell is the same genus as The term “host cell in relation to the present invention 10 includes any cell that comprises either the nucleotide the native host species, i.e. the recombinant gene is re-intro sequence or an expression vector as described above and duced and expressed in a species from the same genus as the which is used in the recombinant production of an enzyme species from which the recombinant gene was isolated. having the specific properties as defined herein. In another embodiment the host cell is the native host Thus, a further embodiment of the present invention pro 15 species, i.e. the recombinant gene is re-introduced and vides host cells transformed or transfected with a nucleotide expressed in the same species from which the recombinant sequence that expresses the enzyme of the present invention. gene was isolated. The cells will be chosen to be compatible with the said vector Organism and may for example be prokaryotic (for example bacterial), The term “organism’ in relation to the present invention fungal, yeast or plant cells. Preferably, the host cells are not includes any organism that could comprise the nucleotide human cells. sequence coding for the enzyme according to the present Examples of Suitable bacterial host organisms are gram invention and/or products obtained therefrom, and/or positive or gram negative bacterial species. wherein a promoter can allow expression of the nucleotide Depending on the nature of the nucleotide sequence encod sequence according to the present invention when present in ing the enzyme of the present invention, and/or the desirabil 25 the organism. ity for further processing of the expressed protein, eukaryotic Suitable organisms may include a prokaryote, fungus, hosts such as yeasts or other fungi may be preferred. In yeast or a plant. general, yeast cells are preferred over fungal cells because The term “transgenic organism’ in relation to the present they are easier to manipulate. However, some proteins are invention includes any organism that comprises the nucle either poorly Secreted from the yeast cell, or in Some cases are 30 otide sequence coding for the enzyme according to the not processed properly (e.g. hyperglycosylation in yeast). In present invention and/or the products obtained therefrom, these instances, a different fungal host organism should be and/or wherein a promoter can allow expression of the nucle selected. otide sequence according to the present invention within the The use of Suitable host cells—such as yeast, fungal and organism. Preferably the nucleotide sequence is incorporated plant host cells—may provide for post-translational modifi 35 in the genome of the organism. cations (e.g. myristoylation, glycosylation, truncation, lapi The term “transgenic organism” does not cover native dation and tyrosine, serine or threonine phosphorylation) as nucleotide coding sequences in their natural environment may be needed to confer optimal biological activity on when they are under the control of their native promoter recombinant expression products of the present invention. which is also in its natural environment. The host cell may be a protease deficient or protease minus 40 Therefore, the transgenic organism of the present invention strain. includes an organism comprising any one of, or combinations The genotype of the host cell may be modified to improve of the nucleotide sequence coding for the enzyme according expression. to the present invention, constructs according to the present Examples of host cell modifications include protease defi invention, vectors according to the present invention, plas ciency, supplementation of rare tRNAs, and modification of 45 mids according to the present invention, cells according to the the reductive potential in the cytoplasm to enhance disulphide present invention, tissues according to the present invention, bond formation. or the products thereof. For example, the host cell E. coli may overexpress rare For example the transgenic organism may also comprise tRNA’s to improve expression of heterologous proteins as the nucleotide sequence coding for the enzyme of the present exemplified/described in Kane (Curr Opin Biotechnol 50 invention under the control of a heterologous promoter. (1995), 6, 494-500 “Effects of rare codon clusters on high Transformation of Host Cells/Organism level expression of heterologous proteins in E. coli). The As indicated earlier, the host organism can be a prokaryotic host cell may be deficient in a number of reducing enzymes or a eukaryotic organism. Examples of Suitable prokaryotic thus favouring formation of stable disulphide bonds as exem hosts include E. coli and Bacillus subtilis. plified/described in Bessette (Proc Natl AcadSci USA (1999), 55 Teachings on the transformation of prokaryotic hosts is 96, 13703-13708 “Efficient folding of proteins with multiple well documented in the art, for example see Sambrook et al disulphide bonds in the Escherichia coli cytoplasm'). (Molecular Cloning: A Laboratory Manual, 2nd edition, In one embodiment the host cell is a bacteria, preferably a 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic gram-positive bacteria, preferably a host cell selected from host is used then the nucleotide sequence may need to be Actinobacteria, such as Biofidobacteria and Aeromonas, par 60 suitably modified before transformation—such as by removal ticularly preferably Aeromonas salmonicida. Still more pre of introns. ferred are Actinomicetales such as Corynebacteria, in par Filamentous fungicells may be transformed using various ticular Corynebacterium glutamicum and Nocardia. methods known in the art—such as a process involving pro Particularly preferred are Streptomycetaceae, such as Strep toplast formation and transformation of the protoplasts fol tomyces, especially S. lividans. 65 lowed by regeneration of the cell wall in a manner known. The A microbial host can be used for expression of the galac use of Aspergillus as a host microorganism is described in EP tolipase gene, e.g. Eubacteria, Archea or Fungi, including O 238 O23. US 8,889,371 B2 39 40 Another host organism can be a plant. A review of the Culturing and Production general techniques used for transforming plants may be found Host cells transformed with the nucleotide sequence of the in articles by Potrykus (Annu Rev Plant Physiol Plant Mol present invention may be cultured under conditions condu Biol (1991 42:205-225) and Christou (Agro-Food-Industry cive to the production of the encoded enzyme and which Hi-Tech March/April 1994.17-27). Further teachings on plant facilitate recovery of the enzyme from the cells and/or culture transformation may be found in EP-A-0449375. medium. General teachings on the transformation of fungi, yeasts The medium used to cultivate the cells may be any conven and plants are presented in following sections. tional medium suitable for growing the host cell in questions Transformed Fungus and obtaining expression of the enzyme. A host organism may be a fungus—such as a filamentous 10 The protein produced by a recombinant cell may be dis fungus. Examples of Suitable such hosts include any member played on the surface of the cell. belonging to the genera Thermomyces, Acremonium, The enzyme may be secreted from the host cells and may Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma conveniently be recovered from the culture medium using and the like. 15 well-known procedures. Teachings on transforming filamentous fungi are reviewed Secretion in U.S. Pat. No. 5,741,665 which states that standard tech Often, it is desirable for the enzyme to be secreted from the niques for transformation of filamentous fungi and culturing expression host into the culture medium from where the the fungi are well known in the art. An extensive review of enzyme may be more easily recovered. According to the techniques as applied to N. crassa is found, for example in present invention, the secretion leader sequence may be Davis and de Serres, Methods Enzymol (1971) 17A: 79-143. selected on the basis of the desired expression host. Hybrid Further teachings on transforming filamentous fungi are signal sequences may also be used with the context of the reviewed in U.S. Pat. No. 5,674,707. present invention. In one aspect, the host organism can be of the genus Typical examples of heterologous secretion leader Aspergillus, Such as Aspergillus niger: 25 sequences are those originating from the fungal amyloglu A transgenic Aspergillus according to the present invention cosidase (AG) gene (glaA—both 18 and 24 amino acid ver can also be prepared by following, for example, the teachings sions e.g. from Aspergillus), the a-factor gene (yeasts e.g. of Turner G. 1994 (Vectors for genetic manipulation. In: Saccharomyces, Kluyveromyces and Hansenula) or the Martinelli S. D., Kinghorn J. R. (Editors) Aspergillus. 50 C.-amylase gene (Bacillus). years on. Progress in industrial microbiology Vol 29. Elsevier 30 By way of example, the Secretion of heterologous proteins Amsterdam 1994. pp. 641-666). Gene expression in filamen in E. coli is reviewed in Methods Enzymol (1990) 182:132 tous fungi has been reviewed in Punt et al. (2002) Trends 43. Biotechnol 2002 May; 20(5):200-6, Archer & Peberdy Crit. Detection Rev Biotechnol (1997) 17 (4):273-306. A variety of protocols for detecting and measuring the Transformed Yeast 35 expression of the amino acid sequence are known in the art. In another embodiment, the transgenic organism can be a Examples include enzyme-linked immunosorbent assay yeast. (ELISA), radioimmunoassay (RIA) and fluorescent activated A review of the principles of heterologous gene expression cell sorting (FACS). in yeast are provided in, for example, Methods Mol Biol A wide variety of labels and conjugation techniques are (1995), 49:341-54, and Curr Opin Biotechnol (1997) Octo 40 known by those skilled in the art and can be used in various ber; 8 (5):554-60 nucleic and amino acid assays. In this regard, yeast—such as the species Saccharomyces A number of companies such as Pharmacia Biotech (Pis cereviseae or Pichia pastoris (see FEMS Microbiol Rev cataway, N.J.), Promega (Madison, Wis.), and US Biochemi (2000 24 (1):45-66), may be used as a vehicle for heterolo cal Corp (Cleveland, Ohio) Supply commercial kits and pro gous gene expression. 45 tocols for these procedures. A review of the principles of heterologous gene expression Suitable reporter molecules or labels include those radio in Saccharomyces cerevisiae and secretion of gene products nuclides, enzymes, fluorescent, chemiluminescent, or chro is given by E Hinchcliffe E Kenny (1993, “Yeast as a vehicle mogenic agents as well as Substrates, cofactors, inhibitors, for the expression of heterologous genes”. Yeasts, Vol 5, magnetic particles and the like. Patents teaching the use of Anthony H Rose and J Stuart Harrison, eds, 2nd edition, 50 such labels include U.S. Pat. No. 3,817,837; U.S. Pat. No. Academic Press Ltd.). 3.850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No. 3,996.345; For the transformation of yeast, several transformation U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149 and U.S. protocols have been developed. For example, a transgenic Pat. No. 4,366,241. Saccharomyces according to the present invention can be Also, recombinant immunoglobulins may be produced as prepared by following the teachings of Hinnen et al., (1978, 55 shown in U.S. Pat. No. 4,816,567. Proceedings of the National Academy of Sciences of the USA Fusion Proteins 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and The amino acid sequence for use according to the present Ito, Hetal (1983, J Bacteriology 153, 163-168). invention may be produced as a fusion protein, for example to The transformed yeast cells may be selected using various aid in extraction and purification. Examples of fusion protein selective markers—such as auxotrophic markers dominant 60 partners include glutathione-S-transferase (GST), 6xHis antibiotic resistance markers. (SEQ ID NO: 33), GAL4 (DNA binding and/or transcrip Transformed Plants/Plant Cells tional activation domains) and (3-galactosidase). It may also A host organism suitable for the present invention may be be convenient to include a proteolytic cleavage site between a plant. A review of the general techniques may be found in the fusion protein partner and the protein sequence of interest articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol 65 to allow removal of fusion protein sequences. (1991 42:205-225) and Christou (Agro-Food-Industry Hi Preferably, the fusion protein will not hinder the activity of Tech March/April 1994 17-27). the protein sequence. US 8,889,371 B2 41 42 Gene fusion expression systems in E. coli have been FIG. 5 shows the structure of the lipolytic enzyme expres reviewed in Curr Opin Biotechnol (1995) 6 (5):501-6. sion vectors pGTK(L131) and pET11 (131-51); In another embodiment of the invention, the amino acid FIG. 6 shows a graph of the effect of a lipolytic enzyme sequence may be ligated to a heterologous sequence to from Streptomyces sp. L130 on digalactosyldiglyceride in encode a fusion protein. For example, for screening of peptide 5 dough; libraries for agents capable of affecting the Substance activity, FIG. 7 shows in graphical form the effect of a lipolytic it may be useful to encode a chimeric Substance expressing a enzyme from Streptomyces sp. L131 on digalactosyldiglyc heterologous epitope that is recognised by a commercially eride in dough; available antibody. FIG. 8 shows in graphical form the effect of a lipolytic Large Scale Application 10 enzyme from Streptomyces on triglyceride in dough; In one preferred embodiment of the present invention, the FIG. 9 shows the pH profile of the lipolytic enzyme amino acid sequence is used for large scale applications. obtained from Streptomyces sp. L131 on galactolipid Sub Strate; Preferably the amino acid sequence is produced in a quan FIG. 10 shows a TLC plate of lipids extracted from dough tity of from 1 g per liter to about 2 g per liter of the total cell 15 treated with a lipolytic enzyme from Streptomyces expressed culture Volume after cultivation of the host organism. in E. coli labelled #236. Lane 1=control; Lane 2=#236, Preferably the amino acid sequence is produced in a quan 0.225PLU-7/g flour; Lane 3=#236, 0.45 PLU-7/g flour; Lane tity of from 100 mg per liter to about 900 mg per liter of the 4=#236, 0.675 PLU-7/g flour; Lane 5=DGDG reference total cell culture volume after cultivation of the host organ material. ism. 20 FIG. 11 shows the construction of expression vector Preferably the amino acid sequence is produced in a quan pRX487 from puC18(L131R) and pIJ48: tity of from 250 mg per liter to about 500 mg per liter of the FIG. 12 shows in graphical form the effect of temperature total cell culture volume after cultivation of the host organ on stability and activity of a lipolytic enzyme from Strepto ism. myces sp L131; Food 25 FIG. 13 shows in graphical form the substrate specificities The composition of the present invention may be used of galactolipases from Streptomyces sp L131, Streptomyces as—or in the preparation of a food. Here, the term “food is avermitillis, Corynebacterium efficiens and Thermobifida used in a broad sense—and covers food for humans as well as fisca, food for animals (i.e. a feed). In a preferred aspect, the food is FIG. 14 shows the structure of an expression vector pCB5 for human consumption. 30 (TF) for expression of Thermobifida fisca lipase in C. The food may be in the form of a solution or as a solid— glutamicum, depending on the use and/or the mode of application and/or FIG.15 shows a sequence alignment of L131 (SEQIDNO: the mode of administration. 4) and homologues S. avermitilis (SEQ ID NO: 14) and T. Food Ingredient fusca (Residues 177-548 of SEQ ID NO. 5). Figure also The composition of the present invention may be used as a 35 discloses a consensus sequence disclosed as SEQID NO:30): food ingredient. FIG. 16 shows a HPTLC plate of reaction products from As used herein the term “food ingredient' includes a for enzyme treatment of crude Soya oil samples. Lane 1 control, mulation, which is or can be added to functional foods or Lane 2=99% crude oil and 1% K371 10% in water, Lane foodstuffs and includes formulations which can be used at 3=98% crude oil and 2% K371 10% in water, Lane 4=97% low levels in a wide variety of products that require, for 40 crude oil and 3% K371 10% in water, Lane 5=99.7% crude oil example, acidifying or emulsifying. and 0.3% Lecitase UltraTM #31081% in water, Lane 6=99% The food ingredient may be in the form of a solution or as crude oil, 0.3% Lecitase UltraTM #31081% in water and 0.7% a solid-depending on the use and/or the mode of application water. As reference phoshpatidylcholine (PC) is analysed; and/or the mode of administration. and Food Products 45 FIG. 17 shows a HPTLC plate of reaction products from The composition of the present invention can be used in the enzyme treatment of crude Soya oil samples. Lane 1 control, preparation of food products Such as one or more of confec Lane 2=99% crude oil and 1% K371 10% in water, Lane tionery products, dairy products, meat products, poultry 3=98% crude oil and 2% K371 10% in water, Lane 4=97% products, fish products and bakery products. crude oil and 3% K371 10% in water, Lane 5=99.7% crude oil The present invention also provides a method of preparing 50 and 0.3% Lecitase UltraTM #31081% in water, Lane 6=99% a food or a food ingredient, the method comprising admixing crude oil, 0.3% Lecitase UltraTM #31081% in water and 0.7% a lipolytic enzyme according to the present invention with water, together with reference lanes of cholesterol ester, another food ingredient. monoglyceride, diglyceride, triglyceride and plant sterol. Further preferable aspects are presented in the accompa nying claims and in the following Figures and examples. 55 EXAMPLE 1. FIG. 1 shows PCR fragment SEQ ID No. 1, which is a partial non-enzyme encoding polynucleotide; this sequence Identification of a Galactolipase Producing Bacterial is a ribosomal 16S RNA gene widely used for taxonomic Strain comparisons; FIG. 2 shows PCR fragment SEQ ID No. 2, which is a 60 Two microbial strains with a similar phenotype coded partial non-enzyme encoding polynucleotide; this sequence L130 and L131 were isolated from soil collected in Southern is a ribosomal 16S RNA gene widely used for taxonomic Finland. The 16S RNA genes of these two strains were 10 comparisons; amplified by standard PCR using oligonucleotide primers FIG.3 shows a polynucleotide encoding a lipolytic enzyme 536f(CAGCMGCCGCGGTAATWC) (SEQID NO: 18) and according to the present invention (SEQID No. 3): 65 1392r-primer (ACGGGCGGTGTGTRC) (SEQID NO:19). FIG. 4 shows an amino acid sequence of a lipolytic enzyme The resulting PCR fragments were partially sequenced. SEQ according to the present invention (SEQID No. 4): ID Nos. 1 and 2 are non-enzyme encoding polynucleotides. US 8,889,371 B2 43 44 These sequences are ribosomal 16S RNA genes widely used TABLE 1-continued for taxonomic comparisons. SEQID No. 1 and SEQID No. 2 were found 15 to have a high similarity. The sequences were ID Organism Label GLU-7 PLU-7 then compared to the 16S RNA gene sequences in GenBank. 182 Streptomyces Lipolytic enzyme 1.21 1.53 For both isolates the highest homology (97%) was observed 5 spp L 130 C 1.8 PLUmL. 183 Streptomyces Lipolytic enzyme O.63 1.29 with the sequence of a 16S RNA gene from Streptomyces spp L 131 A 0.54PLUmL. thermosacchari Thus, the strains were named Streptomyces 184 Streptomyces Lipolytic enzyme O.84 1.16 sp. L130 and Streptomyces sp. L131. spp L 131 B 0.64PLUmL. 185 Streptomyces Lipolytic enzyme 1.35 1.17 EXAMPLE 2 10 spp L 131 C O.8SPLUL. Preparation of Lipolytic Enzyme (Galactolipase) The phospholipase and galactolipase activity of the Samples from Strains Streptomyces sp. L130 and enzymes were assessed using the phospholipase activity L131 15 assay (PLU-7) and the galactolipase activity assay (GLU-7) mentioned herein above. 0.51 of LB medium was inoculated with Streptomyces L130 and cultivated on a rotary shaker at 200 rpm and 30°C. Dough Slurry Experiment for 2 days. This culture was used as inoculum for a 101 0.8 gram Wheat flour was scaled in a 12 ml centrifuge tube fermentor containing the same medium. The cultivation was with lid. 1.5 ml water containing the enzyme was added. The continued for 3 days at 30°C., 600 rpm stirring rate and 0.5 sample was mixed on a Whirley and placed in a heating V/v aeration. The fermentation broth was cleared by centrifu cabinet at 30° C. for 60 minutes. 6 mln-Butanol:Ethanol 9:1 gation (15 min at 5000 rpm) and Triton X-100 was added to was added, and the sample was mixed again until the flour final concentration of 0.1%. The solution was concentrated was finely distributed in the solvent. The tubes were the using Vivaflow 200 ultrafiltration cell (Vivascience AG, Han 25 placed in a water bath at 95°C. for 10 minutes. Then mixed nover, Germany) to 300 ml. The concentrate was dialysed again and placed on a rotation device 45 rpm, for 45 minutes. against 101 of 20 mM Tris HCl buffer, pH 7 containing 2 mM CaCl and 2 mMMgCl, followed by dialysis against 0.51 ml The sample was then centrifuged at 2000 g for 10 minutes. of 85% glycerol. The resulting preparations contained 90U of And 2 ml Supernatant was transferred to a 10 ml dram glass. galactolipase activity assay as defined above (GLU-7). The The solvent was evaporated at 70° C. under a steam of nitro strain Streptomyces L131 was cultivated under the same con 30 gen. ditions and its culture broth was concentrated by the same The isolated lipids are analysed by GLC. procedure. The resulting galactolipase preparation contained Gas Chromatography 70 U of activity. Perkin Elmer 8420 Capillary Gas Chromatography EXAMPLE 3 35 equipped with WCOT fused silica column 12.5 mx0.25 mm IDx0.1 um 5% phenyl-methyl-silicone (CP Sil 8 CB from Baking Experiments Crompack). The galactolipases from bacterial isolates L130 and L131 indicated a high activity on polar lipid substrates, galactolip 40 Carrier: Helium. ids (DGDG) and phospholipids, (galactolipase and phospho Injection: 1.5 L with split. lipase activity), equivalent to that of a Fusarium oxysporum Detector: FID. 385 C. lipase (Lipopan FTMNovozymes A/S Denmark): however the Oven program: 1 2 3 4 galactolipase from bacterial isolates L130 and L131 (i.e. the 45 Oven temperature C. 8O 200 240 360 lipolytic enzyme according to the present invention) were Isothermal, time min 2 O O 10 found to have no significant activity of triglycerides. This Temperature rate C./min 2O 10 12 contrasts sharply with the activity Fusarium oxysporum lipase LipopanFTM. The lipolytic enzymes from bacterial isolates L130 and Sample preparation: Lipid extracted from 0.2 gram flour was L131 were prepared as described in Example 2 and were 50 dissolved in 2 mL heptane:pyridine 2:1 containing an internal analysed for characterisation of their activity on glycolipids, standard heptadecane, 2 mg/mL. 500 uL of the sample was phospholipids and triglycerides, both in standard assay con transferred to a crimp vial. 100 uL MSTFA (N-Methyl-N- ditions and within a dough. trimethylsilyl-trifluoracetamid) was added and the reaction Small scale baking experiments and a model dough sys incubated for 15 minutes at 90° C. tem. Both enzymes are very active on galactolipids in flour. 55 Calculation: Response factors for monoglycerides, diglycer Materials and Methods ides, triglycerides, free fatty acid and galactolipids were Three samples of each enzyme were prepared as in determined from reference mixtures of these components. Example 3. Each sample was labelled as shown in table 1: Based on these response factors the lipids in the dough were calculated. 60 TABLE 1. Results. ID Organism Label GLU-7 PLU-7 The samples of enzyme from Streptomyces were analyzed for phospholipase and galactolipase activity with results 18O Streptomyces Lipolytic enzyme O.9S 1.31 spp L 130 A O.S8PLUL shown in table 2. The activity ratio PLU-7/GLU-7 was also 181 Streptomyces Lipolytic enzyme O.91 1.31 65 calculated. The mean ratio for the samples was 1.4, but with spp L 130 B 0.44PLU/mL. Some deviation in some of the samples, which might be explained by analytical deviations. US 8,889,371 B2 46 TABLE 2 obtain the same effect on galactolipids. The dough slurry experiments also confirmed that the enzymes from Strepto Sample ID Organism GLU-7 PLU-7 Ratio PLU-7GLU-7 myces sp. had no measurable activity on triglycerides. 18O L 13OA O.9S 1.31 1.4 181 L. 130 B O.91 1.31 1.4 EXAMPLE 4 182 L. 130 C 1.21 1.53 1.3 183 L. 131A O.63 1.29 2.0 184 L. 131 B O.84 1.16 1.4 Cloning of the Lipolytic Enzyme Gene from 18S L. 131 C 1.35 1.17 O.9 Streptomyces sp. L131

