title: Efficient and regioselective synthesis of globotriose by a novel α-galactosidase from Bacteroides fragilis.
abstract: Globotriose (Galα1-4Galβ1-4Glc) is an important cell surface epitope that acts as the receptor for Shiga-like toxins, and it is also the core structure of Globo H and SSEA4 that are tumor-associated glycans. Hence, the enzymatic synthesis of globotriose would be necessary for the development of carbohydrate-based therapeutics for bacterial infections and cancers. Here, a novel GH27 α-galactosidase gene (agaBf3S), a 1521-bp DNA encoding 506 amino acids with a calculated molecular mass of 57.7 kDa, from Bacteroides fragilis NCTC9343 was cloned and heterogeneously expressed in Escherichia coli. The recombinant enzyme AgaBf3S preferentially hydrolyzed p-nitrophenyl-α-D-galactopyranoside (pNPαGal) in all tested nitrophenyl glycosides. It showed maximum activity at pH 4.5 and 40 °C, and it was stable at pH 4.0-11.0 below 40 °C and metal-independent. The K m and k cat values for pNPαGal, melibiose, and globotriose were 1.27 mM and 172.97 S(-1), 62.76 mM and 17.74 S(-1), and 4.62 mM and 388.45 S(-1), respectively. AgaBf3S could transfer galactosyl residue from pNPαGal to lactose (Galβ1-4Glc) with high efficiency and strict α1-4 regioselectivity. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation reaction catalyzed by AgaBf3S were studied in detail. AgaBf3S could synthesize globotriose as a single transglycosylation product with a maximum yield of 32.4 % from 20 mM pNPαGal and 500 mM lactose (pH 4.5) at 40 °C for 30 min. This new one-enzyme one-step synthetic reaction is simple, fast, and low cost, which provides a promising alternative to the current synthetic methods for access to pharmaceutically important Galα1-4-linked oligosaccharides.
authors.affiliation: State Key Lab of Microbial Technology, National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gong
authors.affiliation: State Key Lab of Microbial Technology, National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: State Key Lab of Microbial Technology, National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China.
authors.forename: Guofeng
authors.initials: G
authors.lastname: Gu
authors.affiliation: State Key Lab of Microbial Technology, National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China. lililu@sdu.edu.cn.
authors.forename: Lili
authors.initials: L
authors.lastname: Lu
authors.affiliation: State Key Lab of Microbial Technology, National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China. minxiao@sdu.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Xiao
mesh_terms: D000595:Amino Acid Sequence; D001441:Bacteroides fragilis / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008553:Melibiose / Q000096:biosynthesis; D009598:Nitrophenylgalactosides / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014312:Trisaccharides / Q000096:biosynthesis*; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D014312:Trisaccharides; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C113460:globotriose; D008553:Melibiose; D000519:alpha-Galactosidase
keywords: Bacteroides fragilis; Globotriose; Regioselectivity; Transglycosylation; α-Galactosidase