Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Appl Microbiol Biotechnol. 2011 May;90(4):1361-71. doi: 10.1007/s00253-011-3114-9. Epub 2011 Mar 1.
To further enhance repeated batch reactions with immobilized N-carbamoyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T (50) (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met(31), Asn(93), Gln(207), Asn(242), Glu(266), Thr(271), Ala(273)) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T(50) by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k(cat) and K(m) values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of D-p-hydroxyphenylglycine.
为了进一步增强固定化 N-碳酰-D-氨基酸酰胺水解酶（DCase）的重复分批反应，该酶可用于 D-氨基酸的工业生产，我们通过逐步进化提高了来源于恶臭假单胞菌 CGMCC1596 的高可溶性突变体 DCase-M3 的稳定性。在我们之前的报告中，通过易错 PCR 鉴定了六个与热稳定性相关的位点。基于上述结果，通过两轮 DNA 改组获得了一个改良的突变体 B5（Q12L/Q23L/H248Q/T262A/T263S），与亲本酶 DCase-M3 相比，T（50）（定义为热处理 15 分钟后降低初始活性 50%的温度）提高了 10°C。此外，通过氨基酸共识方法，基于同源 DCases 的序列比对，鉴定了 B5 上几个与热稳定性相关的位点（Met（31）、Asn（93）、Gln（207）、Asn（242）、Glu（266）、Thr（271）、Ala（273））。这些位点进一步通过迭代饱和突变（ISM）进行了研究，并获得了一个组合突变体 D1（Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P），其 T（50）比 DCase-M3 提高了约 16°C。氧化稳定性测定表明，最耐热的突变体对过氧化氢的抗性仅略有增加。比较特性表明，D1 不仅保持了其特征的高溶解性，而且与亲本酶具有相似的 k(cat)和 K(m)值以及最适反应 pH 值。固定化形式下显著改善的突变体有望应用于 D-对羟基苯甘氨酸的工业生产。
