高分子量腈水合酶在大肠杆菌中的表达策略及重组菌的细胞催化

Strategy of high molecular mass nitrile hydratase expression in Escherichia coli and the whole-cell catalysis by the recombinant strains

摘要:

【目的】实现红球菌Rhodococcus rhodochrous J1来源的高分子量腈水合酶H-NHase在Escherichia coli BL21(DE3)中过量表达，以提高菌体总酶活，缩短菌体发酵周期，提高生产效率。【方法】将H-NHase中编码α亚基的基因nhhA和调控基因nhhG上游的核糖体结合位点(Shine-Dalgarno sequence，SD)替换成翻译起始强度更高的异源SD，同时优化各亚基之间间隔序列的长度，并根据大肠杆菌密码子偏好性对目的基因进行优化。通过E. coli重组表达系统过表达优化后的腈水合酶基因。采用离子交换层析对H-NHase进行纯化，并通过凝胶过滤层析确定重组酶的相对分子量。优化了全细胞催化条件，并建立底物恒速流加的细胞催化工艺，模拟了烟酰胺的生产工艺。【结果】H-NHase在E. coli中实现了过量表达。重组蛋白粗酶液的活性为85.5±4.3 U/mg，纯酶比活为234.0±11.7 U/mg，H-NHase相对分子质量为504.5±9.8 kD。细胞催化最适pH为7.5，最适温度为25 °C，底物浓度为400 mmol/L。在此条件下，重组菌细胞酶活为256.0±10.4 U/mL，流加工艺最终的产物转化率可达99.9%。【结论】重组H-NHase大肠杆菌细胞生长迅速，发酵周期短，应用此重组菌进行细胞催化可以提高酰胺类物质的生产效率，具有潜在的工业价值。

Abstract:

[Objective] To increase the activity of the whole-cells, reduce the fermentation period and improve production efficiency, high molecular mass nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was over-expressed in Escherichia coli. [Methods] The native SD sequence upstream of the α-subunit gene nhhA and the activator gene nhhG was substituted by a stronger SD sequence, and the length of the intergenic region between the two genes was optimized. In addition, the rare codons within the gene were optimized. The modified nhhBAG was over-produced in recombinant E. coli BL21(DE3). After purification by ion-exchange chromatography, the molecular mass for the recombinant enzyme was determined by Size-exclusion chromatography. The catalytic conditions were optimized, and the process of biosynthesis of nicotinamide was simulated with continuous substrate-flow. [Results] H-NHase was successfully over-expressed in E. coli. The activity in cell-free extracts and the specific activity of the purified enzyme were 85.5±4.3 U/mg and 234.0±11.7 U/mg by using 3-Cyanopyridine as substrate, respectively. The molecular mass for recombinant H-NHase was 504.5±9.8 kD. The optimal catalysis pH, temperature, and substrate concentration using whole cells were 7.5, 25 °C, 400 mmol/L, respectively. The whole-cell activity was up to 256.0±10.4 U/mL. Under this condition, the transformation ratio by whole-cell was 99.9%. [Conclusion] The recombinant E. coli grows fast with a shorter fermentation period. The production efficiency of amides using the recombinant strain would be increased, which exhibits potential value for industrial production.