摘要
低温CPC乙酰化酶在7-ACA的生物合成中具有重要作用和显著的优势,开发低温CPC乙酰化酶具有重大的经济价值。为了获得在低温下具有更高催化活性的CPC乙酰化酶,在前期的研究基础上,以先前获得的CA IIIM为亲本,借助分子对接的手段确定了它的底物结合区域,并利用py MOL软件找出了底物结合区域Loop上关键的脯氨酸残基,分析后将选定的脯氨酸用甘氨酸进行替换。借助p ET32a质粒在E.coli BL21(DE3)中进行了可溶性表达研究,除P272G外,其它突变体均实现了可溶性表达。P238G、P582G和P679G在13℃对CPC的催化活性分别为1.25、1.04和1.38U/mg,较亲本的0.85 U/mg有了显著的提高。此外,分别考察了亲本及突变体的温度稳定性,它们之间无明显的差异。然后,在13℃下进行了7-ACA低温生物合成的研究,结果表明反应24 h后CPC的转化率也能达到80%以上。由此可见在CPC乙酰化酶冷适应性改造方面取得了较为理想的结果,为进一步的改造及应用奠定了坚实的基础,也为其它低温酶的创制提供了可资借鉴的经验。
Cold-active CPC acylase plays an important role and has significant advantage in the biosynthesis of 7-ACA, thus developing cold-active CPC acylase has significant economic value. In order to obtain the CPC acylase with higher catalytic activity at low temperature, on the basis of previous research, the previously obtained CA IIIM was taken as a parent, confirmed its substrate binding region by the method of molecular docking, and found the key proline residues in the loop of CPC acylase substrate binding region by pyMOL. After analyzing, the selected prolines were mutated into glycines, respectively. The study of soluble expression was carried out in E. coli BI21 （ DE3 ） with pET32a plasmid, and soluble expression was achieved in the other mutants except P272G. Catalytic activity of P238G, P582G and P679G to CPC was 1.25, 1.04 and 1.38 U/mg respectively at 13℃, and there was a significant improvement compared with parental 0.85 U/mg. Moreover, the stability of parent and mutants was investigated, and there was no obvious difference between them. Afterwards, the low temperature biosynthesis of 7-ACA was carried out at 13℃, and the results showed that the conversion rate of CPC can reach 80% and above after 24h . The ideal results in cold adaptation improvement of CPC acylase were gained. So a solid foundation for further transformation and application was established, it provided a useful experience for the creating of other low-temperature enzymes.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第12期34-39,共6页
China Biotechnology
基金
国家自然科学基金(31371381)
河南省科技攻关项目(162102210116)
河南省高等学校重点科研项目计划(15A416008)
河南省南水北调中线水源区水生态安全创新型科技团队专项(17454)资助项目
