(19) & (11) EP 2 267 107 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 29.12.2010 Bulletin 2010/52 C11B 3/00 (2006.01) A23J 7/00 (2006.01) C12N 9/20 (2006.01) (21) Application number: 10174040.5 (22) Date of filing: 18.07.2005 (84) Designated Contracting States: • Soe, Jørn AT BE BG CH CY CZ DE DK EE ES FI FR GB GR DK-8381, Tilst (DK) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Mikkelsen, Jørn SK TR DK-2650, Hvidovre (DK) • Povelainen, Mira (30) Priority: 16.07.2004 GB 0416035 FIN-00360, Helsinki (FI) 26.07.2004 US 591185 P • Pitkanen, Virve 07.07.2005 GB 0513859 02320 Espoo (FI) (83) Declaration under Rule 32(1) EPC (expert (74) Representative: Williams, Aylsa solution) D Young & Co LLP 120 Holborn (62) Document number(s) of the earlier application(s) in London EC1N 2DY (GB) accordance with Art. 76 EPC: 05773820.5 / 1 776 455 Remarks: This application was filed on 25-08-2010 as a (71) Applicant: Danisco A/S divisional application to the application mentioned 1001 Copenhagen K (DK) under INID code 62. (72) Inventors: • Miasnikov, Andrei 10160 Degerby (FI) (54) Lipolytic Enzyme. Uses Thereof In The Food Industry (57) The invention also encompases theuse of a lipo- a galactolipid to one or more acyl acceptor substrates, lytic enzyme obtainable from one of the following genera: wherein the enzyme is obtainable fromSteptomyces Streptomyces, Corynebacterium and Thermobifida in species and includes a lipolytic enzyme comprising an various methods and uses, wherein said lipolytic enzyme amino acid sequence as shown in SEQ ID No. 4 or an is capable of hydrolysing a glycolipid or a phospholipid amino acid sequence which has at least 60% identity or transferring an acyl group from a glycolipid or phos- thereto. The enzyme may be encoded by a nucleic acid pholipids to a acyl acceptor. Preferably, the lipolytic en- selected from the group consisting of: zyme for use in these methods and uses comprises an a nucleic acid comprising a nucleotide sequence shown amino acid sequence as shown in any one of SEQ. ID. in SEQ ID No. 3; No.s 4, 5, 7, 8, 12, 14 or 16 or an amino acid sequence a nucleic acid which is related to the nucleotide sequence having at least 70% identity therewith or comprises a of SEQ ID No. 3 by the degeneration of the genetic code; nucleotide sequence shown as SEQ ID No. 3, 6, 9, 13, and 15 or 17 or a nulceotide seqeunce which has at least a nucleic acid comprising a nucleotide sequence which 70% identity therewith. The present invention also relates has at least 70% identity with the nucleotide sequence to a lipolytic enzyme capable of hydrolysing at least a shown in SEQ ID No. 3. galactolipid or capable of transferring an acyl group from EP 2 267 107 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 267 107 A2 Description FIELD OF INVENTION 5 [0001] The present invention relates to a novel lipolytic enzyme, in particular a novel lipolytic enzyme, and nucleotide sequences encoding same. The present invention also relates to methods of production of the novel lipolytic enzyme and to uses thereof. The present invention also relates to methods and uses of a lipolytic enzyme. TECHNICAL BACKGROUND 10 [0002] The beneficial use of lipolytic enzymes active on glycolipids in bread making was taught in EP 1 193 314. It was taught that the partial hydrolysis products the lysoglycolipids were found to have very high emulsifier functionality. However, the enzymes taught in EP 1 193 314 were also found to have significant non- selective activity on triglycerides which resulted in unnecessarily high free fatty acid. 15 [0003] A lipolytic enzyme from Fusarium oxysporum having phospholipase activity has been taught in EP 0 869 167. This lipolytic enzyme has high triacylglyceride hydrolysing (lipase) activity. This enzyme is now sold by Novozymes A/S (Denmark) as Lipopan F™. [0004] WO02/00852discloses five lipase enzymes and their encoding polynucleotides, isolated from Fusarium venena- tum, F. sulphureum, Aspergillus berkeleyanum, F. culmorum and F. solani. All five enzymes are described as having 20 triacylglycerol hydrolysing activity, phospholipase and galactolipase activity. [0005] Lipolytic enzyme variants, with specific amino acid substitutions and fusions, have been produced; some of which have an enhanced activity on the polar lipids compared to the wildtype parent enzymes. WO01/39602 describes such a variant, referred to as SP979, which is a fusion of theThermomyces lanuginosus lipase, and the Fusarium oxysporum lipase described in EP 0 869 167. This variant has been found to have a significantly high ratio of activity 25 on phospholipids and glycolipids compared to triglycerides. [0006] In WO02/094123 it was discovered that by selecting lipolytic enzymes which were active on the polar lipids (glycolipids and phospholipids) in a dough, but substantially not active on triglycerides or 