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Molecular cloning, and optimized production and characterization of recombinant cyclodextrin glucanotransferase from Bacillus sp. T1

文献类型： 外文期刊

作者： Liu, Zhenyang 1 ; Wu, Guogan 2 ; Wu, Huawei 1 ;

作者机构： 1.Yangtze Univ, Coll Life Sci, 1 South Loop Rd, Jingzhou 434025, Peoples R China

2.Shanghai Acad Agr Sci, Biotechnol Res Inst, 2901 Bei Zhai Rd, Shanghai 201106, Peoples R China

关键词： Cyclodextrin glucanotransferase (CGTase); beta-Cyclodextrin (beta-CD); 16S rDNA; Heterologous expression; Purification

期刊名称：3 BIOTECH （ 影响因子：2.893； 五年影响因子：3.446 ）

ISSN： 2190-572X

年卷期： 2022 年 12 卷 3 期

页码：

收录情况： SCI

摘要： Cyclodextrin glucosyltransferase (CGTase) is an enzyme which degrades starch to produce cyclodextrins (CDs). In this study, the beta-CGTase producing strain T1 was identified as Bacillus sp. by its morphological characteristics and 16S rDNA sequence analysis. The cgt-T1 gene was cloned and expressed in Escherichia coli. CGTase-T1 was purified by Ni-nitrilotri-acetic acid agarose column and the molecular weight was determined as approximately 75 kDa using SDS-PAGE analysis. For the expression of soluble proteins, the optimal induction conditions were 10 h at 25 degrees C with OD600 at 0.8. The purified CGTase-T1 exhibited maximum activity with an optimal pH and temperature of 6.0 and 65 degrees C. The enzyme was stable in a pH range of 7.0-10.0, retaining over 85% relative activity for 1 h. CGTase-T1 activity can be significantly enhanced by adding 1 mM Ba2+. Using a soluble starch substrate, the kinetic parameters were revealed with K-M and k(cat)/K-M values of 2.75 mg mL(-1) and 1253.97 s(-1) mL mg(-1), respectively. Additionally, the four enzyme activities of CGTase-T1 were determined. The highest conversion rate to CDs (40.9%) was achieved from soluble starch after 8 h of enzyme reaction, where mainly beta-CD was produced (79.1% of the total CDs yield), indicating that CGTase-T1 potentially has industrial application prospect.

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