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Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity.

ABSTRACT: Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among ?-, ?-, and ?-CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased Km value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the -5, -6, and -7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths.IMPORTANCE The low water solubility of sophoricoside seriously limits its applications in the food and pharmaceutical industries. Long-chain glycosylated sophoricosides show greatly improved water solubility. Here, the product specificity of cyclodextrin glycosyltransferase (CGTase) for long-chain glycosylated sophoricosides was significantly affected by pH. Our results reveal the pH-regulated mechanism of the glycosylated product specificity of CGTase. This work adds to our understanding of the synthesis of long-chain glycosylated sophoricosides and provides guidance for exploring related product specificity of CGTase based on pH regulation.

SUBMITTER: Han R

PROVIDER: S-EPMC7082579 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity.

Han Ruizhi R Ni Jie J Zhou Jieyu J Dong Jinjun J Xu Guochao G Ni Ye Y

Applied and environmental microbiology 20200318 7

Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from <i>Paenibacillus macerans</i> (<i>Pm</i>CGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, β-, and γ-CGTases with different product specificit ...[more]

PMID: 32005733

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Project description:BackgroundCompared with steviol glycosides, the taste of glucosylated steviol glycosides is better and more similar to that of sucrose. At present, cyclodextrin glucanotransferase (CGTase) is primarily used to catalyze the conversion of steviol glycosides to glucosylated steviol glycosides, with soluble starch serving as a glycosyl donor. The main disadvantages of enzymatic transglycosylation are the limited number of enzymes available, the low conversion rates that result in low yields, and the lack of selectivity in the degree of glycosylation of the products. In order to fill these gaps, the proteome of Alkalihalobacillus oshimensis (also named Bacillus oshimensis) was used for mining novel CGTases.ResultsHere, CGTase-15, a novel β-CGTase with a wide pH adaptation range, was identified and characterized. The catalyzed product of CGTase-15 tasted better than that of the commercial enzyme (Toruzyme® 3.0 L). In addition, two amino acid sites, Y199 and G265, which play important roles in the conversion of steviol glycosides to glucosylated steviol glycosides were identified by site-directed mutagenesis. Compared with CGTase-15, CGTase-15-Y199F mutant significantly increased the conversion rate of rebaudioside A (RA) to glucosylated steviol glycosides. Compared with CGTase-15, the content of short-chain glycosylated steviol glycosides catalyzed by CGTase-15-G265A mutant was significantly increased. Moreover, the function of Y199 and G265 was verified in other CGTases. The above mutation pattern has also been applied to CGTase-13 (a CGTase discovered by our laboratory with great potential in the production of glycosylated steviol glycosides), confirming that the catalytic product of CGTase-13-Y189F/G255A mutant has a better taste than that of CGTase-13.ConclusionsThis is the first report on the improvement of the sensory profiles of glycosylated steviol glycosides through site-directed mutagenesis of CGTase, which is significant for the production of glycosylated steviol glycosides.

Dataset Information

Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity.

Publications

Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity.

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