Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Enzyme Microb Technol. 2022 Sep;159:110066. doi: 10.1016/j.enzmictec.2022.110066. Epub 2022 May 17.
The γ-cyclodextrin glycosyltransferase (γ-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to γ-cyclodextrins (CDs). Maximum cyclization activity of 2.28 ± 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 ºC. Surface expression of γ-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed γ-CGTase revealed optima at pH 10.0 and 40 ºC along with a t of 24.75 min at 50ºC. The K and V values on soluble potato starch were 10.94 mg/ml and 4.33 µmoles min g DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed γ-CGTase was further utilized for CD production and specifically, γ-CD conversion was obtained. The maximum conversion was achieved at 50 ºC, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed γ-CGTase was able to convert soluble potato starch (2.5%) into γ-CDs with a 72.7% specific yield and no other peaks corresponding to α- and β-CDs were observed on HPLC. The enzyme was found to be ~100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at - 20 ºC even after 7 days.
埃文斯氏乳酪杆菌的γ-环糊精糖基转移酶（γ-CGTase）通过 pAIDA-I 自转运蛋白在大肠杆菌的细胞表面表达，并进一步用于将淀粉转化为γ-环糊精（CDs）。在 37℃下用 0.1mM IPTG 诱导 3 小时后，获得了 2.28±0.46 U/g 生物质的最大环化活性。使用 FITC 缀合的抗 HIS 抗体通过流式细胞术证实了 γ-CGTase 的表面表达。表面展示的 γ-CGTase 的生化特性研究表明，最佳 pH 值为 10.0，最佳温度为 40℃，在 50℃下的 t 值为 24.75 分钟。在可溶性马铃薯淀粉上的 K 和 V 值分别为 10.94mg/ml 和 4.33 µmoles min g DCW，活化能计算为 89.8 kJ/mol。表面展示的 γ-CGTase 进一步用于 CD 生产，特别是获得了 γ-CD 的转化。在 50℃、pH 9.0 下，使用可溶性马铃薯淀粉（2.5%；w/v），最终酶浓度为 0.6 U/g 淀粉，达到最大转化率。表面展示的 γ-CGTase 能够将可溶性马铃薯淀粉（2.5%）转化为 γ-CDs，特异性产率为 72.7%，在 HPLC 上未观察到其他对应于 α-和 β-CDs 的峰。该酶在连续 24 小时的 2 个循环中具有约 100%的操作稳定性，即使在 -20℃下储存 7 天，也具有 >75%的储存稳定性。