Dough Experiment. 10 The chromosomal DNA was isolated from Streptomyces The activity of the enzyme on wheat lipids was tested in the sp. L131 using a modification of a standard method. Bacteria dough slurry experiment as mentioned under materials and were grown on a rotary shaker in LB medium at 30° C. and high aeration (100 ml of medium per 0.51 baffled flask, 200 Methods. The isolated lipids from the dough were analysed rpm) to early stationary phase. From 500 ml bacterial culture by GLC as shown in table 3 15 cells were collected with centrifugation and washed once TABLE 3 with lysis buffer (550 mM glucose, 100 mM Tris, 2 mM EDTA, pH 8.0). GLC analysis of dough lipids (90 based on flour weight). Cell pellet was re-suspended in 10 ml of lysis buffer and Enzyme lysozyme was added to 1 mg/ml. Cells were incubated at 37° Sam- dosage C. for at least 15 min. The progress of lysozyme digestion was ple PLUg followed by transferring aliquots of bacterial Suspension into ID flour FFA MGMG DGMG MGDG DGDG TRI 1% SDS solution and measuring the absorption of the result 85 O.10S 0.1642 O.OO42 O.O380 O.O345 0.152O O.S.S15 ing mixture at 600 nm. The amount of lysozyme and incuba 85 O.263 O.1687 O.O130 OO67O O.O239 O.O941 O.S470 25 tion time were adjusted so that at least 70-90% of all cells 85 O.S26 O.2096 O.O121 O.O664 OO158 0.0617 O.S460 85 1.OS O.2597 O.OO36 0.0546 O.OO68 O.O3O3 O.S301 were lysed as evidenced by the decrease in Acco. At this point 82 O.O97 O.1542 O.OOS1 O.OS63 O.O313 O.1148 O.S475 of time, SDS was added to the bacterial suspension to 1% and 82 O.244. O.1687 OO159 0.0785 O.O2OO O.OS 66 O.S280 proteinase K to 0.1 mg/ml. The Suspension was incubated at 82 O.488 O.2095 O.OOSS O.0646 O.OO98 0.0219 O.S418 56° C. for 30 min followed by extractions with phenol and 82 O.976 O.2581 O.OO92 O.0439 O.OO43 O.OO4S O.SS79 30 Con- O O1529 OOOO6 O.O188 O.O44O O.1443 O.S.054 chloroform. After chloroform extraction, DNA was precipi trol tated with sodium acetate (0.5M final concentration) and Lipo- 1.47 O.23 O.O3 O.10 O.O1 O.O7 0.44 isopropanol (0.6 vol/vol) and the DNA pellet was washed 8 with 70% ethanol, dried in vacuum and dissolved in TE buffer TM (10 mM Tris, 1 mM EDTA) containing RNAse A (0.01 FFA = free fatty acids. MGMG = monogalactosylmonoglyceride, DGMG = digalactosyl 35 mg/ml). diglyceride. MGDG = monogalactosyldiglyceride, DGDG = digalactosyldiglyceride. TRI= The DNA was partially digested with restriction endonu triglyceride, clease Sau3A and the hydrolysates fractionated on a 0.8% The results from table 3 and table 4 confirm that the agarose gel. The 3-10 kb fraction of the Sau3A was isolated enzymes isolated in the Supernatant from fermentation of from agarose gels by electroelution. This DNA preparation Streptomyces Sp L130 and L131 are very active on galacto 40 was used to construct a gene library using Stratagene's lipids in a dough. The diesters DGDG and MGDG are hydro (LaJolla, USA) ZAP Express/Predigested Vector/Gigapack lyzed to the corresponding monoesters DGMG and MGMG. Cloning Kit (product #239615). Ligation, packaging, ampli The results are also illustrated graphically in FIGS. 6 and 7. fication of library and its conversion to the phagemid form These results confirm that both enzymes are very active at low were carried out according to the protocols provided by Strat dosage 0-0.2 Units/g flour and corresponding amount of 45 agene. Plasmid form of the resulting gene library was monoester is produced. At higher dosage 0.4-1 Units/gram screened on indicator plates prepared as follows. 80 ml of flour DGDG is further degraded but also some hydrolysis of sterile LBagar containing 25 mg/l of kanamycin was placed the monoesters are observed. This may indicate the enzymes into each 15 cm Petri dish and allowed to solidify. Subse are not specific to the position of the fatty acid in the galac quently, 10 ml top agar layer was added containing 0.5% tolipid molecule. 50 DGDG and 0.0005% Safranine O. The gene library was The activity of the enzymes on triglyceride, as illustrated in plated at a density of approximately 5000 colonies per 15 cm FIG. 8, is almost not existent. It is therefore concluded that the plate. The plates were incubated at 37°C. for 24h followed by enzymes tested have no significant effect on triglyceride. This a four-day incubation at room temperature. A clone forming is also in agreement with some experiments conducted on red halo on indicator plate was selected from the library and tributyrin as substrate, where no activity was observed. 55 purified by cloning on a new indicator plate. Summary The plasmid isolated from this clone (named pBK(L131)) A lipolytic enzyme was isolated in the Supernatant from was used to re-transform E. coli strain XL1-Blue MRF" to fermentation of Streptomyces sp. kanamycin resistance. All Such transformants displayed The lipolytic enzyme was found to have both phospholi galactolipase-positive phenotype. pBK(L131) contained an pase and galactolipase activity, but no significant activity on 60 approximately 7.5 kb insert. This insert was sequenced. One triglycerides. The ratio of phospholipase:galactolipase activ sequenced region (SEQ ID No. 3) was found to contain an ity was approx. 1.4 for the samples tested. open reading frame encoding a protein (SEQID No. 4) show Dough slurry experiments confirms that the enzymes were ing homology to a known lipase from Streptomyces rimosus. active on galactolipids in the flour. The enzymes were active This lipase, a member of so-called GDSX (SEQID NO: 31) in dough at a very low dosage 0-0.2 Units/g flour. Commer 65 family of lipases/esterases/acyltransferases is only known to cial phospholipases like Lipopan FTM (Novozymes A/S, Den be able to hydrolyse neutral lipids and artificial lipase sub mark) need to be dosed in 3-4 times higher dosage in order to Strates. US 8,889,371 B2 47 48 A series of deletions and Sub-clones of the original insert GAGCGGCGCCACCGTGACGTACA, anti-sense primer) were constructed and tested for galactolipase activity. It was (SEQID NO: 21). The amplified DNA fragment was digested found that a deletion derivative carrying 3 kb EcoRI SacI with EcoRI and Xbal and cloned into a B. subtilis-E. coli fragment of the original insert still retains full DGDGse activ shuttle vector pGTK44. This vector has been constructed by ity. This data correlated well with the results of partial DNA. substituting the Sall-EcoRI fragment of plasmid pGTK44 One area demonstrated homology to known lipases. This area (Povelainen et al., Biochem J.371, 191-197 (2003)) contain was Subsequently sequenced completely. Comparison of this ing degQ36 promoter with EcoRI-SalI fragment of pCT44 (KeroVuo J. et al. Biotechnology Letters 22, 1311-1317 sequence with the GenBank revealed that the closest homo (2000)). logue (58.5%) of the L131 galactolipase that has been bio Galactolipase activity was detected in E. coli transformed chemically characterised is a lipase from S. rimosus, and 10 with the resulting plasmid pGTK44(L131) (FIG. 5) using identified as a lipidiacyl transferase in WOO4/064987 and indicator plates. Control transformants (containing parent WOO4/O64537. plasmid pGTK44) were galactolipase-negative. Thus, protein Expression of L131 Galactolipase in E. coli. sequence represented by SEQ ID No 4 indeed possesses The standard pET-System, in which the gene is under con galactolipase activity. The same pair of primers amplified a trol of the T7 phage promoter, was used into express the L131 15 fragment of the same size (by agarose gel electrophoresis) galactolipase in E. coli. with chromosomal DNA of Streptomyces sp. L130 further Expression of L131 Galactolipase in Streptomyces lividans. confirming earlier observations about close similarity of the The shuttle vector pRX487-5 (FIG. 11) (derived from two isolated Strains and their galactolipase genes. pIJ4987: Kieser T. etal Practical Streptomyces genetics. The For expression in E. coli under control of the T7 phage John Innes Foundation, Crowes, Norwich, England (2000)) promoter, the deduced galactolipase coding region was used for expression of L131 galactolipase in S. lividans com amplified by PCR using chromosomal DNA of the Strepto bines E. coli plasmid puC18 and the S. lividans plasmid myces sp. L131 as template and the two oligonucleotide prim IJ487. In pRX487-5, the lac promoter of puC18 is placed ers (oL131-51 GGTCATGCTAGCATGAGATTGAC upstream of promoter-less kanamycin phosphotransferase CCGATCCCTGTCGG (SEQ ID NO: 22) and oL131-31 25 GCATGGATCCGCGGCGCCACCGTGACGTACA) (SEQ gene of plJ487. Indeed, the plasmid transformed E. coli not ID NO. 23). The PCR product was digested with Nhel and only to amplicillin but also to at least a low level (5 mg/l) of BamHI and ligated with pBT11a (Novagen, USA) vector kanamycin resistance. The vector contains unmodified digested with the same restriction . The liga EcoRI-Xbal fragment of the chromosomal DNA of S. ther tion mixture was used to transform the E. coli strain XL mosacchari comprising the complete coding sequence of the Blue1 MRF" and 12 different plasmid clones with restriction galactolipase gene, about 160 bp of upstream non-coding 30 patterns corresponding to the structure of pET11 (131-51) sequence and 420 bp of downstream non-coding sequence. (FIG. 4) were isolated. Each plasmid clone was used to sepa All transformants displayed similar levels of galactolipase rately transform the E. coli strain BL21 (DE3) and the result activity as judged by the halo formation on indicator plates. ing transformants were grown on LB-amplicilin containing Similarly, when S. lividans carrying pRX487-5 was culti galactolipase activity indicator layer (Example 4). Most vated at 101 level and the resulting culture was cloned on 35 clones did express active galactolipase. One clone (pET11 indicator plates, all clones appeared to produce equal (131-51)-12) was selected as a source of recombinant galac amounts of galactolipase activity. In Small shake-flask cul tolipase for Subsequent characterisation. tures inoculated by vegetative cells directly from plates, the The enzyme expressed in E. coli (labelled #236) was analy transformants typically produced about 10-20 mU/ml of sed and found to have: 0.33 GLU/ml and 0.36 PLU/ml, when galactolipase activity after 3 days of cultivation. When shake 40 analysed using the GLU-7 assay and PLU-7 assay taught flask cultures were inoculated with spores of the recombinant herein. S. lvividans, higher galactolipase activities were measured In liquid culture E. coli BL21 (DE3) expressed about 2 (about 30 ml J/ml), correlating with higher biomass accumu mu/ml of galactolipase activity after 40 h cultivation in LB lation. In an experiment where S. lividans carrying pRX487-5 ampicillin broth (37°C., 200 rpm shaking). Essentially all of 45 the activity was found in the culture broth. No galactolipase was grown in fermentor under high aeration conditions and activity was detected in E. coli BL21 (DE3) transformed with fed-batch mode (see material and methods for details) biom pET11a (Novagen, USA) and cultivated under the same con ass accumulation reached 170 g/l (wet weight). Accordingly, ditions. much higher galactolipase activity—about 1 U/ml was About four liters of galactolipase-containing culture broth detected. culture was concentrated on a rotary evaporator to about 300 Biochemical Properties of L131. 50 ml and dialysed against 151 of 20 mM Tris HCl buffer, pH 7 Some biochemical properties of L131 were tested. The pH containing 2 mM CaCl and 2 mM MgCl2. The dialysed optimum of the enzyme was found to be around 6.5-7.5 (FIG. material was again concentrated on a rotary evaporator to 9). The enzyme exhibited maximum activity towards DGDG about 30 ml and dialysed against 2 1 of 50% glycerol. The at a temperature ~50° C. Above this temperature inactivation resulting preparation (18 ml) contained about 100 ml J/ml of occurred, but not sharply, and after 20 min incubation in 90° 55 galactolipase activity. C., ~10% of residual activity was detected (FIG. 12). The enzyme expressed in E. coli (labelled #236) was also tested in dough. High activity on galactolipids was observed EXAMPLE 5 in dough as can be seen from FIG. 10, which shows a TLC plate. Expression of Streptomyces L131 Lipolytic Enzyme 60 EXAMPLE 6 According to the Present Invention Gene in E. coli Expression of the Lipolytic Enzyme According to The open reading frame of pBK(L131) encoding presump the Present Invention Gene from Streptomyces sp. tive lipolytic enzyme according to the present invention was L131 in Different Hosts amplified by PCR using primers ol.131-5 65 (GGTGAATTCATGAGATTGACCCGATCCCTGTCGG, Construction of the vector pGTK44(L131) has been out sense primer) (SEQ ID NO: 20) and oL131-3 (ACTTCTA lined in the Example 5. Besides E. coli, this vector can be used US 8,889,371 B2 49 50 to produce Streptomyces L131 lipolytic enzyme according to expression cassette (including promoter, lipolytic enzyme the present invention in Bacillus. Using this vectoris only one gene coding region and an optional transcription terminator) of many possible ways to express the L131 lipolytic enzyme into a chromosome is also feasible. In particular, multi-copy according to the present invention in Bacillus. For example, integration of the expression cassette into the host chromo the pst promoter employed in pGTK44(L131) may be some would be efficient. replaced by any other strong constitutive or regulated pro The recombinant hosts expressing the lipolytic enzyme moter active in Bacillus. Many such promoters are known in gene can be, advantageously, mutated to reduce the level of the art. For example, degQ36 promoter (Yang M et al. J. protease activity in the culture broth. The cultivation of any of Bacteriol. 