Double mutations enhance β-cyclization activity of cyclodextrin glycosyltransferase from Bacillus circulans

Q2 Chemical Engineering

Journal of Molecular Catalysis B-enzymatic Pub Date : 2016-11-01 DOI:10.1016/j.molcatb.2016.12.002

Min Huang , Junyan Ren , Caiming Li , Zhengbiao Gu , Yan Hong , Li Cheng , Zhaofeng Li

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引用次数: 1

Abstract

A major drawback to the industrial production of β-cyclodextrin is the limited β-cyclization activity of cyclodextrin glycosyltransferase (CGTase). Here, we construct mutants of the β-CGTase from Bacillus circulans strain STB01 that contain single substitutions at Tyr89 and double substitutions at Tyr89 and Asp577. The results show that the double mutants Y89G/D577R, Y89D/D577R, and Y89N/D577R display enhanced β-cyclization activity, and have higher β-cyclization activity than that of the three single Tyr89 mutants. The double mutant Y89D/D577R exhibited the highest β-cyclization activity and β-cyclodextrin production, increasing 35.1% and 12.4% compared with those of the wild-type CGTase, respectively. The β-cyclization activity of double mutant Y89D/D577R is also higher than that of the single mutant D577R, which had the highest β-cyclization activity among the mutants prepared in our previous studies. The enhanced β-cyclization activity of these mutants may be a result of intermolecular interactions that stabilize intermediates in the β-cyclization reaction. Thus, double mutant Y89D/D577R is much more suitable for industrial β-cyclodextrin production than the wild-type enzyme.

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双突变增强环状芽孢杆菌环糊精糖基转移酶β-环化活性

工业生产β-环糊精的一个主要缺点是环糊精糖基转移酶(CGTase)的β环化活性有限。在这里，我们从环状芽孢杆菌菌株STB01中构建了β-CGTase突变体，该突变体含有Tyr89的单取代，Tyr89和Asp577的双取代。结果表明，双突变体Y89G/D577R、Y89D/D577R和Y89N/D577R均表现出增强的β环化活性，且其β环化活性高于3个Tyr89单突变体。双突变体Y89D/D577R表现出最高的β-环化活性和β-环糊精产量，分别比野生型CGTase提高35.1%和12.4%。双突变体Y89D/D577R的β-环化活性也高于单突变体D577R，是我们前期制备的突变体中β-环化活性最高的。这些突变体的β-环化活性增强可能是分子间相互作用的结果，这种相互作用稳定了β-环化反应中的中间体。因此，双突变体Y89D/D577R比野生型酶更适合于工业生产β-环糊精。

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来源期刊

Journal of Molecular Catalysis B-enzymatic 生物-生化与分子生物学

CiteScore

2.58

自引率

0.00%

发文量

审稿时长

3.4 months

期刊介绍： Journal of Molecular Catalysis B: Enzymatic is an international forum for researchers and product developers in the applications of whole-cell and cell-free enzymes as catalysts in organic synthesis. Emphasis is on mechanistic and synthetic aspects of the biocatalytic transformation. Papers should report novel and significant advances in one or more of the following topics; Applied and fundamental studies of enzymes used for biocatalysis; Industrial applications of enzymatic processes, e.g. in fine chemical synthesis; Chemo-, regio- and enantioselective transformations; Screening for biocatalysts; Integration of biocatalytic and chemical steps in organic syntheses; Novel biocatalysts, e.g. enzymes from extremophiles and catalytic antibodies; Enzyme immobilization and stabilization, particularly in non-conventional media; Bioprocess engineering aspects, e.g. membrane bioreactors; Improvement of catalytic performance of enzymes, e.g. by protein engineering or chemical modification; Structural studies, including computer simulation, relating to substrate specificity and reaction selectivity; Biomimetic studies related to enzymatic transformations.