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Cellobiose 2-Epimerase, Process for Producing

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Cellobiose 2-Epimerase, Process for Producing (19) TZZ ¥ZZ_T (11) EP 2 395 080 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/00 (2006.01) C12N 1/15 (2006.01) 06.08.2014 Bulletin 2014/32 C12N 1/19 (2006.01) C12N 1/21 (2006.01) C12N 5/10 (2006.01) C12N 9/90 (2006.01) (2006.01) (2006.01) (21) Application number: 10738433.1 C12N 15/09 C12P 19/00 (22) Date of filing: 25.01.2010 (86) International application number: PCT/JP2010/050928 (87) International publication number: WO 2010/090095 (12.08.2010 Gazette 2010/32) (54) CELLOBIOSE 2-EPIMERASE, PROCESS FOR PRODUCING SAME, AND USE OF SAME CELLOBIOSE 2-EPIMERASE, HERSTELLUNGSVERFAHREN DAFÜR UND VERWENDUNG CELLOBIOSE 2-ÉPIMÉRASE, PROCÉDÉ DE PRODUCTION DE CELLE-CI ET UTILISATION DE CELLE-CI (84) Designated Contracting States: (74) Representative: Daniels, Jeffrey Nicholas AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Page White & Farrer HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL Bedford House PT RO SE SI SK SM TR John Street London WC1N 2BF (GB) (30) Priority: 05.02.2009 JP 2009025070 (56) References cited: (43) Date of publication of application: WO-A1-2008/062555 14.12.2011 Bulletin 2011/50 • PARK CHANG-SU ET AL: "Characterization of a (73) Proprietor: Hayashibara Co., Ltd. recombinant cellobiose 2-epimerase from Okayama-shi, Okayama (JP) Caldicellulosiruptor saccharolyticus and its application in the production of mannose from (72) Inventors: glucose.", APPLIED MICROBIOLOGY AND • WATANABE Hikaru BIOTECHNOLOGY DEC 2011 LNKD- PUBMED: Okayama-shi 21691788,vol. 92, no. 6, December 2011 (2011-12), Okayama 700-0907 (JP) pages 1187-1196, XP002683252, ISSN: 1432-0614 •YAGIMasahiro • DATABASE PROTEIN [Online] NCBI 2007 Okayama-shi XP008164001 Database accession no. YP_ Okayama 700-0907 (JP) 001179132 • NISHIMOTO Tomoyuki • VAN DE WERKEN H.J. ET AL.: ’Hydrogenomics Okayama-shi of the extremely thermophilic bacterium Okayama 700-0907 (JP) Caldicellulosiruptor saccharolyticus’ APPL. • CHAEN Hiroto ENVIRON. MICROBIOL. vol. 74, no. 21, 2008, Okayama-shi pages 6720 - 6729 Okayama 700-0907 (JP) • TAGUCHI H. ET AL.: ’Cloning and sequencing of • FUKUDA Shigeharu the gene for cellobiose 2-epimerase from a Okayama-shi ruminal strain of Eubacterium cellulosolvens.’ Okayama 700-0907 (JP) FEMS MICROBIOL LETT. vol. 287, no. 1, 2008, pages 34 - 40 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 395 080 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 395 080 B1 Description Field of the Invention 5 [0001] The present invention relates to a cellobiose 2-epimerase, its preparation and uses, in particular, a cellobiose 2-epimerase which catalyzes aldose-ketose conversion as well as 2-epimerization, a process of producing thereof, a DNA encoding the enzyme, a recombinant DNA and a transformant thereof, and a process for producing isomerized saccharides using the enzyme. 10 Background of the Invention [0002] "Isomerase" is a generic term meaning a enzyme catalyzing conversion of isomers. According to "Enzyme Nomenclature", Academic Press Inc., USA, 1992, it includes the following six groups: (1) EC5.1; racemasesandepime- rases catalyzing optical isomerization, (2) EC 5.2; enzymes catalyzing geometric conversion ofcis-trans isomers, (3) 15 EC 5.3; enzymes catalyzing aldose-ketose conversion, keto-enol tautomerization, and intramolecular rearrangement of double bond, (4) EC 5.4; enzymes catalyzing intramolecular rearrangement of substituent to produce structural isomers, (5) EC 5.5; enzymes catalyzing intramolecular lyase-reaction, and (6) EC 5.99; enzymes catalyzing other isomerization. Among these isomerases, for example, the following enzymes are well known as isomerases catalyzing isomerization of neutral saccharides: xylose isomerase (EC 5.3.1.5) catalyzing conversion between D-xylose and D-xylulose, or be- 20 tween D-glucose and D-fructose (aldose-ketose conversion), aldose 1-epimerase (EC 5.1.3.3) catalyzing conversion between α and β anomer of aldose, ketose 3-epimerase catalyzing epimerization of C-3 position of ketopentoses and ketohexoses to produce the corresponding epimers (q.v. Japanese Patent Kokai No. 125776/1994 or International Patent Publication No. WO 2007/058086). These enzymes are widely used for industrial production of isomerized saccharides, quantitative determination of saccharides, and preparation of rare saccharides. 