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: Wei Gong, Li Xu, Guofeng Gu, Lili Lu, Min Xiao
• Journal: Applied microbiology and biotechnology (Appl Microbiol Biotechnol) Vol. 100 Issue 15 Pg. 6693-6702 (Aug 2016) ISSN: 1432-0614 [Electronic] Germany
• PMID: 27020280 (Publication Type: Journal Article)

Chemical References

• Trisaccharides
• Nitrophenylgalactosides
• 4-nitrophenylgalactoside
• globotriose
• Melibiose
• alpha-Galactosidase

Topics

• Amino Acid Sequence
• Bacteroides fragilis (enzymology, genetics)
• Escherichia coli (genetics, metabolism)
• Melibiose (biosynthesis)
• Nitrophenylgalactosides (metabolism)
• Sequence Alignment
• Substrate Specificity
• Trisaccharides (biosynthesis)
• alpha-Galactosidase (genetics, metabolism)