期刊
JOURNAL OF BIOTECHNOLOGY
卷 322, 期 -, 页码 74-78出版社
ELSEVIER
DOI: 10.1016/j.jbiotec.2020.06.023
关键词
Leloir glycosyltransferase; Solid enzyme formulation; Enzyme cascade; C-glycosylation; Sucrose synthase
Sugar nucleotide-dependent (Leloir) glycosyltransferases are powerful catalysts for glycoside synthesis. Their applicability can be limited due to elaborate production of enzyme preparations deployable in biocatalytic processes. Here, we show that efficient enzyme formulation promotes glycosyltransferases for the synthesis of the natural C-glycoside nothofagin. Adding Brij-35 detergent (1 %, w/v) during sonication of the E. coli. BL21-Gold (DE3) expression strain, recovery of Oryza sativa C-glycosyltransferase was enhanced by similar to 3-fold, partly due to the release of enzyme activity trapped in insoluble pellet. Freeze drying of the resulting cell-free extract ( similar to 17 U ml(-1)) reduced the volume similar to 20-fold and gave similar to 55 mg solids ml(-1) liquid processed, with 83 % retention of the original activity and a specific activity of 0.20 U mg(-1) solids. The Glycine max sucrose synthase was processed analogously, giving a solid enzyme preparation of 0.28 U mg(-1) in 63 % yield. Both enzyme formulations were stable for several weeks. The glycosyltransferase cascade reaction for 3'-beta-C-glucosylation of phloretin (60 mM; as inclusion complex with hydroxypropyl-beta-cyclodextrin) from UDP-glucose (generated in situ by sucrose synthase from 500 mM sucrose and 0.5 mM UDP) showed excellent performance metrics (>= 98 % yield; 3.2 g l(-1) h(-1) space-time yield; similar to 90 regeneration cycles for UDP). Collectively, our study demonstrates a facile procedure for solid glycosyltransferase formulations practically usable in glycoside synthesis.
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