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https://doi.org/10.1007/s00253-016-7464-1
Copy DOIPublication Date: Mar 28, 2016 | |
Citations: 13 |
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 57.7kDa, 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 pH4.5 and 40°C, and it was stable at pH4.0-11.0 below 40°C and metal-independent. The K m and k cat values for pNPαGal, melibiose, and globotriose were 1.27mM and 172.97S(-1), 62.76mM and 17.74S(-1), and 4.62mM and 388.45S(-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 20mM pNPαGal and 500mM lactose (pH4.5) at 40°C for 30min. 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.
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