Abstract: Weak transglycosylation is a common problem with β-galactosidases from the glycoside hydrolase family 42 (GH42). The β-galactosidase BgaB from Geobacillus stearothermophilus, a typical thermostable enzyme of the GH42 family was investigated. Glu303 was predicted to be the catalytic nucleophile of BgaB. To improve the transglycoside activity of BgaB, functional studies and molecular modifications were carried out on the Glu303. Using site-directed mutagenesis and chemical modification to replace the carboxyl group of the Glu303 with a cysteine sulfinate (—SOO-), the Ox-E303C mutant was generated. Compared with the wild-type enzyme, the Ox-E303C mutant was found to increase galactooligosaccharides (GOS) synthesis from 0% to 11.5%. The result shows that introduction of a—SOO- group on to the Glu303 could improve transglycoside activity, and the catalytic nucleophile was involved in the transglycosylation regulation of BgaB. The results presented here have significant implications for the molecular modification of the transglycoside activity of GH42 β-galactosidases.
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