1-mono-glycerides an improved functionality could be achieved. [0007] In co-pending PCT application number PCT/IB2005/000875, wild-type lipolytic enzymes having a higher ratio 30 of activity on polar lipids as compared with triglycerides are taught. However, this document does not teach lipolytic enzymes from Streptomyces, Thermobifida or Corynebacterium species. [0008] Prior to the present invention no lipolytic enzymes having activity or significant activity on glycolipids had been published from Streptomyces species. Likewise, no lipolytic enzymes having activity or significant activity on glycolipids had been published from Thermobifida species or Corynebacterium species. Although lipases, i.e. triacylglycerol hy- 35 drolysing enzymes, have been isolated from Streptomyces species (see Vujaklija et al Arch Microbiol (2002) 178: 124-130 for example), these enzymes have never been identified as having glycolipid hydrolysing activity. ASPECTS OF THE INVENTION 40 [0009] The present invention in predicated upon the seminal finding of a lipolytic enzyme having significant galactolipid activity from the genus Streptomyces. In particular the lipolytic enzyme from the genusStreptomyces has significant galactolipid hydrolysing activity and/or significant galactolipid acyltransferase activity, particularly when used in the methods and uses according to the present invention. [0010] In addition, the present invention in predicated upon the seminal finding that lipolytic enzymes from the genera 45 Thermobifida or Corynebacterium have significant galactolipid activity. In particular the lipolytic enzymes from the genera Thermobifida or Corynebacterium have significant galactolipid hydrolysing activity and/or significant galactolipid acyl- transferase activity, particularly when used in the methods and uses of the present invention. [0011] In a broad aspect the present invention relates to a lipolytic enzyme capable of hydrolysing at least glycolipids and/or capable of transferring an acyl group from at least a glycolipid to one or more acyl acceptor substrates, wherein 50 the enzyme is obtainable, preferably obtained, from Streptomyces species. [0012] In a further aspect the present invention relates to a lipolytic enzyme capable of hydrolysing at least galactolipids and/or capable of transferring an acyl group from at least a galactolipid to one or more acyl acceptor substrates, wherein the enzyme is encoded by a nucleic acid selected from the group consisting of: 55 a) a nucleic acid comprising a nucleotide sequence shown in SEQ ID No. 3; b) a nucleic acid which is related to the nucleotide sequence of SEQ ID No. 3 by the degeneration of the genetic code; and c) a nucleic acid comprising a nucleotide sequence which has at least 70% identity with the nucleotide sequence 2 EP 2 267 107 A2 shown in SEQ ID No. 3. [0013] The present invention yet further provides a lipolytic enzyme comprising an amino acid sequence as shown in SEQ ID No. 4 or an amino acid sequence which has at least 60% identity thereto. 5 [0014] In another aspect the present invention provides a lipolytic enzyme capable of hydrolysing at least a galactolipid and/or capable of transferring an acyl group from at least a galactolipid to one or more acyl acceptor substrates, wherein the enzyme comprises an amino acid sequence as shown in SEQ ID No. 4 or an amino acid sequence which has at least 60% identity thereto. [0015] In a further aspect the present invention provides a nucleic acid encoding a lipolytic enzyme comprising an 10 amino acid sequence as shown in SEQ ID No. 4 or an amino acid sequence which has at least 60% identity therewith. [0016] SEQ ID No. 3 is shown in Figure 3 and SEQ ID No. 4 is shown in Figure 4. [0017] The present invention yet further provides a nucleic acid encoding a lipolytic enzyme, which nucleic acid is selected from the group consisting of: 15 a) a nucleic acid comprising a nucleotide sequence shown in SEQ ID No. 3; b) a nucleic acid which is related to the nucleotide sequence of SEQ ID No. 3 by the degeneration of the genetic code; and c) a nucleic acid comprising a nucleotide sequence which has at least 70% identity with the nucleotide sequence shown in SEQ ID No. 3. 20 [0018] The present invention yet further provides the use of a lipolytic enzyme according to the present invention in a substrate (preferably a foodstuff) for preparing a lysoglycolipid, for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monoga- lactosyl diglyceride (MGDG)) with the lipolytic enzyme according to the present invention to produce the partial hydrolysis 25 product, i.e. the lysoglycolipid. [0019] In a further aspect, the present invention provides the use of a lipolytic enzyme according to the present invention in a substrate (preferably a foodstuff) for preparing a lyso-phospholipid, for example lysolecithin, by treatment of a phospholipid (e.g.