166, 113-119 (1986)), cdd promoter, also known as Such recombinant hosts can be carried out in the presence of p43 (Wang P Z, Doi R H. J. Biol. Chem. 259, 8619-8625 10 compounds stabilising the enzyme. Such compounds may be (1984), amylase or neutral protease promoters etc. In addition various proteins (e.g. casein, peptone of serum albumin) or to pGTK44(L131) and other Bacillus vectors based on pTZ12 different lipids, lysolipids or detergents (e.g. galactolipids, replicon (Aoki T. et al., Mol. Gen. Genet. 208, 348-352 mono- and diacylglycerols or Triton X-100). (1987)) any other plasmid vector (e.g. pUB110, Gryczan T Jet 15 al. J. Bacteriol. 134,318-29 (1978) and its derivatives) can be used. EXAMPLE 7 Other preferred hosts for expression of the Streptomyces L131 lipolytic enzyme according to the present invention Acyl-Transferase Activity of Streptomyces L131 gene are high-GC Gram positive bacteria, in particular, Strep Lipolytic Enzyme and its Derivatives tomyces, (for example, S. lividans, S. coelicolor, S. griseus, S. natalensis, S. rubiginosus, S. Olivaceus, S. Olivochromogenes, Some lipases may also possess acyl-transferase activity. In S. violaceOruber). In Such hosts, the lipolytic enzyme accord particular, some members of the GDSX (SEQ ID NO: 31) ing to the present invention gene can be introduced under its family, for example, Aeromonas hydrophila acyltransferase own promoter on a multi-copy vector (e.g. using plJ110 25 (P10480) (taught in copending International Application No. derivatives such as plJ486, Ward et al. Mol. Gen. Genet. 203, PCT/IB2004/000655) have high acyl-transferase activity. 468-478 (1986)) or placed under control of a strong Strepto Thus, Streptomyces L131 lipolytic enzyme may be predicted myces promoter, for example ermE* (Schmitt-John T. Engels to have also the acyl-transferase activity as well. This activity J. W. Appl. Microbiol. Biotechnol. 36, 493-498 (1992)) or 30 can be further enhanced through random mutagenesis/di thiostreptone-inducinbe tipA promoter (Kieser T et al. in rected evolution. Moreover, since A. hydrophila acyl-trans Practical Streptomyces Genetics, p. 386, The John Innes ferase and Streptomyces L131 lipolytic enzyme share the Foundation, Norwich UK (2000)). same overall protein fold, combining the Substrate specificity In addition to prokaryotic hosts, L131 lipolytic enzyme of Streptomyces L131 lipolytic enzyme with high transferase gene may be expressed in one of the many Suitable fungal 35 efficiency of the Aeromonas enzyme is possible. This combi species. In particular, yeasts such as Saccharomyces cerevi nation may be achieved through the known techniques of siae, Schizosaccharomyces pombe, Pichia pastoris, targeted mutagenesis/protein design or by gene shuffling. Hansenula polymorpha are Suitable. In yeast, the lipolytic enzyme gene may be placed under control of any of the EXAMPLE 8 known strong yeast promoters, such glycolytic promoters 40 (PGK, GDH, ENO etc) phosphate starvation induced promot ers such as PHO5, the promoters of ethanol/methanol Identification of Alternative Lipolytic Enzymes from metabolism such as ADH1 promoter in S. cerevisiae or Other Streptomyces Species methanol-inducible promoters in H. polymorpha or P pas toris. 45 The GDSX (SEQ ID NO:31) family of esterase's (Upton When expressing the lipolytic enzyme gene in any host, C, Buckley JT. Trends Biochem. Sic. 20, 178-179 (1995), construction of a synthetic or semi-synthetic gene encoding pfamO0657.11) is a group of esterases/lipases/acyl trans the sequence of SEQID 4 would be advantageous. Likewise, ferases sharing a specific sequence motif around the active partly or completely synthetic genes may be designed based site serine (GDSX (SEQID NO:31) where X is a hydropho on sequences available through homology searches in silico 50 bic amino acid residue). This group of enzymes is also known as explained in Example 4. Such sequences, may incorporate as lipase family 11 (Arpigny J. L. Jaeger K-E. Biochem. J. a number ofuseful features that are absent in wild-type lipoly 343, 177-183 (1999)). Although this family includes many tic enzyme genes. For, example, the codon bias can be cor different types of esterases, lipases and acyl-transferases, the rected to better correspond codon preferences of the expres lipolytic enzyme according to the present invention is a sion hosts. One special case of codon bias correction useful 55 GDSX (SEQID NO:31) enzyme. for all hosts is to convert the GTG initiation codon of SEQID Thus, the sequences taught in the present invention of the No 3 into ATG. Another typical modification obvious for a Streptomyces sp. L131 lipolytic enzyme (galactolipase) can be used in silico to identify other galactolipases from other man skilled in the art is to exchange the native Streptomyces species of Streptomyces. signal sequence of the L131 lipolytic enzyme with a signal 60 sequence native to or known to be functional in the chosen To determine if a protein has the GDSX (SEQ ID NO:31) expression host. motif according to the present invention, the sequence is Previous examples of useful expression systems for L131 preferably compared with the hidden markov model profiles lipolytic enzyme focused on using plasmid vectors for the (HMM profiles) of the pfam database. introduction of the lipolytic enzyme gene into the expression 65 Pfam is a database of protein domain families. Pfam con host. This is indeed the preferred mode to implement current tains curated multiple sequence alignments for each family as invention. However, an alternative approach of integrating the well as profile hidden Markov models (profile HMMs) for US 8,889,371 B2 51 52 identifying these domains in new sequences. An introduction EXAMPLE 9 to Pfam can be found in Bateman A et al. (2002) Nucleic Acids Res. 30: 276-280. Hidden Markov models are used in a Identification of Galactolipases for Use in the number of databases that aim at classifying proteins, for Methods and Uses of the Present Application review see Bateman A and Haft D H (2002) Brief Bioinform 3:236-245. As mentioned above, the sequence of the novel Streptomy For a detailed explanation of hidden Markov models and ces thermosacchari L131 offers for the possibility for in silico how they are applied in the Pfam database see Durbin R, Eddy identification of new family II galactolipases. In this regard, S, and Krogh A (1998) Biological sequence analysis; proba one particular region which may be of particularinterest is the bilistic models of proteins and nucleic acids. Cambridge Uni 10 GDSX (SEQ ID NO:31) motif. versity Press, ISBN 0-521-62041-4. The Hammer software The GDSX (SEQ ID NO: 31) motif is comprised of four package can be obtained from Washington University, St conserved amino acids. Preferably, the serine within the motif Louis, USA. is a catalytic serine of the lipid acyltransferase enzyme. Suit Alternatively, the GDSX (SEQ ID NO: 31) motif can be 15 ably, the serine of the GDSX (SEQID NO:31) motif may be identified using the Hammer Software package, the instruc in a position corresponding to Ser-16 in Aeromonas hydro tions are provided in Durbin R, Eddy S, and Krogh A (1998) phila lipolytic enzyme taught in Brumlik & Buckley (Journal Biological sequence analysis; probabilistic models of pro of Bacteriology April 1996, Vol. 178, No. 7, p 2060-2064). teins and nucleic acids. Cambridge University Press, ISBN To determine if a protein has the GDSX (SEQ ID NO:31) 0-521-62041-4 and the references therein, and the HMMER2 motif, the sequence is preferably compared with the hidden profile provided within this specification. markov model profiles (HMM profiles) of the pfam database. The PFAM database can be accessed, for example, through As mentioned in Example 8, pfam is a database of protein several servers which are currently located at websites main domain families. Thus, the pfam database may also be used to tained by the Sanger Institute (UK) in conjunction with identify Suitable enzymes from genera other than Streptomy Wellcome Trust Institute, the Institut National de la Recher 25 CéS. che Agronomique, and the Center for Genomics and Bioin Alternatively, the GDSX (SEQ ID NO: 31) motif can be formatics of the Karolinska Institutet, among others. identified using the Hammer Software package, the instruc The database offers a search facility where one can entera tions are provided in Durbin R, Eddy S, and Krogh A (1998) protein sequence. Using the default parameters of the data Biological sequence analysis; probabilistic models of pro base the protein sequence will then be analysed for the pres 30 teins and nucleic acids. Cambridge University Press, ISBN ence of Pfam domains. The GDSX (SEQID NO:31) domain 0-521-62041-4 and the references therein, and the HMMER2 is an established domain in the database and as Such its profile provided within this specification. presence in any query sequence will be recognised. The data Preferably, the lipolytic enzyme in accordance with the base will return the alignment of the PfamO0657 consensus 35 present invention comprises the GDSX (SEQ ID NO: 31) sequence to the query sequence. motif. Preferably when aligned with the PfamO0657 consensus When aligned to either the pfam PfamO0657 consensus sequence the lipolytic enzyme for use in the compositions/ sequence (as described in WOO4/064987) and/or the L131 methods of the invention have at least one, preferably more sequence herein disclosed (SEQID No 4) than one, preferably more than two, of the following, a GDSX 40 i) The galactolipase/lipid acyl-transferase enzyme enzyme (SEQID NO:31) block, a GANDY (SEQID NO:35) block, of the invention, or for use in methods of the invention, a HPT block. Suitably, the lipolytic enzyme may have a has preferably a GDSX (SEQ ID NO: 31) motif, more GDSx (SEQ ID NO:31) block and a GANDY (SEQID NO: preferably a GDSY (SEQID NO:34) motif. 35) block. Alternatively, the enzyme may have a GDSX (SEQ and/or ID NO: 31) block and a HPT block. Preferably the enzyme 45 ii) The galactolipase/lipid acyl-transferase enzyme comprises at least a GDSX (SEQ ID NO:31) block. enzyme of the invention, or for use in methods of the The pfamO0657 GDSX (SEQ ID NO: 31) domain is a invention, has preferably a GANDY (SEQID NO: 35) unique identifier which distinguishes proteins possessing this block, more preferably a GANDY (SEQ ID NO: 35) domain from other enzymes. block comprising amino GGNDX (SEQ ID NO: 36), In addition or as an alternative thereto, alternative lipolytic 50 more preferably GGNDA (SEQID NO:37) or GGNDL enzymes from other Streptomyces species can be identified by (SEQ ID NO:38). conducting a sequence identity comparison and/or hybridisa and/or tion with one or more of the PCR sequence fragments shown iii) The enzyme of the invention, or for use in methods of as SEQID No. 1 or SEQID No. 2. Suitably, the comparisons the invention, has preferable an HPT block. may be carried out with fragments comprising over 15 nucle 55 and preferably otides of SEQ ID No. 1 or SEQ ID No. 2, preferably with iv) The galactolipase/lipid acyl-transferase enzyme of the fragments comprising over 20 nucleotides of SEQID No. 1 or invention, or for use in methods of the invention, has SEQ ID No. 2. Suitably, the complete sequences shown as preferably a GDSY motif and a GANDY block compris SEQID No. 1 or SEQID No. 2 could be used. Preferably, the 60 ing amino GGNDX, preferably GGNDA or GGNDL, hybridisation is carried out at high or very high Stringency and a HPT block (conserved histadine). conditions. Nucleotide sequences having at least 80%, pref In this regard, the inventors identified a homologous erably at least 85%, at least 90%, at least 95%, at least 97%, sequence to Streptomyces L131 which did not comprise a at least 98% or at least 99% identity to SEQID No. 1 or SEQ GDSX (SEQ ID NO: 31) motif: namely Novosphingobium ID No. 2 indicate strains of Streptomyces which may be 65 aromaticivorans (NAL) Sources of the lipolytic enzyme, i.e. the galactolipase, accord Novosphingobium\aromaticivorans\GDSx (SEQID NO:31) ing to the present invention. 284 aa US 8,889,371 B2 53 54

