Glycosyltransferase Co-Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of the C-Glucoside Nothofagin with Efficient Reuse of Enzymes

Hui Liu, Gregor Tegl, Bernd Nidetzky*

*Korrespondierende/r Autor/-in für diese Arbeit

Publikation: Beitrag in einer FachzeitschriftArtikelBegutachtung

Abstract

Sugar nucleotide-dependent (Leloir) glycosyltransferases are synthetically important for oligosaccharides and small molecule glycosides. Their practical use involves one-pot cascade reactions to regenerate the sugar nucleotide substrate. Glycosyltransferase co-immobilization is vital to advance multi-enzyme glycosylation systems on solid support. Here, we show glycosyltransferase chimeras with the cationic binding module Zbasic2 for efficient and well-controllable two-enzyme co-immobilization on anionic (ReliSorb SP400) carrier material. We use the C-glycosyltransferase from rice (Oryza sativa; OsCGT) and the sucrose synthase from soybean (Glycine max; GmSuSy) to synthesize nothofagin, the natural 3’-C-β-d-glucoside of the dihydrochalcone phloretin, with regeneration of uridine 5’-diphosphate (UDP) glucose from sucrose and UDP. Exploiting enzyme surface tethering via Zbasic2, we achieve programmable loading of the glycosyltransferases (∼18 mg/g carrier; 60%–70% yield; ∼80% effectiveness) in an activity ratio (OsCGT:GmSuSy=∼1.2) optimal for the overall reaction rate (∼0.2 mmol h−1 g−1 catalyst; 30 °C, pH 7.5). Using phloretin solubilized at 120 mM as inclusion complex with 2-hydroxypropyl-β-cyclodextrin, we demonstrate complete substrate conversion into nothofagin (∼52 g/L; 21.8 mg product h−1 g−1 catalyst) at 4% mass loading of the catalyst. The UDP-glucose was recycled 240 times. The solid catalyst showed excellent reusability, retaining ∼40% of initial activity after 15 cycles of phloretin conversion (60 mM) with a catalyst turnover number of ∼273 g nothofagin/g protein used. Our study presents important progress towards applied bio-catalysis with immobilized glycosyltransferase cascades. (Figure presented.).

Originalspracheenglisch
Seiten (von - bis)2157-2169
Seitenumfang13
FachzeitschriftAdvanced Synthesis and Catalysis
Jahrgang363
Ausgabenummer8
DOIs
PublikationsstatusVeröffentlicht - 13 Apr. 2021

ASJC Scopus subject areas

  • Katalyse
  • Organische Chemie

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