Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis.
Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis.
复制标题
通过无偏系统发育共识引导诱变提高粪产碱菌肌酸酶的热稳定性
DOI：
10.1186/s12934-020-01451-9
复制
发表时间：
2020-10-17
影响因子：
6.4
通讯作者：
Yang G
中科院分区：
文献类型：
--
作者：
Bai X;Li D;Ma F;Deng X;Luo M;Feng Y;Yang G
肌酐的酶法定量已成为临床评估肾功能的重要方法。尽管肌酸酶（CR）经常用于此目的，但其较差的热稳定性严重限制了工业应用。在此，我们报道了一种来自粪产碱杆菌（afCR）的新型肌酸酶，它比目前使用的肌酸酶具有更高的催化活性和更低的Km值。此外，我们开发了一种无偏系统发育共识方法来提高afCR的热稳定性。
我们应用无偏系统发育共识方法从24种肌酸酶家族同源物中鉴定出59个候选共识残基，用于筛选热稳定性提高的afCR突变体。选择afCR的21种氨基酸进行诱变，其中11种与亲本酶（afCR - M0）相比表现出热稳定性提高。在连续筛选中对单点突变进行组合，得到了四重突变体D17V/T199S/L6P/T251C（M4 - 2），其在57°C下的半衰期提高了约1700倍，T5015比afCR - M0高4.2°C。该突变体保留了与afCR - M0相当的催化活性，因此在肌酐检测应用中显示出很大的潜力。结构同源建模揭示了与各个突变相关的广泛潜在分子相互作用，这些相互作用有助于提高afCR的热稳定性。
这项研究的结果清楚地表明，用于提高afCR热稳定性的无偏系统发育共识设计在提高更多酶的热稳定性方面是有效且有前景的。
Background
Enzymatic quantification of creatinine has become an essential method for clinical evaluation of renal function. Although creatinase (CR) is frequently used for this purpose, its poor thermostability severely limits industrial applications. Herein, we report a novel creatinase from Alcaligenes faecalis (afCR) with higher catalytic activity and lower KM value, than currently used creatinases. Furthermore, we developed a non-biased phylogenetic consensus method to improve the thermostability of afCR.
Results
We applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening afCR mutants with improved thermostability. Twenty-one amino acids of afCR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme (afCR-M0). Combination of single-site mutations in sequential screens resulted in a quadruple mutant D17V/T199S/L6P/T251C (M4-2) which showed ~ 1700-fold enhanced half-life at 57 °C and a 4.2 °C higher T5015 than that of afCR-M0. The mutant retained catalytic activity equivalent to afCR-M0, and thus showed strong promise for application in creatinine detection. Structural homology modeling revealed a wide range of potential molecular interactions associated with individual mutations that contributed to improving afCR thermostability.
Conclusions
Results of this study clearly demonstrated that the non-biased-phylogenetic consensus design for improvement of thermostability in afCR is effective and promising in improving the thermostability of more enzymes.
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DOI：
10.1073/pnas.0808220106
发表时间：
2009-02-24
影响因子：
11.1
作者：
Gribenko, Alexey V.;Patel, Mayank M.;Makhatadze, George I.
通讯作者：
Makhatadze, George I.
影响因子：
4.3
作者：
Bloom JD;Glassman MJ
通讯作者：
Glassman MJ
影响因子：
2.4
作者：
Heselpoth, Ryan D.;Yin, Yizhou;Nelson, Daniel C.
通讯作者：
Nelson, Daniel C.
通过一致性诱变提高短乳杆菌谷氨酸脱羧酶的热稳定性
影响因子：
3
作者：
Hua, Yujiao;Lyu, Changjiang;Mei, Lehe
通讯作者：
Mei, Lehe
影响因子：
--
作者：
Edwardraja, Selvakumar;Sriram, Sokalingam;Lee, Sun-Gu
通讯作者：
Lee, Sun-Gu