title: Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.
abstract: Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for β-gal I, and 543 and 25 mM-1s-1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria; Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines.
authors.forename: Sheryl Lozel
authors.initials: SL
authors.lastname: Arreola
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Montira
authors.initials: M
authors.lastname: Intanon
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Suljic
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Roman
authors.initials: R
authors.lastname: Kittl
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria; School of Food Biotechnology and Food Technology, Hanoi University of Science and Technology, Hanoi, Vietnam.
authors.forename: Ngoc Hung
authors.initials: NH
authors.lastname: Pham
authors.affiliation: Division of Organic Chemistry, Department of Chemistry, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Kosma
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007223:Infant; D007422:Intestines / Q000382:microbiology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000096:biosynthesis*; D055503:Protein Multimerization; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D007527:Isoenzymes; D009844:Oligosaccharides; C001970:allolactose; D001616:beta-Galactosidase; D005947:Glucose; D007785:Lactose; D005690:Galactose
references.citation: J Agric Food Chem. 2007 Aug 8;55(16):6772-7
references.pmid: 17630761
references.citation: Carbohydr Res. 2010 Jul 2;345(10):1408-16
references.pmid: 20385377
references.citation: Adv Food Nutr Res. 1995;38:1-102
references.pmid: 15918291
references.citation: Appl Microbiol Biotechnol. 2009 Oct;84(5):899-907
references.pmid: 19434405
references.citation: N Biotechnol. 2011 Oct;28(6):806-13
references.pmid: 21888996
references.citation: Biotechnol J. 2007 Apr;2(4):480-5
references.pmid: 17285679
references.citation: Appl Microbiol Biotechnol. 2007 Oct;76(6):1365-72
references.pmid: 17684740
references.citation: J Food Sci. 2011 May;76(4):R103-11
references.pmid: 22417365
references.citation: Bioinformatics. 2007 Nov 1;23(21):2947-8
references.pmid: 17846036
references.citation: Biotechnol Bioeng. 2000 Jul 20;69(2):140-9
references.pmid: 10861393
references.citation: Biotechnol Bioeng. 1987 Dec 20;30(9):1019-25
references.pmid: 18581545
references.citation: FEMS Microbiol Ecol. 2005 Apr 1;52(2):145-52
references.pmid: 16329901
references.citation: Appl Environ Microbiol. 2000 Apr;66(4):1379-84
references.pmid: 10742215
references.citation: Appl Microbiol Biotechnol. 2011 Feb;89(3):645-54
references.pmid: 20852995
references.citation: FEMS Microbiol Lett. 2007 Apr;269(1):136-44
references.pmid: 17227458
references.citation: Curr Opin Biotechnol. 2002 Oct;13(5):490-6
references.pmid: 12459343
references.citation: J Agric Food Chem. 2012 Feb 22;60(7):1713-21
references.pmid: 22283494
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Appl Environ Microbiol. 1999 Oct;65(10):4506-12
references.pmid: 10508082
references.citation: Protein Sci. 2012 Dec;21(12):1792-807
references.pmid: 23011886
references.citation: J Appl Microbiol. 2008 Feb;104(2):305-44
references.pmid: 18215222
references.citation: Biotechnol Lett. 2003 Oct;25(20):1769-74
references.pmid: 14626424
references.citation: J Nutr. 1995 Jun;125(6):1401-12
references.pmid: 7782892
references.citation: Appl Environ Microbiol. 2001 Sep;67(9):4256-63
references.pmid: 11526031
references.citation: J Agric Food Chem. 2006 Jul 12;54(14):4989-98
references.pmid: 16819907
references.citation: Biotechnol J. 2010 Aug;5(8):838-47
references.pmid: 20669255
references.citation: Folia Microbiol (Praha). 2000;45(1):29-34
references.pmid: 11200668
references.citation: Appl Microbiol Biotechnol. 2004 Dec;66(3):276-84
references.pmid: 15480628
references.citation: J Agric Food Chem. 2012 Jan 25;60(3):740-8
references.pmid: 22148735
references.citation: Nutr Res Rev. 2004 Dec;17(2):259-75
references.pmid: 19079930
references.citation: Appl Microbiol Biotechnol. 2002 Mar;58(4):439-45
references.pmid: 11954789
references.citation: Am J Clin Nutr. 2004 Dec;80(6):1658-64
references.pmid: 15585783
references.citation: J Agric Food Chem. 2013 Mar 6;61(9):2213-23
references.pmid: 23383752
references.citation: J Agric Food Chem. 2006 Jul 12;54(14):4999-5006
references.pmid: 16819908
references.citation: Appl Environ Microbiol. 2001 Jun;67(6):2526-30
references.pmid: 11375159
references.citation: Appl Environ Microbiol. 2001 May;67(5):2276-83
references.pmid: 11319112
references.citation: J Agric Food Chem. 2011 Apr 27;59(8):3803-11
references.pmid: 21405014
references.citation: Appl Microbiol Biotechnol. 2009 Apr;82(6):1079-88
references.pmid: 19099301
references.citation: Microbiologica. 1986 Jan;9(1):39-45
references.pmid: 3951366