TY - JOUR
T1 - Glycosyltransferase Co-Immobilization for Natural Product Glycosylation
T2 - Cascade Biosynthesis of the C-Glucoside Nothofagin with Efficient Reuse of Enzymes
AU - Liu, Hui
AU - Tegl, Gregor
AU - Nidetzky, Bernd
N1 - Publisher Copyright:
© 2021 The Authors. Advanced Synthesis & Catalysis published by Wiley-VCH GmbH
PY - 2021/4/13
Y1 - 2021/4/13
N2 - 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.).
AB - 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.).
KW - C-glycosylation
KW - cascade bio-catalysis
KW - co-immobilization
KW - Leloir glycosyltransferase
KW - nothofagin
KW - sugar nucleotide regeneration
UR - http://www.scopus.com/inward/record.url?scp=85101873647&partnerID=8YFLogxK
U2 - 10.1002/adsc.202001549
DO - 10.1002/adsc.202001549
M3 - Article
AN - SCOPUS:85101873647
SN - 1615-4150
VL - 363
SP - 2157
EP - 2169
JO - Advanced Synthesis and Catalysis
JF - Advanced Synthesis and Catalysis
IS - 8
ER -