SEQ ID No. 10 ZP 00094165 mggvklfarr capvillalag lapaatvare aplaegaryv algs Sfaagp gvgpnapgsp 61 ercqrgtliny phillaealkl dilvdatcsga tthhvlgpwn evppoidsvn, gdtrl vtilti 121 gogndvisfvgn ifaaacekma spapricgkWr eiteeewqad eermir Sivro iharaplarv 181 vvvdyitvlp psgtcaamai sparlaqsrs aakirlarita rvareegasl likfishis rrh. 241 hpcsakpwsn gllsapaddgi pvhpnirlgha eaaaalvklv kmk A

SEQ ID No. 11 tgc.cggaact caa.gcggcgt ctago.cgaac tocatgc.ccga aag.cgc.gtgg cactatoccg 61 aagaccaggt Ctcggacgcc agcgagcgcc tatggcc.gc cgaaatcacg cgcgaac agc 121 totaccgc.ca gct coacgac gagctg.ccct atgacagtac cgtacgt.ccc gagaagtacc 181 t ccatcgcaa ggacggttcg atcgagat.cc accagcagat cgtgattgcc cgc.gaga cac 241 agcgt.ccgat C9tgctgggc aagggtggcg cgaagat Caa ggcgatcgga gaggcc.gcac 3 O1 goaaggaact titcgcaattig citcgacacca aggtgcacct gttcc tigcat gtgaaggtog 361 acgagcgctg. g.gc.cgacgcc aaggaaatct acgaggaaat cggccticgaa tgggit Caagt 421 gaagct ctitc gcgc.gc.cgct gcgc.cccagt acttct cqcc Cttgc.cgggc tggct Coggc 481 ggctacggtc gcgcgggaag Caccgctggc cqaaggcgcg cgttacgttg cgctgggaag 541 ct cott CCC gcaggt ccgg gcgtggggcc Caacgc.gc.cc ggat.cgc.ccg aacgctg.cgg 601 ccggggcacg Ctcaact acc cqc acctgct cqc.cgaggcg citcaa.gct cq atctogt cqa 661 togac Ctgc agcggcgcga Cacccacca C9tgctgggc C cctggaacg aggttcc ccc 721 t cagat.cgac agcgtgaatg gcgacacccg cct cqtcacc citgac catcg gcggaaacga 781 tdtgtcgttc gtcggcaa.ca tott.cgcc.gc cqcttgcgag aagatggcgt. cgc.ccgatcc 841 gogotgcggc aagtggcggg agat Caccga ggaagagtgg Caggc.cgacg aggagcggat 901 gcgctic catc gtacgc.ca.ga t coacgc.ccg cdc.gc.ct citc gcc.cgggtgg tggtggtc.ga 961 ttacat cacg gtc.ctg.ccgc catcaggcac ttgcgctgcc atggcgattt cgc.cggaccg 1021 gotggcc.cag agcc.gcagcg cc.gcgaaacg gCttgc.ccgg attaccgcac ggg.tc.gc.gc.g 1081 agaa.gagggit gcatcgctgc ticaagttctic gcatat ct cq cgc.cggCacc atc catgctic 1141 tocaa.gc.cc tigagcaacg gcc titt cogC ccc.ggc.cgac gacggcatcC cgg to catcc 1201 gaac.cggctic gga catgctgaag.cggcago ggcgctggtc. aagcttgttga aattgatgaa 1261 gtagct actg. cactgatttic aaatag tatt gcc tdtcago titt coagcc.c ggattgttgc 1321 agcgcaa.cag aaacttgtcc gtaatggatt gatggittitat gtc.gct cqca aattgcc.gtc 1381 gaagggaacg ggcgcgtc.gc ticgittaacgt. Cctgggtgca gcagtgacgg agcgc.gtgga 1441 tagtgatac toggtgtc atcggtgtac gcgcc.gc.cat tcc catgcct gtacgc.gc.cg A /

