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通过半理性工程提高SceCPR的催化效率,以实现D-pantolactone的不对称减少
Man Zhao1, Bin Zhou1, Xiaoli Jia1
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
来自Saccharomyces cerevisiae (SceCPR) 的基因工程基托潘托拉克 (KPL) 减少酶显示出显著增强的活性,用于产生D-潘托拉克 (D-PL),这是一个关键的性中间体. 突变SceCPR ((S158A/Y298H) 突变使催化效率提高了18.5倍.
科学领域:
- 生物催化剂是一种生物催化剂.
- 酵素工程是什么? 酶工程是什么
- 有机合成 有机合成
背景情况:
- D-panttolactone (D-PL) 是D-pantothenic酸合成的一个关键的性中间体.
- 来自Saccharomyces cerevisiae (SceCPR) 的原生基antolactone (KPL) 减少酶对不对称的KPL减少到D-PL的活性较低.
研究的目的:
- 为了提高SceCPR的催化活性,以提高D-PL的生产效率.
- 通过计算分析指导的半理性设计来设计SceCPR.
主要方法:
- 计算机辅助设计包括植物遗传学分析和分子动力学模拟,确定了关键残留物 (Ser158,Asn159,Gln180,Tyr208,Tyr298,Trp299).
- 对已识别的残留物进行了局部定向突变发生 (单一和组合).
- 进行了酶活性测定和3D结构分析.
主要成果:
- 突变SceCPR ((S158A/Y298H) 与野生类型SceCPR相比显示了18.5倍更高的催化效率 (kcat/Km).
- 结构分析显示,突变物体的催化口袋扩大,更具水友性,并增强了 π-π 相互作用.
- 使用SceCPR (S158A/Y298H) 和葡萄糖脱酶 (GDH) 的优化全细胞系统实现了490.21毫米的D-PL降低,具有>99%的反反分子过量和98%的转化.
结论:
- 半理性工程显著改善了SceCPR用于D-PL合成的催化性能.
- 突变SceCPR ((S158A/Y298H) 为工业D-PL生产提供了一个高效的生物催化剂.
- 工程酶和全细胞系统代表了一种最先进的方法,用于奇拉中间体合成.
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