Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation
Kai Chen, Lei Lin, Ruiqi Ma, Jiajie Ding, Huayi Pan, Yehui Tao, Yan Li, Honghua Jia
Abstract
Sucrose synthase (SuSy, EC 2.4.1.13) is a unique glycosyltransferase (GT) for developing cost-effective glycosylation processes. Up to now, some SuSys derived from plants and bacteria have been used to recycle uridine 5′-diphosphate glucose in the reactions catalyzed by Leloir GTs. In this study, after sequence mining and experimental verification, a SuSy from Micractinium conductrix ( Mc SuSy), a single-cell green alga, was overexpressed in Escherichia coli , and its enzymatic properties were characterized. In the direction of sucrose cleavage, the specific activity of the recombinant Mc SuSy is 9.39 U/mg at 37°C and pH 7.0, and the optimum temperature and pH were 60°C and pH 7.0, respectively. Its nucleotide preference for uridine 5′-diphosphate (UDP) was similar to plant SuSys, and the enzyme activity remained relatively high when the DMSO concentration below 25%. The mutation of the predicted N -terminal phosphorylation site (S31D) significantly stimulated the activity of Mc SuSy. When the mutant S31D of Mc SuSy was applied by coupling the engineered Stevia glycosyltransferase UGT76G1 in a one-pot two-enzyme reaction at 10% DMSO, 50 g/L rebaudioside E was transformed into 51.06 g/L rebaudioside M in 57 h by means of batch feeding, with a yield of 76.48%. This work may reveal the lower eukaryotes as a promising resource for SuSys of industrial interest.