Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation.

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 (McSuSy), 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 McSuSy 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 McSuSy. When the mutant S31D of McSuSy 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.

The rapid high-throughput screening of ω-transaminases via a colorimetric method using aliphatic α-diketones as amino acceptors.

ω-Transaminases (ω-TAs) are widely available for the production of chiral amines and unnatural amino acids. Herein, a rapid spectrophotometric method was developed for screening ω-TAs based on the colored products that can be generated from transamination reactions between aliphatic α-diketones and amino donors catalyzed by ω-TAs. The possible mechanism of the formation of the colored product was investigated according to LC-Q-TOF-MS analysis. Among seven diketones, 2,3-butanedione was selected as the most suitable amino acceptor for colorimetric screening of ω-TAs with high efficiency, high sensitivity, and low background interference. Meanwhile, the absorbance of the colored product generated by 2,3-butanedione catalyzed by ω-TAs in this method was linearly correlated with the results by HPLC analysis. This method was also confirmed to effectively screen ω-TA mutants with high activity towards isopropylamine.

Screening UDP-Glycosyltransferases for Effectively Transforming Stevia Glycosides: Enzymatic Synthesis of Glucosylated Derivatives of Rubusoside.

Five plant-derived uridine diphosphate glycosyltransferases (UGTs) that catalyzed the glucosylation of stevia glycosides (SGs) were uncovered as the result of sequence mining considering the catalytic residues and conserved motifs of the known UGTs. Thereinto, LbUGT from Lycium barbarum with high activity toward rubusoside has been enzymatically characterized. The recombinant LbUGT was demonstrated to catalyze the ß-1,6-glucosylation at C19 of rubusoside, producing a monoglucosyl derivative 13-[(O-ß-d-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) ester], which was then submitted to a ß-1,2-glucosylation by LbUGT, resulting in a diglucosyl derivative 13-[(O-ß-d-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(2-O-ß-d-glucopyranosyl-6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) ester]. The di-glycosylated product of rubusoside showed an obvious increase in sweetness intensity (134 times sweeter than 5% sucrose) and almost eliminated the unpleasant bitter taste. This work will provide a reference for the taste improvement of SGs.

Bioconversion of Stevioside to Rebaudioside E Using Glycosyltransferase UGTSL2.

Rebaudioside E, one of the minor components of steviol glycosides, was first isolated and identified from Stevia rebaudiana in 1977. It is a high-intensity sweetener that tastes about 150-200 times sweeter than sucrose and is also a precursor for biosynthesis of rebaudioside D and rebaudioside M, the next-generation Stevia sweeteners. In this work, new unknown steviol glycosides were enzymatically synthesized from stevioside by coupling UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Rebaudioside E was speculated to be the main product of glucosylation of the Glc(ß1→C-19) residue of stevioside along with the formation of a (ß1→2) linkage based on the analysis of the regioselectivity and stereoselectivity of UGTSL2, and verified afterwards by LC-MS/MS with standard. In a 20-ml bioconversion reaction of 20 g/l stevioside by UGTSL2 and StSUS1, 15.92 g/l rebaudioside E was produced for 24 h.