Dataset Information

Enhancing the ?-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7.

ABSTRACT: Cyclodextrin glycosyltransferases (CGTases) (EC 2.4.1.19) catalyze the conversion of starch or starch derivates into mixtures of ?-, ?-, and ?-cyclodextrins. Because time-consuming and expensive purification procedures hinder the widespread application of single-ingredient cyclodextrins, enzymes with enhanced specificity are needed. In this study, we tested the hypothesis that the ?-cyclodextrin selectivity of Paenibacillus macerans ?-CGTase could be augmented by masking subsite -7 of the active site, blocking the formation of larger cyclodextrins, particularly ?-cyclodextrin. Five single mutants and three double mutants designed to remove hydrogen-bonding interactions between the enzyme and substrate at subsite -7 were constructed and characterized in detail. Although the rates of ?-cyclodextrin formation varied only modestly, the rate of ?-cyclodextrin formation decreased dramatically in these mutants. The increase in ?-cyclodextrin selectivity was directly proportional to the increase in the ratio of their kcat values for ?- and ?-cyclodextrin formation. The R146A/D147P and R146P/D147A double mutants exhibited ratios of ?-cyclodextrin to total cyclodextrin production of 75.1% and 76.1%, approximately one-fifth greater than that of the wild-type enzyme (63.2%), without loss of thermostability. Thus, these double mutants may be more suitable for the industrial production of ?-cyclodextrin than the wild-type enzyme. The production of ?-cyclodextrin by these mutants was almost identical to their production of ?-cyclodextrin, which was unaffected by the mutations in subsite -7, suggesting that subsite -7 was effectively blocked by these mutations. Further increases in ?-cyclodextrin selectivity will require identification of the mechanism or mechanisms by which these small quantities of larger cyclodextrins are formed.

SUBMITTER: Wang L

PROVIDER: S-EPMC4959485 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Publications

Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7.

Wang Lei L Duan Xuguo X Wu Jing J

Applied and environmental microbiology 20160404 8

Cyclodextrin glycosyltransferases (CGTases) (EC 2.4.1.19) catalyze the conversion of starch or starch derivates into mixtures of α-, β-, and γ-cyclodextrins. Because time-consuming and expensive purification procedures hinder the widespread application of single-ingredient cyclodextrins, enzymes with enhanced specificity are needed. In this study, we tested the hypothesis that the α-cyclodextrin selectivity of Paenibacillus macerans α-CGTase could be augmented by masking subsite -7 of the active ...[more]

PMID: 26850297

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Project description: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.

Dataset Information

Enhancing the ?-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7.

Publications

Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7.

Similar Datasets

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