This enzyme comprises the sequence “GSSF (SEQ ID fusca), the GANDY (SEQ ID NO: 35) box, which is either NO:39) as opposed to GDSX (SEQ ID NO:31). GGNDA (SEQID NO:37) or GGNDL (SEQID NO:38), and When tested it was found that this enzyme does not com the HPT block (considered to be the conserved catalytic his prise glycolipase activity in accordance with the present 35 tadine). These three conserved blocks are highlighted in FIG. invention. 15. Therefore, the GDSX (SEQ ID NO: 31) motif may be When aligned to either the pfam PfamO0657 consensus important when attempting to identify other Suitable galacto sequence (as described in WOO4/064987) and/or the L131 lipases. 40 sequence herein disclosed (SEQ ID No. 4) it is possible to Notably, the enzyme from S. rimosus that has been purified identify three conserved regions, the GDSX block, the and characterised biochemically and shows about 56% GANDY block and the HPT block (see WOO4/064987 for sequence homology to Streptomyces L131 (Abramic M., et further details). al. (1999); Vujaklija D. et al. (2002)) is known to hydrolyse neutral lipids such as triolein or nitrophenyl esters of fatty. 45 EXAMPLE 10 The enzyme from S. rimosus may also hydrolyse galactoli pase in accordance with the present invention. Similarly, two Gene Cloning and Construction of Expression other Streptomyces species for which genome sequence data Vectors is available—S. coelicolor A2(3) and S. avermitilis may con tain enzymes having galactolipase activity, for example 50 Corynebacterium efficiens DSM 44549. Thermobifida (NP 625998 and NP 827753) are currently annotated in fusca DSM 43792 and Streptomyces avermitilis DSM46492 GenBank as “putative secreted hydrolases”. were used for isolating the genes homologous to the galacto Many other useful homologues of Streptomyces L131 lipase gene of S. thermosacchari L131. galactolipase can be identified by a similar approach. Suitable The strains accorded with a DSM number are deposited galactolipase/lipid acyl-transferase enzyme enzymes for use 55 and publicly available with Deutsche Sammlung von Mikro in the methods of the invention may be identified by align organismen and Zellkulturen GmbH (DSM). ment to the L131 sequence using Align X, the Clustal W Escherichia coli strains XL-Blue MRF, BL21 (DE3) pairwise alignment algorithm of VectorNTI using default set (Novagen) and S17-1 (Simon R. et al., 1983), Bacillus subtilis tings. BD170, Streptomyces lividans strain 1326 (John Innes Cen Alternatively, Suitable galactolipase for use in the methods 60 tre), Corynebacterium glutamicum DSM20300 were used as of the invention may be identified by alignment to the pfam the hosts for heterologous expression. The strain of Aeromo PfamO0657 consensus sequence (as described in WOO4/ nas salmonicida (DSM 12609) was also used as an expression 064987). host. FIG. 15 shows an sequence alignment of the L131 and S. thermosacchari L131, Citrobacter freundii P3-42 and homologues from S. avermitilis and T. fusca. FIG. 15 illus 65 Enterobacter nimipressuralis P1-60 were isolated in our trates the conservation of the GDSX (SEQ ID NO:31) motif laboratory from natural environment and taxonomically iden (GDSY (SEQ ID NO:34) in L131 and S. avermitilis and T. tified by 16S rRNA gene sequencing. US 8,889,371 B2 55 56 The following culture media were used in this study. LB (5 E. coli the resistance to at least 3 mg/l kanamycin. The pro g/l yeast extract, 10 g/l tryptone, 10 g/l NaCl, pH 7.0), 2xYT toplasts of S. lividans 1326 were transformed with 0.1-10 ug (10 g/l NaCl, 10 g/l yeast extract, 16 g/l tryptone) were used of pRX487-5 to thiostreptone (1.2 mg/l) and kanamycin (5 for cultivation of E. coli and other Gram-negative bacteria. mg/l) resistance. These transformants produced active galac Nutrient broth (3 g/l beef extract, 5 g/l peptone, pH 7.0) was tolipase as judged by the DGDG-safrainine indicator plate used for growing C. efficiens and N. aromaticivorans, YM assay. The transformants were plated on SM plates (Kieseret broth (3 g/l yeast extract, 3 g/1 malt extract, 5 g/l peptone, 10 al., 2000) supplemented with 5 mg/ml of kanamycin and g/l dextrose, pH 7.0) was used for cultivation of S. avermitilis, allowed to sporulate. The resulting spores were used for Medium 65 (4 g/l glucose, 4 g/l tryptone, 10 g/l malt extract, inoculating shake flask and fermentor cultures. 2 g/l CaCO, pH 7.2) was used for T. fusca. 10 Construction of Expression Vectors for Corynebacterium DNA Isolation. glutamicum. Standard alkaline lysis procedure combined with Qiagen All expression vectors used in this work are based on the column purification method was used for plasmid isolation. plasmid pCB5 which is a shuttle vector carrying C. One exception was the preparative isolation of plasmid DNA glutamicum replicon from plasmid pSR1 (Yoshihama et al., from Streptomyces. In this case, equilibrium centrifugation in 15 1985) and ColE1 replicon from E. coli. The promoter that is CsCl gradient was used as the final purification step. used in this vector is derived from the cop1 gene encoding the Methods for Introduction of DNA into Microbial Strains. major secreted protein of C. glutamicum—PS1. Enzymes Both E. coli and C. glutamicum Strains were transformed were expressed from their native genes including unmodified by electroporation using 1 mm cuvettes and the following signal peptides, e.g. T. fusca (FIG. 14). electroporation parameter settings: 1800V. 25 F., 200 ul B. Fermentation Conditions subtilis BD170 was transformed by “Paris' method based on Fermentation of Lipase-Producing Streptomyces Strains. natural competence (Harwood C. R. and Cutting S.M., 1990). In shake flasks, lipase-producing recombinant S. lividans Streptomyces lividans was transformed by protoplast method strains were grown in a medium containing (per liter) 10 g (Kieser T. et al., 2000). DNA was introduced into A. salmo peptone, 5g yeast extract, 2 g KHPO and 10g glucose (pH nicida by conjugation with E. coli using filter mating method 25 7.0) supplemented with appropriate antibiotics: thiostreptone of Harayama et al. (1980). was used at 1.2 mg/l, kanamycin at 20 mg/l, chloramphenicol Construction of Rifampicin-Resistant Mutant of A. salmoni at 1.5 mg/l and erythromycin at 1.5 mg/l. Spore Suspensions cida. produced by growing the transformants on SM plates were About 10 cells from overnight culture of A. salmonicida used to start the cultivations. DSM12609 were plated on a series of LBagar plates contain 30 For fed-batch fermentations, Braun Biostat E. fermentor ing 5-30 mg/l rifampicin. The plates were irradiated by short (101) was used. The initial medium (71), contained (per liter): wave UV light using SpectroLinker XL-1500 device (Spec peptone 20g, yeast extract, 10g, glucose 20g and appropriate tronics Corp. USA). The radiation dose was 4-6 J/M. The antibiotics as described above (except for thiostreptone, plates were incubated at 30° C. for 2 days. Several colonies which was not used in 10 1 cultures). The cultivation was growing on 30 mg/l rifampicin were selected and additionally 35 conducted at 30°C., constant 10 l/min aeration and 600 rpm tested on 50 mg/l rifampicin. One clone resistant to 50 mg/1 stirring rate. Inocula (2x250 ml perfermentation) were grown rifampicin (named R1) was chosen for Subsequent work. in 21 Erlenmeyer flasks as described in the previous para Construction of E. coli Expression Vectors for L131 Galac graph. The fermentation was carried out in batch mode for tolipase Homologues. 18-20 h after which time, a solution containing 30% glucose The lipase gene of Streptomyces avermitilis was amplified 40 and 12.5% peptone was fed to the fermentor culture at a rate by PCR using chromosomal DNA as template and the two of 0.5 ml/min. Samples (30 ml) of the culture were withdrawn oligonucleotide primers oSAL-5 (GGGAATTCCATAT aseptically twice a day. GAGACGTTCCCGAATTACG) (SEQ ID NO: 24) and Fermentation of Recombinant C. glutamicum Strains. OSAL-3 (GCATGGATCCGGTGACCTGTGCGACGG) Shake-flask cultures of C. glutamicum were grown in LB (SEQID NO: 25). For amplification of lipase genes of Ther 45 containing 50 mg/l kanamycin at 30° C. and 200 rpm agita mobifida fisca and Corynebacterium efficiens the oligonucle tion rate. otide primers used were oTFL-5 (GGGAATTC Fermentation of Recombinant A. Salmonicida Strains. CATATGGGCAGCGGACCACGTG) (SEQID NO: 26) and In shake flasks, the recombinant A. Salmonicida Strains oTFL-3 (GCATGGATCCGACACGCACGGCTCAACG) were cultivated in 2xYT medium supplemented with strep (SEQ ID NO: 27), OCEL-5 (GGGAATTCCATATGAGGA 50 tomycin and kanamycin (at 25 mg/l). To induce tac promoter, CAACGGTCATCG) (SEQ ID NO: 28) and OCEL-3 IPTG (1-5 mM) or lactose (1-10%) were added to the growth (GCATGGATCCGGCATCGGGCTCATCC) (SEQ ID NO: medium. 29), respectively. The PCR products were digested with Ndel Two sets of conditions for production of recombinant acyl and BamHI and ligated with plT11 a (Novagen, USA) vector transferase in A. Salmonicida were tested at fermentor scale. digested with the same restriction endonucleases. 55 In the first experiment, the initial medium (71) was 2xYT L131 galactolipase expression vector for S. lividans was supplemented with 2% glucose, 50 mg/l of kanamycin and 50 constructed as follows. Plasmid pUC18(L131 RX) that con mg/l of streptomycin and the feeding solution (31) contained tains the 1.37 kb EcoRI-Xbal fragment of the original cloned 25% glucose, 10% tryptone and 5% yeast extract, 100 mg/l of DNA fragment carrying L 131 lipase gene (pBK(L131)) was both kanamycin and streptomycin. Cultivation was carried digested with EcoRI and ligated with EcoRI digested plJ487 60 out at 101/min aeration, 600 rpm stirring rate and 28°C. The (Kieseret al., 2000). This ligation leads to the formation of the pH was adjusted to 7.5 by 25% NH and 10% phosphoric two recombinant plasmids differing in relative orientation of acid. The fermentor was inoculated with 0.51 of overnight pIJ487 and puC18(L131 RX). For subsequent work a variant culture of A. Salmonicida and grown in batch mode for 24 h. where lac promoter of the puC18 is flanking the promoter At this point IPTG was added to 5 mM and the feeding was less neo' gene of plJ487 has been selected based on restric 65 started at a rate of 50 ml/h. tion analysis. This construction was named pRX487-5 (FIG. In the second experiment, the initial medium was modified 11). Besides amplicillin resistance, this plasmid also confers by Substituting glucose with lactose. Feeding solution was 2 US 8,889,371 B2 57 58 1 of 20% lactose. The fermentation temperature was increased Determination of Effects of pH and Temperature on Lipase to 30° C. and the pH of the culture medium decreased to 7.0. Activity. Inoculation was done as in the first experiment and the feed For the determination of the effect of pH on enzymatic ing (100 ml/h) was started after 20 h of cultivation in the activity, it was measured over a range of pH 2-10 by using the initial medium galactolipase activity assay except that the buffers used in the Enzyme Assays experiment were as follows: pH 2-3.5 Glycine-HCl; pH 4-5 Safrainine Plate Screening Method. NaOAc; pH 5.5-7.5 Tris-Maleate; pH 7.5-9 Tris-HCl; pH 10 In Safrainine plate screening the bottom layer contained CAPS. culture medium+additive, 1.5% agarose and 0.002% safra The effect of temperature on galactolipase stability was nine (0.2% stock solution in water, sterile filtered) and the top 10 determined by incubating aliquots of enzyme for 20 min at layer 0.7% agarose, 1% DGDG and 0.002% safrainine. various temperatures (22°C.-90° C.) following incubation on Determination of Galactolipase Activity (Glycolipase Activ ice for 60 min. Residual activity was analysed by galactoli ity Assay (GLU-7)): pase activity assay. Substrate: For detection of optimal temperature for galactolipase 0.6% digalactosyldiglyceride (Sigma D 4651), 0.4% Tri 15 activity, the usual assay mixture was equilibrated at the ton-X 100 (Sigma X-100) and 5 mM CaCl was dissolved in required temperature (the range 20°C.-70°C.) and 2 or 4 ul of 0.05M HEPES buffer pH 7. enzyme was added to start the reaction. The activity was Assay Procedure: analysed by galactolipase activity assay, but using a shorter 400 uL substrate was added to an 1.5 mL Eppendorf tube period of time (20 min). and placed in an Eppendorf Thermomixer at 37° C. for 5 minutes. At time t—0 min, 50 uL enzyme solution was added. EXAMPLE 11 Also a blank with water instead of enzyme was analyzed. The sample was mixed at 10x100 rpm in an Eppendorf Thermo Characterisation of Galactolipase Candidates from mixer at 37°C. for 10 minutes. At time t—10 min the Eppen 25 Biodiversity Study dorf tube was placed in another thermomixer at 99 C. for 10 minutes to stop the reaction. The sequence of Streptomyces thermosacchari L131 Free fatty acid in the samples was analyzed by using the galactolipase offers for the possibility for in silico identifica NEFA C kit from WAKO GmbH. tion of new family II galactolipases. Enzyme activity GLU at pH 7 was calculated as micromole 30 Many other useful homologues of Streptomyces L131 fatty acid produced per minute under assay conditions galactolipase can be identified, for example, "hypothetical Determination of Phospholipase Activity (Phospholipase protein' from Thermobifida fusca (ZP 00058717) and Activity Assay (PLU-7)): “hypothetical protein' from Corynebacterium efficiens (NP Substrate 738716). 0.6% L-C. Phosphatidylcholine 95% Plant (Avanti 35 We cloned and expressed 3 homologues of Streptomyces #441601), 0.4% Triton-X 100 (Sigma X-100) and 5 mM L131 galactolipase: the genes of Streptomyces avermitilis CaCl was dispersed in 0.05M HEPES buffer pH 7. (SAL). Thermobifida fisca (TFL), and Corynebacterium effi Assay Procedure: ciens (CEL). All genes were expressed in E. coli by using plT 400 uL substrate was added to an 1.5 mL Eppendorf tube 40 expression system. The recombinant E. coli strains were first and placed in an Eppendorf Thermomixer at 37° C. for 5 analysed using DGDG indicator plates with Safrainine and minutes. At time t—0 min, 50 uL enzyme solution was added. the enzymes of S. avermitilis, T. fusca and C. efficiens were Also a blank with water instead of enzyme was analyzed. The found to have galactolipase activity. sample was mixed at 10x100 rpm in an Eppendorf Thermo The enzymes showing galactolipase activity were further mixer at 37°C. for 10 minutes. At time t—10 min the Eppen 45 examined. Substrate specificities of those galactolipase can dorf tube was placed in another thermomixer at 99 C. for 10 didates were studied (FIG. 13). The activity of candidate minutes to stop the reaction. enzymes towards DGDG, lecithin, olive oil, nitrophenyl Free fatty acid in the samples was analyzed by using the butyrate, nitrophenyl decanoate (NP-D) and nitrophenyl NEFA C kit from WAKO GmbH. palmitate was tested. The enzymes were found to have very Enzyme activity PLU-7 at pH 7 was calculated as micro 50 different substrate specificity profiles. Acyl-transferase activ mole fatty acid produced per minute under assay conditions. ity was tested in an assay using NP-D as Substrate and quan Spectrophotometric Assay with p-nitrophenyl Palmitate tifying both the release of nitrophenol and free fatty acids by (pNPP). NEFA kit. Preliminary data suggests that at least the enzyme Lipase activity was measured with a spectrophotometric from Thermobifida fisca has transferase activity towards 55 glycerol and glucose. assay at 30° C. with pNPP as substrate, by using 50 mM Thermo-stability of galactolipase candidates was tested. It Tris-Maleate buffer (pH 6.5) with 0.4% Triton X-100 and was found that the Corynebacterium efficiens enzyme was the 0.1% gum Arabic. The substrate stock solution (100 mM) was most thermostable while the enzyme of Streptomyces aver prepared in dioxane. The kinetic measurement was started by mitilis was the most thermo-sensitive. addition of enzyme to the reaction mixture. To evaluate the 60 initial hydrolytic activity, the increase in absorption at 410 nm EXAMPLE 12 was followed with Spectramax plate reader every 20s for 20 min. One unit of lipase activity was defined as the amount of Streptomyces thermosacchari L131 Degumming enzyme that liberated 1 umol of p-nitrophenol per min. The Trial activity toward other p-NP esters was measured in the same 65 manner, by using 1 mM each Substrate. (Abramic M. et al. A phospholipase from Streptomyces thermosacchari L131 (1999)) was tested in crude Soya oil. US 8,889,371 B2 59 60 Materials and Methods thermosacchari L131 to the oil. Only the lowest dosage (lane K371: Streptomyces thermosacchari L131 enzyme expressed 2) did not completely hydrolyse the phospholipids. Lecitase in S. lividans freeze dried on starch. UltraTM also hydrolysed the phospholipids in the oil when 5% (Activity: 108 PLU-7/g). water was available (Lane 6) but without adding extra water (Lane 5) only part of the phospholipids were hydrolysed. Lecitase Ultra (#3108) from Novozymes, Denmark FIG. 17 shows TLC (Buffer 5) of reaction products from Cholesterolester, Fluka 26950 enzyme treatment of crude Soya oil samples according to Plant Sterol Generol 122N from Henkel, Germany table 4. As referenced, cholesterolester, monoglyceride, dig Crude soya oil from The Solae Company, Aarhus Denmark lyceride, triglyceride and plant sterol. Free fatty acid (FFA) is Lecithin: L-O. Phosphatidylcholine 95% Plant (Avanti also indicated #441601) 10 Phospholipase Activity The results shown in FIG. 17 indicate that the hydrolysis of The phospholipase assay was the same as that used in phospholipids is coincident with the formation of free fatty Example 10. acid. HPTLC CONCLUSION Applicator: Automatic TLC Sampler 4, CAMAG 15 HPTLC plate: 20x10 cm, Merck no. 1.05641. Activated 30 The results confirm that Streptomyces thermosacchari minutes at 160° C. before use. L131 effectively hydrolyses phospholipids in crude soya oil Application: 1 ul of a 8% solution of oil in buffer was applied and is a suitable alternative enzyme for degumming of plant to the HPTLC plate using Automatic TLC applicator. oils. Running buffer 4: Chloroform:Methanol:Water 75:25:4 All publications mentioned in the above specification are Running buffer 5: P-ether:Methyl-tert-butyl ketone: Acetic herein incorporated by reference. Various modifications and acid 70:30:1 variations of the described methods and system of the present Application/Elution Time: invention will be apparent to those skilled in the art without Running buffer 4: 20 min departing from the scope and spirit of the present invention. Running buffer 5: 10 min 25 Although the present invention has been described in connec TLC Development tion with specific preferred embodiments, it should be under The plate was dried in an oven for 10 minutes at 160° C. stood that the invention as claimed should not be unduly cooled, and dipped into 6% cupri acetate in 16% HPO. limited to such specific embodiments. Indeed, various modi Dried additionally 10 minutes at 160° C. and evaluated fications of the described modes for carrying out the invention directly. 30 which are obvious to those skilled in biochemistry and bio Degumming Experiment technology or related fields are intended to be within the Streptomyces thermosacchari L131 (K371) was used for Scope of the following claims. degumming studies in the formulations shown in table 4. The samples were placed at 40° C. for 18 hours with REFERENCES agitation, after which time a sample was collected for HPTLC 35 analysis by dissolving the sample in Chloroform:Methanol Abramic M. Lescic I. Korica T., Vitale L., Saenger W. Pigac 2:1 J. Purification and properties of extracellular lipase from Streptomyces rimosus. Enzyme and Microbial Technology TABLE 4 40 25522-529 (1999) Degumming of crude Soya oil with Streptomyces thermosacchari Harayama S., Masataka T., Iino T. High-frequency mobilisa L131 And Lecitase Ultra TM tion of the chromosome of Escherichia coli by a mutant of plasmid RP4 temperature sensitive for maintenance. Mol. Lane 1 2 3 4 5 6 Gen. Genet. 180, 47-56 (1980). Crude soya oil % 99 99 98 97 99.7 99 45 Harwood C. R. and Cutting S.M. Molecular biological meth K371, 10% in water % 1 2 3 ods for Bacillus. John Wiley & Sons Ltd., West Sussex, Lecitase Ultra TM #3108, % O.3 O.3 England (1990) 1% in water Kieser T., Bibb M.J., Buttner M.J., Chater K. F., Hopwood D. Water % 1 O O O 0.7 A. Practical Streptomyces genetics. The John Innes Foun dation, Crowes, Norwich, England (2000) The results from the HPTLC analysis are shown in FIG.16 50 Vujaklija D., Schroder W. Abramic M., Zou P. Lescic I. and FIG. 17. Franke P. Pigac J. A novel Streptomycete lipase: cloning, FIG. 16 shows TLC plate (Buffer 4) of reaction products sequencing and high-level expression of the Streptomyces from enzyme treatment of crude Soya oil samples according rimosus GDS(L)-lipase gene. Arch. Microbiol. 178, 124 to table 4. As referenced, phosphatidylcholine (PC) was also 130 (2002) analysed. Phosphatydylethanolamine (PE) and lysophos 55 Yoshihama M, Higashiro K, Rao EA, Akedo M. Shanabruch phatidylcholine (LPC) are also indicated. WG, Follettie M T. Walker G. C. Sinskey A. J. Cloning The TLC results in FIG. 16 clearly show that phosphati vector System for Corynebacterium glutamicum. J. Bacte dylcholine was completely removed by adding Streptomyces riol. 162 (2):591-597 (1985).

