College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, China.
Bontec Bio-engineering (Shenzhen) Co. Ltd, Shenzhen, 518101, Guangdong, China.
Appl Biochem Biotechnol. 2019 Oct;189(2):448-458. doi: 10.1007/s12010-019-02996-1. Epub 2019 May 2.
For efficiently enhancing the activity of isoeugenol monooxygenase, a whole cell overproducing active aggregate IEM720-18A was successfully fabricated via the fusion of amphiphilic short peptide 18A (EWLKAFYEKVLEKLKELF) and isoeugenol monooxygenase and then efficiently expressed in E. coli BL21 (DE3). Such resulting bacteria, E. coli BL21 (DE3) harboring pET30a-IEM720-18A was applied in the biotransformation of isoeugenol to vanillin with the optimization of cultivation conditions. Our results revealed that the vanillin concentration reached to the highest level (14.5 mmol/L) under the optimized reaction conditions including 1.5-g cells containing active aggregate of IEM720-18A, 10% (v/v) dimethyl sulfoxide (DMSO), 100 mmol/L isoeugenol, 50 mmol/L glycine-sodium hydroxide buffer (pH 10.5) in 10 mL reaction volume, and 200 rpm at 25 °C for 36 h. Moreover, the active aggregate IEM720-18A was immobilized with 100 mmol/L glutaraldehyde at 4 °C to improve the operational stability. The highest activity could be achieved when the reactions were carried out at 25 °C and the relative activity of the immobilized enzyme maintained over 60% after seven recycles. Our study provides a new approach to the biotransformation of isoeugenol into vanillin.
为了有效提高异丁香酚单加氧酶的活性，通过融合两亲性短肽 18A（EWLKAFYEKVLEKLKELF）和异丁香酚单加氧酶，成功构建了高效表达的全细胞异丁香酚单加氧酶活性聚集体 IEM720-18A，并在大肠杆菌 BL21（DE3）中高效表达。将含有 pET30a-IEM720-18A 的大肠杆菌 BL21（DE3）应用于异丁香酚向香草醛的生物转化，优化了培养条件。结果表明，在优化的反应条件下，包括 1.5 克含有 IEM720-18A 活性聚集体的细胞、10%（v/v）二甲基亚砜（DMSO）、100mmol/L 异丁香酚、50mmol/L 甘氨酸-氢氧化钠缓冲液（pH 10.5）和 10mL 反应体积中，200rpm 于 25°C 下反应 36 小时，香草醛浓度达到最高水平（14.5mmol/L）。此外，通过 100mmol/L 戊二醛在 4°C 下固定 IEM720-18A 活性聚集体，以提高操作稳定性。当反应在 25°C 下进行时，可获得最高的酶活，固定化酶的相对酶活在 7 次循环后仍保持在 60%以上。本研究为异丁香酚向香草醛的生物转化提供了一种新方法。
