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

Abstract

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, MgSO4 4.19 mmol L−1, NiSO4 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.

Economical synthesis of γ-cyclodextrin catalyzed by oriented cyclodextrin glycosyltransferase displayed on bacterial polyhydroxyalkanoate nanogranules

“…The E. coli BL21 (DE3) strain harboring the recombinant plasmid pET22b (+)-cgt was synthesized according to the biased codons of E. coli BL21 (DE3) and induced to produce free γ-CGTase by the addition of Isopropyl-β-d-1-thiogalactopyranoside (IPTG) as described in our previous study [ 24 ]. After the supernatant of cell lysis was precipitated by ammonium sulfate, the free γ-CGTase was purified by an α-CD-Sepharose 6B affinity column (Fig.…”

“…Further analyses of the products converted from starch revealed that the majority (90.44%) of product CDs were the γ form, nearly 11% higher than the wild enzyme. However, the free γ-CGTase showed poor thermal stability, exceeding a 50% loss of hydrolysis and cyclization activities after only 20 min treatment at the optimal temperature (55 °C) [ 24 ]. Thus, our purpose in this study was to develop a robust and reused biocatalytic system for cost-effective production of γ-CD.…”

Molecular strategies to enhance the keratinase gene expression and its potential implications in poultry feed industry

“…For CGTase expression, 10 g/L and 15 g/L of yeast and peptone extract were the optimal concentrations. Additionally, 15 g/L was the optimal glucose concentration for CGTase expression so the glucose concentration had been optimized ( Duan et al, 2020 ). Additionally, the impact of metal ions on CGTase expression was examined.…”

Microbial Cyclodextrin Glycosyltransferases: Sources, Production, and Application in Cyclodextrin Synthesis

“…T1 at 25 °C postinduction with 0.3 mM IPTG and OD600 = 0.8 after 10 h incubation in E. coli BL21 (DE3) host. Moreover, Duan et al [63] optimized recombinant production of γ-CGTase from B. clarkii 7364 by using the Placket-Burman and response surface methodologies in E. coli BL21 (DE3) host. They reported a 2.8-fold enhancement in γ-CGTase activity (53992 U/mL; hydrolysis activity) after medium optimization [63].…”

“…Moreover, Duan et al [63] optimized recombinant production of γ-CGTase from B. clarkii 7364 by using the Placket-Burman and response surface methodologies in E. coli BL21 (DE3) host. They reported a 2.8-fold enhancement in γ-CGTase activity (53992 U/mL; hydrolysis activity) after medium optimization [63]. Kim et al [64] reported a 6-fold enhancement in the soluble expression of B. macerans CGTase in E. coli upon the coexpression of both DnaK-DnaJ-GrpE and GroEL-GroES.…”