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High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits
Published on: September 20, 2016
Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product
Chaoning Liang1, Yi Zhang1,2, Yan Jia3
1CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Abstract:
Glycodiversification broadens the scope of natural product-derived drug discovery. The acceptor substrate promiscuity of glucosyltransferase-D (GTF-D), a carbohydrate-processing enzyme from Streptococcus mutans, was expanded by protein engineering. Mutants in a site-saturation mutagenesis library were screened on the fluorescent substrate 4-methylumbelliferone to identify derivatives with improved transglycosylation efficiency. In comparison to the wild-type GTF-D enzyme, mutant M4 exhibited increased transglycosylation capabilities on flavonoid substrates including catechin, genistein, daidzein and silybin, using the glucosyl donor sucrose. This study demonstrated the feasibility of developing natural product glycosyltransferases by engineering transglycosidases that use donor substrates cheaper than NDP-sugars, and gave rise to a series of α-glucosylated natural products that are novel to the natural product reservoir. The solubility of the α-glucoside of genistein and the anti-oxidant capability of the α-glucoside of catechin were also studied.
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