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 39

<21 Os SEQ ID NO 1 &211s LENGTH: 435

US 8,889,371 B2 63 - Continued cc.gc.ccacgg att cogattic ggcgatgtc.c gcc.cgaccitt caacaac cac gaactgttct 84 O tcggcaacga Ctggctgcac to act CaCCC tgc.cggtgttg ggagt cqtac CaCCC Cacca 9 OO gcacgggc.ca t cagagcggc tat ctg.ccgg t cct caacgc caa.ca.gctcg acctgat caa 96.O cgcacggc.cg gcgcgtcacg Ctcgg.cgcgg gcgcc.gcagc gcgttgatca gcc cacagtg t cccaccgt.c acggtC9agg gtgtacgt.ca 108 O gctic Cagaag tggaacgt.ca gCagg accgt. ggagc.cgt.cc ctgacct cqt cgaagaactic 114 O cggggt cagc gtgat caccc ctic ccc.cgta gcc.gggggcg aaggcggcgc cgaactic citt 12 OO gtaggacgt.c Cagtcgtgcg gcc.cggcgtt gcc accqtcc gcgtaga.ccg citt coatggit 126 O

tcc.ccg.cgga acticggtggg gatgtc.cgtg Cccalaggtgg tcc.cggtggit 132O gtcc.gaga.gc accgggggct cgt accggat gatgtgcaga tccaaagaat t 1371

SEQ ID NO 4 LENGTH: 267 TYPE : PRT ORGANISM: Streptomyces sp.

< 4 OOs SEQUENCE: 4

Met Arg Lieu. Thir Arg Ser Lieu. Ser Ala Ala Ser Wall Ile Wall Phe Ala 1. 5 1O 15

Lell Luell Luell Ala Lieu. Lieu. Gly Ile Ser Pro Ala Glin Ala Ala Gly Pro 25 3 O

Ala Wall Ala Lieu. Gly Asp Ser Tyr Ser Ser Gly Asn Gly Ala Gly 35 4 O 45

Ser Tyr Ile Asp Ser Ser Gly Asp Cys His Arg Ser Asn Asn Ala Tyr SO 55 60

Pro Ala Arg Trp Ala Ala Ala Asn Ala Pro Ser Ser Phe Thir Phe Ala 65 70 8O

Ala Ser Gly Ala Val Thr Thr Asp Val Ile Asn Asn Glin Lieu. Gly 85 90 95

Ala Luell Asn Ala Ser Thr Gly Lieu. Wall Ser Ile Thir Ile Gly Gly Asn 105 11 O

Asp Ala Gly Phe Ala Asp Ala Met Thir Thr Cys Wall Thir Ser Ser Asp 115 12 O 125

Ser Thir Lieu. Asn Arg Lieu Ala Thir Ala Thr Asn Ile Asn. Thir 13 O 135 14 O

Thir Luell Luell Ala Arg Lieu. Asp Ala Val Tyr Ser Glin Ile Ala Arg 145 150 155 160

Ala Pro Asn Ala Arg Val Val Val Lieu. Gly Tyr Pro Arg Met Tyr Lieu. 1.65 17O 17s

Ala Ser Asn Pro Trp Tyr Cys Lieu Gly Lieu. Ser Asn Thir Lys Arg Ala 18O 185 19 O

Ala Ile Asn Thir Thr Ala Asp Thr Luell Asn. Ser Wall Ile Ser Ser Arg 195

Ala Thir Ala His Gly Phe Arg Phe Gly Asp Wall Arg Pro Thir Phe Asn 21 O 215 22O

Asn His Glu Leu Phe Phe Gly Asn Asp Trp Lieu. His Ser Luell Thir Lieu. 225 23 O 235 24 O

Pro Wall Trp Glu Ser Tyr His Pro Thir Ser Thir Gly His Glin Ser Gly 245 250 255

Luell Pro Wall Lieu. Asn Ala Asn Ser Ser Thr 26 O 265 US 8,889,371 B2 65 66 - Continued

<210s, SEQ ID NO 5 &211s LENGTH: 548 212. TYPE : PRT &213s ORGANISM: Thermobifida Fusca

<4 OOs, SEQUENCE:

