Efficient enzyme formulation promotes Leloir glycosyltransferases for glycoside synthesis

Markus Mikl, Alexander Dennig, Bernd Nidetzky*

*Korrespondierende/r Autor/in für diese Arbeit

Publikation: Beitrag in einer FachzeitschriftArtikel

Abstract

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 ∼3-fold, partly due to the release of enzyme activity trapped in insoluble pellet. Freeze drying of the resulting cell-free extract (∼17 U ml−1) reduced the volume ∼20-fold and gave ∼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′-β-C-glucosylation of phloretin (60 mM; as inclusion complex with hydroxypropyl-β-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; ∼90 regeneration cycles for UDP). Collectively, our study demonstrates a facile procedure for solid glycosyltransferase formulations practically usable in glycoside synthesis.

Originalspracheenglisch
Seiten (von - bis)74-78
Seitenumfang5
FachzeitschriftJournal of Biotechnology
Jahrgang322
DOIs
PublikationsstatusVeröffentlicht - 10 Okt 2020

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • !!Applied Microbiology and Biotechnology

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