25 [0003] On the other hand, Tyleret al., Archives of Biochemistry and Biophysics, Vol.119, pp.363-367 (1967), reported that Ruminococcus albus, an anaerobic bacteria, produces a cellobiose 2-epimerase, and it epimerizes C-2 position of reducing-terminal glucose in cellosiose to produce epicellobiose (4-O-β-D-Glucosyl D-mannose), which enzyme has been assigned a enzyme number of EC 5.1.3.11 in Enzyme Nomenclature referred to above. Ito et al., Biochemical and Biophysical Research Communication, Vol.360, pp.640-645 (2007) and Ito et al., Applied Microbiology and Biotechnol- 30 ogy, Vol.79, pp.433-441 (2008) disclosed the amino-acid sequence of the cellobiose 2-epimerase, the DNA sequence encoding the amino-acid sequence, and that the cellobiose 2-epimerase acts on cellooligosaccharide or lactose, as well as cellobiose, to produce epicelllooligosaccharide or epilactose (4-O- β-D-calactosyl D-mannnose). Furthermore, Taguchi et al., FEMS Microbiology Letters, Vol.287, pp.34-40 (2008), disclosed cellobiose 2-epimerase produced by Eubacterium cellulosolvens, also an anaerobic bacteria. 35 [0004] Nishimukai et al., Journal of Agricultural and Food Chemistry, Vol.56, pp.10340-10345 (2008) disclosed that when epilactose, converted from lactose by cellobiose 2-epimerase, was ingested in rat, it exerted physiological functions of promoting calcium absorption in the small intestine, increasing the amount of short-chain fatty acid in the intestine, and lowering the plasma cholesterol level, suggesting that epilactose is expected to be developed for a prebiotic material. [0005] However, the above known cellobiose 2-epimerase have problems that they are hard to use for industrial 40 production of epilactose or epicellobiose because of their low heat-resistance. Heat-resistance is an important property for practical application of enzyme reaction, and a highly heat-resistant enzyme is economically beneficial because a longtime reaction can be carried out with a small amount of the enzyme, resulting in low consumption of the enzyme. In consideration of industrial use, enzyme reaction is preferable to be conducted at 55°C or more, preferably, 60°C or more. In the above regard, a cellobiose 2-epimerase with higher heat-resistance is desired. 45 NCBI Database protein; 2007, Accession YP_001179132 discloses the amino acid sequence of N-acylglucosamine 2- epimerase. Vande Werken H.J.et al: "Hydrogenomics ofthe extremely thermophilic bacterium (Caldicellulosiruptor saccharolyticus ", appl. Environ. Microbial., Vol. 74, No. 21, 2008, p.p. 6720-6729 discloses the genomic sequence of a Caldicellulosiruptor sacchorolyticus. 50 Disclosure of the Invention [0006] An object of the present invention is to provide a use of a highly thermostable cellobiose 2-epimerase. [0007] Under the circumstances mentioned above, to attain the object, the inventors of the present invention screened 55 many thermophilic microorganisms to obtain a thermostable cellobiose 2-epimerase. As the result, the inventors found that a liquid disrupted-cell extract of Caldicellusiruptor saccharolyticus ATCC43494 in the genus of Caldicellusiruptor, had an enzyme activity of epimerizing lactose to form epilactose and producing a presumable isomerized saccharide from cellobiose. The inventors purified the epimerase as an electrophoretically single protein, investigated its properties, 2 EP 2 395 080 B1 and found that the enzyme had thermostability up to 70°C. However, the yield of the purified enzyme is too small to investigate its substrate specificity in detail. [0008] According to the amino-acid sequence of the epimerase, the inventors cloned the DNA encoding the epimerase from the genomic DNA of Caldicellusiruptor saccharolyticus, transformed E. coli with the recombinant DNA, and the 5 transformant was cultured to prepare the recombinant enzyme. The inventors investigated the substrate specificity of the recombinant enzyme and found that the enzyme has wide substrate specificity, such as unexpectedly act on D- glucose or D-fructose among monosaccharides, maltose among disaccharides, and maltooliosaccharides and cellooli- gosaccharides with glucose polymerization degree of 3 or more among oligosaccharides, to produce the corresponding epimers, i.e., D-mannose, D-talose, epimaltose (4-O-α-D-glucosyl D-mannose) and epimaltooligosaccharides or epi- 10 cellooligosaccharides with glucose polymerization degree of 3 or more, respectively, as well as epimerising cellobiose and lactose to form epicellobiose and epilactose, respectively. The inventors also found that in addition to 2-epimerization, the enzyme also catalyzes aldose-ketose conversion in higher enzyme dosage, to convert D-glucose or D-mannose into D-fructose, D-galactose or D-talose into D-tagatose, maltose or epimaltose into maltulose (4-O-α-D-glucosyl D- fructose), cellobiose or epicellobiose
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