High-level production of γ-cyclodextrin glycosyltransferase in recombinant Escherichia coli BL21 (DE3): culture medium optimization, enzymatic properties characterization, and product specificity analysis

• Published in: Annals of microbiology 2020-12, Vol.70 (1), Article 70
• Main Authors: Duan, Menglu, Wang, Yan, Yang, Guowu, Li, Jiao, Wan, Yi, Deng, Yuan, Mao, Yong
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Biomedical and Life Sciences; Biotechnology; Biotransformation; Cyclodextrin; E coli; Escherichia coli; Food industry; Glycosyltransferase; High-performance liquid chromatography; Hydrolysis; Life Sciences; Medical Microbiology; Microbial Ecology; Microbial Genetics and Genomics; Microbiology; Mycology; Original Article; pH effects; Pharmaceutical industry; Starch
• ISSN: 1590-4261; 1869-2044
• DOI: 10.1186/s13213-020-01610-8

Purpose γ-Cyclodextrin glycosyltransferase (γ-CGTase) catalyzes the biotransformation of low-cost starch into valuable γ-cyclodextrin (γ-CD), which is widely applied in biotechnology, food, and pharmaceutical industries. However, the low specificity and activity of soluble γ-CGTase increase the production cost of γ-CD, thereby limiting its applications. Therefore, the present study aimed at optimizing an economical medium for high production of γ-CGTase by the recombinant Escherichia coli ( E. coli ) BL21 (DE3) and evaluating its enzymatic properties and product specificity. Methods The γ-CGTase production was optimized using the combination of Plackett-Burman experimental design (PBD) and Box-Behnken design-response surface methodology (BBD-RSM). The hydrolysis and cyclization properties of γ-CGTase were detected under the standard assay conditions with buffers of various pHs and different reaction temperatures. The product specificity of γ-CGTase was investigated by high-performance liquid chromatography (HPLC) analysis of three CDs (α-, β-, γ-CD) in the biotransformation product of cassava starch. Results The γ-CGTase activity achieved 53992.10 U mL −1 under the optimum conditions with the significant factors (yeast extract 38.51 g L −1 , MgSO 4 4.19 mmol L −1 , NiSO 4 0.90 mmol L −1 ) optimized by the combination of PBD and BBD-RSM. The recombinant γ-CGTase exhibited favorable stability in a wide pH and temperature range and maintained both the hydrolysis and cyclization activity under the pH 9.0 and 50 °C. Further analysis of the products from cassava starch catalyzed by the γ-CGTase reported that the majority (90.44%) of product CDs was the γ form, which was nearly 11% higher than the wild enzyme. Cyclododecanone added to the transformation system could enhance the γ-CD purity to 98.72%, which is the highest purity value during the transformation process reported so far. Conclusion The yield of γ-CGTase activity obtained from the optimized medium was 2.83-fold greater than the unoptimized medium, and the recombinant γ-CGTase exhibited a favorable thermal and pH stability, and higher γ-cyclization specificity. These results will provide a fundamental basis for the high productivity and purity of γ-CD in the industrial scale.