Met Lieu. Pro His Pro Ala Gly Glu Arg Gly Glu Wall Gly Ala Phe Phe 1. 1O 15

Ala Luell Luell Wall Gly Thir Pro Glin Asp Arg Arg Lell Arg Luell Glu 25 3 O

His Glu Thir Arg Pro Lell Arg Gly Arg Gly Gly Glu Arg Arg 35 4 O 45

Wall Pro Pro Luell Thir Lell Pro Gly Asp Gly Wall Lell Thir Thir Ser SO 55 60

Ser Thir Arg Asp Ala Glu Thir Wall Trp Arg Lys His Lell Glin Pro Arg 65 70

Pro Asp Gly Gly Phe Arg Pro His Luell Gly Wall Gly Luell Luell Ala 85 90 95

Gly Glin Gly Ser Pro Gly Wall Luell Trp Gly Arg Glu Gly Cys Arg 105 11 O

Phe Glu Wall Arg Asp Thir Pro Gly Luell Ser Arg Thir Arg Asn 115 12 O 125

Gly Asp Ser Ser Pro Pro Phe Arg Ala Gly Trp Ser Lell Pro Pro 13 O 135 14 O

Cys Gly Glu Ile Ser Glin Ser Ala Arg Thir Pro Ala Wall Pro Arg 145 150 155 16 O

Ser Luell Luell Arg Thir Asp Arg Pro Asp Gly Pro Arg Gly Arg Phe 1.65 17O

Wall Gly Ser Gly Pro Arg Ala Ala Thir Arg Arg Arg Lell Phe Luell Gly 18O 185 19 O

Ile Pro Ala Luell Wall Lell Wall Thir Ala Luell Thir Lell Wall Luell Ala Wall 195

Pro Thir Gly Arg Glu Thir Lell Trp Arg Met Trp Cys Glu Ala Thir Glin 21 O 215 22O

Asp Trp Luell Gly Wall Pro Wall Asp Ser Arg Gly Glin Pro Ala Glu 225 23 O 235 24 O

Asp Gly Glu Phe Lell Lell Lell Ser Pro Wall Glin Ala Ala Thir Trp Gly 245 250 255

Asn Tyr Ala Lell Gly Asp Ser Tyr Ser Ser Gly Asp Gly Ala Arg 26 O 265 27 O

Asp Tyr Pro Gly Thir Ala Wall Gly Gly Trp Arg Ser Ala 27s 285

Asn Ala Pro Glu Lell Wall Ala Glu Ala Asp Phe Ala Gly His 29 O 295 3 OO

Lell Ser Phe Luell Ala Cys Ser Gly Glin Arg Gly Tyr Ala Met Luell Asp 3. OS 310 315

Ala Ile Asp Glu Wall Gly Ser Glin Luell Asp Trp Asn Ser Pro His Thir 3.25 330 335

Ser Luell Wall Thir Ile Gly Ile Gly Gly Asn Asp Lell Gly Phe Ser Thir 34 O 345 35. O

Wall Luell Lys Thir Met Wall Arg Wall Pro Luell Lell Asp Ser Ala 355 360 365

Thir Asp Glin Glu Asp Ala Ile Arg Arg Met Ala Phe Glu 37 O 375 38O

US 8,889,371 B2 71 72 - Continued

Asp Trp Luell Gly Wall Pro Wall Asp Ser Arg Gly Glin Pro Ala Glu SO 55 60

Asp Gly Glu Phe Lell Lell Lell Ser Pro Wall Glin Ala Ala Thir Trp Gly 65 70 7s

Asn Ala Lell Gly Asp Ser Tyr Ser Ser Gly Asp Gly Ala Arg 85 90 95

Asp Pro Gly Thir Ala Wall Lys Gly Gly Trp Arg Ser Ala 105 11 O

Asn Ala Tyr Pro Glu Lell Wall Ala Glu Ala Asp Phe Ala Gly His 115 12 O 125

Lell Ser Phe Luell Ala Ser Gly Glin Arg Gly Tyr Ala Met Luell Asp 13 O 135 14 O

Ala Ile Asp Glu Wall Gly Ser Glin Lieu. Asp Trp Asn Ser Pro His Thir 145 150 155 160

Ser Luell Wall Thir Ile Gly Ile Gly Gly Asn Asp Lell Gly Phe Ser Thir 1.65 17O 17s

Wall Luell Thir Met Wall Arg Wall Pro Luell Lell Asp Ser Ala 18O 185 19 O

Thir Asp Glin Glu Asp Ala Ile Arg Llys Arg Met Ala Phe Glu 195

Thir Thir Phe Glu Glu Lell Ile Ser Glu Wall Arg Thir Arg Ala Pro Asp 21 O 215 22O

Ala Arg Ile Luell Wall Wall Gly Tyr Pro Arg Ile Phe Pro Glu Glu Pro 225 23 O 235 24 O

Thir Gly Ala Tyr Tyr Thir Lell Thir Ala Ser ASn Arg Trp Luell Asn 245 250 255

Glu Thir Ile Glin Glu Phe Asn Glin Gln Lieu. Ala Ala Wall Ala Wall 26 O 265 27 O

His Asp Glu Glu Ile Ala Ala Ser Gly Gly Wall Ser Wall Glu Phe 28O 285

Wall Asp Wall His Ala Lell Asp Gly His Glu Gly Ser Asp Glu 29 O 295

Pro Trp Wall Asn Gly Wall Glin Luell Arg Asp Luell Thir Gly Wall Thir 3. OS 310 315

Wall Asp Arg Ser Thir Phe His Pro Asn Ala Ala His Arg Ala Wall 3.25 330 335

Gly Glu Arg Wall Ile Glu Glin Ile Glu Thir Gly Pro Gly Arg Pro Luell 34 O 345 35. O

Ala Thir Phe Ala Wall Wall Ala Gly Ala Thir Wall Asp Thir Luell Ala 355 360 365

Gly Glu Wall Gly 37 O

SEQ ID NO 8 LENGTH: TYPE : PRT ORGANISM: Corynebacterium Efficiens

< 4 OOs SEQUENCE: 8

Met Arg Thr Thr Val Ile Ala Ala Ser Ala Lieu Lleu Lleu Lieu. Ala Gly 1. 5 1O 15

Cys Ala Asp Gly Ala Arg Glu Glu Thir Ala Gly Ala Pro Pro Gly Glu 25 3 O

Ser Ser Gly Gly Ile Arg Glu Glu Gly Ala Glu Ala Ser Thr Ser Ile US 8,889,371 B2 73 74 - Continued

35 4 O 45

Thir Asp Wall Tyr Ile Ala Lieu. Gly Asp Ser Tyr Ala Ala Met Gly Gly SO 55 60

Arg Asp Glin Pro Lieu. Arg Gly Glu Pro Phe Cys Lell Arg Ser Ser Gly 65 70 7s 8O

Asn Pro Glu Lieu. Lieu. His Ala Glu Wall. Thir Asp Lell Thir Cys Glin 85 90 95

Gly Ala Wall Thr Gly Asp Lieu. Lieu Glu Pro Arg Thir Lell Gly Glu Arg 105 11 O

Thir Luell Pro Ala Glin Val Asp Ala Lieu. Thir Glu Asp Thir Thir Lieu Wall 115 12 O 125

Thir Luell Ser Ile Gly Gly Asn Asp Leu Gly Phe Gly Glu Wall Ala Gly 13 O 135 14 O

Cys Ile Arg Glu Arg Ile Ala Gly Glu Asn Ala Asp Asp Val Asp 145 150 155 160

Lell Luell Gly Glu Thir Ile Gly Glu Glin Lieu. Asp Glin Lell Pro Pro Glin 1.65 17O 17s

Lell Asp Arg Wal His Glu Ala Ile Arg Asp Arg Ala Gly Asp Ala Glin 18O 185 19 O

Wall Wall Wall Thr Gly Tyr Lieu Pro Lieu Wal Ser Ala Gly Asp Cys Pro 195

Glu Luell Gly Asp Val Ser Glu Ala Asp Arg Arg Trp Ala Wall Glu Lieu. 21 O 215

Thir Gly Glin Ile Asn. Glu Thir Wall Arg Glu Ala Ala Glu Arg His Asp 225 23 O 235 24 O

Ala Luell Phe Val Lieu Pro Asp Asp Ala Asp Glu His Thir Ser Cys Ala 245 250 255

Pro Pro Glin Glin Arg Trp Ala Asp Ile Glin Gly Glin Glin Thir Asp Ala 26 O 265 27 O

Pro Luell His Pro Thir Ser Ala Gly His Glu Ala Met Ala Ala Ala 285

Wall Arg Asp Ala Lieu. Gly Lieu. Glu Pro Wall Glin Pro 29 O 295 3 OO

<210s, SEQ ID NO 9 &211s LENGTH: 3 OOO &212s. TYPE: DNA <213> ORGANISM: Corynebacterium Efficiens

<4 OOs, SEQUENCE: 9 ttctggggtg titatggggtt gttatcggct cgt.cctgggit ggat.ccc.gc.c aggtggggta 6 O ttcacggggg actitttgttgt cCaac agc.cg agaatgagtg CCCtgagcgg tgggaatgag 12 O gtgggcgggg atgagggggc ggcgggctict gcgaccc.ccg 18O gcc.ccggtga aaatc.cggct gtaat cagca accc.cgt.cgg 24 O ggaggt cago gcc.cggagtg tctacgcagt cggat.cct ct cggacticggc Catgctgtcg 3OO gcagcatcgc gct cocgggit Cttggcgt.cc Ctcggctgtt Ctgcctgctg tcc ctggaag 360 gcgaaatgat Caccggggag tgatacaccg gtggit ct cat cc.cggatgcc Cactt.cggcg c catc.cggca att C9gcag Ctc.cgggtgg aagtaggtgg catc.cgatgc gtcggtgacg 48O c cat agtggg cgaagat citc atcctgct cq agggtgctica ggccact ct c cggat.cgata 54 O tCggggg.cgt. CCttgatggc gtc.cttgctg aalaccgaggit gcagottgttg ggct tccaat titcgcaccac ggagcgggac gaggctggaa tgacggc.cga agagc.ccgtg gtggacctica 660

US 8,889,371 B2 77 78 - Continued

<210s, SEQ ID NO 10 &211s LENGTH: 284 212. TYPE : PRT <213> ORGANISM: Novosphingobium Aromaticivorans

<4 OOs, SEQUENCE: 10

Met Gly Glin Val Llys Lieu. Phe Ala Arg Arg Cys Ala Pro Wall Lieu. Luell 1. 5 1O 15

Ala Luell Ala Gly Lieu Ala Pro Ala Ala Thir Wall Ala Arg Glu Ala Pro 25 3 O

Lell Ala Glu Gly Ala Arg Tyr Val Ala Lieu. Gly Ser Ser Phe Ala Ala 35 4 O 45

Gly Pro Gly Val Gly Pro Asn Ala Pro Gly Ser Pro Glu Arg SO 55 60

Arg Gly Thir Lieu. Asn Tyr Pro His Lieu. Lieu Ala Glu Ala Luell Llys Lieu. 65 70 7s 8O

Asp Luell Wall Asp Ala Thr Cys Ser Gly Ala Thr Thir His His Wall Lieu 85 90 95

Gly Pro Trp Asn. Glu Wall Pro Pro Glin Ile Asp Ser Wall Asn Gly Asp 105 11 O

Thir Arg Luell Wall. Thir Lieu. Thir Ile Gly Gly Asn Asp Wall Ser Phe Wall 115 12 O 125

Gly Asn Ile Phe Ala Ala Ala Cys Glu Lys Met Ala Ser Pro Asp Pro 13 O 135 14 O

Arg Gly Llys Trp Arg Glu Ile Thr Glu Glu Glu Trp Glin Ala Asp 145 150 155 16 O

Glu Glu Arg Met Arg Ser Ile Val Arg Glin Ile His Ala Arg Ala Pro 1.65 17O 17s

Lell Ala Arg Val Val Val Val Asp Tyr Ile Thr Wall Lell Pro Pro Ser 18O 185 19 O

Gly Thir Cys Ala Ala Met Ala Ile Ser Pro Asp Arg Lell Ala Glin Ser 195

Arg Ser Ala Ala Lys Arg Lieu Ala Arg Ile Thr Ala Arg Wall Ala Arg 21 O 215

Glu Glu Gly Ala Ser Lieu Lleu Lys Phe Ser His Ile Ser Arg Arg His 225 23 O 235 24 O

His Pro Ser Ala Lys Pro Trip Ser Asn Gly Lell Ser Ala Pro Ala 245 250 255

Asp Asp Gly Ile Pro Wal His Pro Asn Arg Lieu. Gly His Ala Glu Ala 26 O 265 27 O

Ala Ala Ala Lieu Val Llys Lieu Val Llys Lieu Met 27s 28O

SEQ ID NO 11 LENGTH: 15 OO TYPE: DNA ORGANISM: Novosphingobium Aromaticivorans

< 4 OOs SEQUENCE: 11 tgc.cggaact caa.gcggcgt. Ctagc.cgaac t catgcc.cga cactatoccg 6 O alagaccaggt Ctcggacgc.c agcgagcgc.c cgaaatcacg cgcgaac agc 12 O tctaccgc.ca gct coacgac gagctgcc ct atgacagtac cgitacgt.ccc gagaagtacc 18O tccatcgcaa ggacggttcg atcgagat.co accagcagat cgtgattgcc cgc.gaga cac 24 O agcgt.ccgat aagggtgg.cg cgaagat Caa ggcgatcgga gaggcc.gcac 3OO

US 8,889,371 B2 83 - Continued c cccaccgcg gcc.ggc.cagt ccggtggcta Cctgc.cggtc Ctcaacggcg cc.gc.ctgacc 1560 t caggcggaa ggagaagaag aaggagcgga gggaga.cgag gagtgggagg cc.ccgc.ccga 162O cggggit cocc gtc.ccc.gt ct cogtc.t.ccgt. Caagt caccg agaacgc.cac 168O cgcgt.cggac gtggc.ccgca ccggactic.cg cacct coacg cgcacggcac t ct cqaacgc 1740

tcqtgcgt.cg tdaccaccac cgc.gagcgct cgc.cgc.ccga 18OO cgggalaggac agcgt.ccgcc accc.cggatc ggaga.ccgac t cacccaccg 1860 gtagcc.gacc tcc.gcgggca gcc.gc.ccgac cgtgaacgtc cgggtgc.ccg 1920

ggcgga Cagg ccc.ccgagta gag.cccacca cggtcacctic 198O caccgactgc 2 OOO

SEQ ID NO 14 LENGTH: 269 TYPE : PRT ORGANISM: Streptomyces avermitilis

< 4 OOs SEQUENCE: 14

Met Arg Arg Ser Arg Ile Thir Ala Wall Thir Ser Lell Luell Lieu Ala 1. 5 15

Wall Gly Cys Ala Lieu. Thr Gly Ala Ala Thir Ala Glin Ala Ser Pro Ala 2O 25 3 O

Ala Ala Ala Thr Gly Tyr Val Ala Luell Gly Asp Ser Tyr Ser Ser Gly 35 4 O 45

Wall Gly Ala Gly Ser Tyr Lieu Ser Ser Ser Gly Asp Arg Ser 5 O 55 60

Ser Ala Tyr Pro Tyr Lieu. Trp Glin Ala Ala His Ser Pro Ser Ser 65 8O

Phe Ser Phe Met Ala Cys Ser Gly Ala Arg Thir Gly Asp Wall Lieu Ala 85 90 95

Asn Glin Luell Gly Thr Lieu. Asn. Ser Ser Thir Gly Lell Wall Ser Lieu. Thir 1OO 105 11 O

Ile Gly Gly Asn Asp Ala Gly Phe Ser Asp Wall Met Thir Thir Cys Val 115 12 O 125

Lell Glin Ser Asp Ser Ala Cys Lieu. Ser Arg Ile Asn Thir Ala Lys Ala 13 O 135 14 O

Tyr Wall Asp Ser Thir Lieu Pro Gly Glin Luell Asp Ser Wall Thir Ala 145 150 155 160

Ile Ser Thir Lys Ala Pro Ser Ala His Wall Ala Wall Lell Gly Tyr Pro 1.65 17O 17s

Arg Phe Llys Lieu Gly Gly Ser Cys Luell Ala Gly Lell Ser Glu Thir 18O 185 19 O

Arg Ser Ala Ile Asn Asp Ala Ala Asp Lell Asn Ser Ala Ile 195 2OO 2O5

Ala Lys Arg Ala Ala Asp His Gly Phe Thir Phe Gly Asp Wall Llys Ser 21 O 215 22O

Thir Phe Thir Gly His Glu Ile Cys Ser Ser Ser Thir Trp Luell His Ser 225 23 O 235 24 O

Lell Asp Luell Luell Asn Ile Gly Glin Ser Tyr His Pro Thir Ala Ala Gly 245 250 255

Glin Ser Gly Gly Tyr Leu Pro Wall Met Asn Ser Wall Ala 26 O 265

<210s, SEQ ID NO 15

US 8,889,371 B2 87 88 - Continued

Met Gly Ser Gly Pro Arg Ala Ala Thir Arg Arg Arg Lell Phe Luell Gly 15

Ile Pro Ala Luell Wall Lell Wall Thir Ala Luell Thir Lell Wall Luell Ala Wall 25 3 O

Pro Thir Gly Arg Glu Thir Lell Trp Arg Met Trp Glu Ala Thir Glin 35 4 O 45

Asp Trp Luell Gly Wall Pro Wall Asp Ser Arg Gly Glin Pro Ala Glu SO 55 60

Asp Gly Glu Phe Lell Lell Lell Ser Pro Wall Glin Ala Ala Thir Trp Gly 65 70

Asn Ala Lell Gly Asp Ser Ser Ser Gly Asp Gly Ala Arg 85 90 95

Asp Pro Gly Thir Ala Wall Lys Gly Gly Trp Arg Ser Ala 105 11 O

Asn Ala Tyr Pro Glu Lell Wall Ala Glu Ala Asp Phe Ala Gly His 115 12 O 125

Lell Ser Phe Luell Ala Ser Gly Glin Arg Gly Tyr Ala Met Luell Asp 13 O 135 14 O

Ala Ile Asp Glu Wall Gly Ser Glin Luell Asp Trp Asn Ser Pro His Thir 145 150 155 160

Ser Luell Wall Thir Ile Gly Ile Gly Gly Asn Asp Lell Gly Phe Ser Thir 1.65 17O 17s

Wall Luell Thir Met Wall Arg Wall Pro Luell Lell Asp Ser Ala 18O 185 19 O

Thir Asp Glin Glu Asp Ala Ile Arg Arg Met Ala Phe Glu 195

Thir Thir Phe Glu Glu Lell Ile Ser Glu Wall Arg Thir Arg Ala Pro Asp 21 O 215 22O

Ala Arg Ile Luell Wall Wall Gly Pro Arg Ile Phe Pro Glu Glu Pro 225 23 O 235 24 O

Thir Gly Ala Tyr Thir Lell Thir Ala Ser ASn Arg Trp Luell Asn 245 250 255

Glu Thir Ile Glin Glu Phe Asn Glin Glin Luell Ala Ala Wall Ala Wall 26 O 265 27 O

His Asp Glu Glu Ile Ala Ala Ser Gly Gly Wall Ser Wall Glu Phe 28O 285

Wall Asp Wall His Ala Lell Asp Gly His Glu Gly Ser Asp Glu 29 O 295

Pro Trp Wall Asn Gly Wall Glin Luell Arg Asp Luell Thir Gly Wall Thir 3. OS 310 315

Wall Asp Arg Ser Thir Phe His Pro Asn Ala Ala His Arg Ala Wall 3.25 330 335

Gly Glu Arg Wall Ile Glu Glin Ile Glu Thir Gly Pro Gly Arg Pro Luell 34 O 345 35. O

Ala Thir Phe Ala Wall Wall Ala Gly Ala Thir Wall Asp Thir Luell Ala 355 360 365

Gly Glu Wall Gly 37 O

<210s, SEQ ID NO 17 &211s LENGTH: 96.8 &212s. TYPE: DNA <213> ORGANISM: Thermobifida Fusca

US 8,889,371 B2 91 92 - Continued ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

SEQUENCE: 21 acttctagag cqgcgccacc gtgacgtaca 3 O

SEQ ID NO 22 LENGTH: 37 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

SEQUENCE: 22 ggtcatgcta gcatgagatt gaccc.gatcc Ctgtcgg 37

SEQ ID NO 23 LENGTH: 31 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

SEQUENCE: 23 gcatggat.cc gcggcgc.cac cqtgacgtac a 31

SEQ ID NO 24 LENGTH: 32 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

SEQUENCE: 24 gggaatticca tatgagacgt t cc.cgaatta C9 32

SEO ID NO 25 LENGTH: 27 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

SEQUENCE: 25 gcatggat.cc ggtgacctgt gcgacgg 27

SEQ ID NO 26 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

SEQUENCE: 26 gggaatticca tatgggcagc ggaccacgtg 3 O

SEO ID NO 27 LENGTH: 28 TYPE: DNA ORGANISM: Artificial Sequence US 8,889,371 B2 93 94 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 27 gcatggat.cc gacacgcacg gct caacg 28

<210s, SEQ ID NO 28 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 28 gggaatticca tatgaggaca acggt catcg 3 O

<210s, SEQ ID NO 29 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 29 gcatggat.cc ggcatcgggc ticatcC 26

<210s, SEQ ID NO 3 O & 211 LENGTH 225 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 30

Met Arg Arg Ser Arg Phe Lieu Ala Ala Lieu. Ile Lieu. Lieu. Thir Lieu. Ala 1. 5 1O 15

Ala Lieu. Gly Ala Ala Ala Arg Ala Ala Pro Ala Ala Tyr Val Ala Luell 2O 25 3 O Gly Asp Ser Tyr Ser Ser Gly Gly Ala Gly Ser Tyr Ser Ser Gly Asp 35 4 O 45

Arg Ser Thir Lys Ala Tyr Pro Ala Lieu. Trp Ala Ala Ala His Ala SO 55 60

Ser Ser Phe Ser Phe Ala Cys Ser Gly Ala Arg Thr Tyr Asp Val Luell 65 70 7s

Ala Gln Leu Lieu. Asn Ser Thr Lieu Val Ser Ile Thir Ile Gly Gly Asn 85 90 95

Asp Ala Gly Phe Ala Asp Met Thir Thr Cys Val Lieu Ser Asp Ser Ala 1OO 105 11 O

Lieu. Arg Ile Ala Ala Lys Tyr Ile Thr Lieu Pro Ala Arg Lieu Asp 115 12 O 125

Ser Val Tyr Ser Ala Ile Thr Arg Ala Pro Ala Arg Val Val Val Luell 13 O 135 14 O

Gly Tyr Pro Arg Ile Tyr Ser Gly Lieu. Gly Lieu. Ser Thr Lys Arg Ala 145 150 155 160

Ala Ile Asin Asp Ala Ala Asp Lieu. Asn. Ser Val Ile Ala Lys Arg Ala 1.65 17O 17s

Ala Asp His Gly Phe Thr Phe Gly Asp Val Thr Phe Gly His Glu Luell US 8,889,371 B2 95 - Continued

18O 185 19 O

Cys Ser Ala Pro Trp Lieu. His Ser Lieu. Thir Lieu Pro Val Ser Tyr His 195 2OO 2O5

Pro Thr Ala Gly His Ala Ala Gly Tyr Lieu Pro Val Lieu. Asn. Ser Ile 21 O 215 22O

Thir 225

<210s, SEQ ID NO 31 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) <223> OTHER INFORMATION: Wariable amino acid 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 31 Gly Asp Ser Xaa 1.

<210s, SEQ ID NO 32 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) <223> OTHER INFORMATION: Tyr, Ala or Lieu. 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 32 Gly Ala Asn Asp Xaa 1. 5

<210s, SEQ ID NO 33 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic 6xHis tag

<4 OOs, SEQUENCE: 33

His His His His His His 1. 5

<210s, SEQ ID NO 34 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 34 Gly Asp Ser Tyr 1. US 8,889,371 B2 97 98 - Continued

<210s, SEQ ID NO 35 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 35 Gly Ala Asn Asp Tyr 1. 5

<210s, SEQ ID NO 36 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) <223> OTHER INFORMATION: Wariable amino acid 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 36 Gly Gly Asn Asp Xaa 1. 5

<210s, SEQ ID NO 37 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 37 Gly Gly Asn Asp Ala 1. 5

<210s, SEQ ID NO 38 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 38 Gly Gly Asn Asp Lieu. 1. 5

<210s, SEQ ID NO 39 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 39 Gly Ser Ser Phe 1. US 8,889,371 B2 99 100 The invention claimed is: with or comprises a nucleotide sequence shown as SEQ 1. A method of preparing a lysoglycolipid comprising ID NO: 9 or a nucleotide sequence which has at least treating a Substrate comprising a glycolipid with at least one 70% identity therewith and which encode the said lipolytic enzyme to produce said lysoglycolipid, wherein said lipolytic enzyme. lipolytic enzyme has glycolipase activity and wherein said 2. A method according to claim 1 wherein said lipolytic lipolytic enzyme is obtainable from the genus Corynebacte enzyme is capable of transferring an acyl group from a gly rium and wherein said lipolytic enzyme comprises at least colipid to one or more acyl acceptor Substrates. one GDSX motif, wherein G is glycine, D is aspartic acid, S is 3. A method according to claim 1 wherein said lipolytic serine and X is a hydrophobic amino acid residue selected enzyme comprises an amino acid sequence shown as SEQID from the group consisting of F. W.Y. H. K. M. I. L. V. A and 10 G: NO: 8 or an amino acid sequence having at least 90% identity or a GANDY block, wherein the GANDY block comprises therewith. the amino acid motif GGNDA or GGNDL, wherein G is 4. The method according to claim 1 wherein said lysogly glycine, N is asparagine, D is aspartic acid, A is alanine colipid is DGMG or MGMG. and L is leucine; 15 5. The method according to claim 1 wherein said substrate or a HPT block, wherein H is histidine, P is proline and T is an edible oil. is threonine wherein said lipolytic enzyme comprises an 6. The method according to claim 1 whereinx is the hydro amino acid sequence shown as SEQ ID NO: 8 or an phobic amino acid residue Y. amino acid sequence having at least 70% identity there k k k k k