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. 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. 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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通过无偏系统进化共识引导的诱变提高粪便产碱杆菌肌酸酶的热稳定性

肌酐的酶法定量已成为临床评估肾功能的重要方法。尽管肌酐酶（CR）通常用于此目的，但其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菌（afCR）的新型肌酸酐酶，它比目前使用的肌酸酐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差的系统进化共识方法，以提高afCR的热稳定性。我们应用了一种无偏见的系统发育共有方法，从24个肌酐酶家族同源物中鉴定出59个候选共有残基，以筛选具有改进的热稳定性的afCR突变体。选择了afCR的21个氨基酸进行诱变，与亲本酶（afCR-M0）相比，其中11个具有更好的热稳定性。顺序筛选中单点突变的组合产生了四重突变体D17V / T199S / L6P / T251C（M4-2），在57°C时半衰期延长了约1700倍，T5015比T5015高出4.2°C。 afCR-M0。该突变体保留了与afCR-M0相当的催化活性，因此显示出在肌酐检测中的应用前景。结构同源性建模揭示了与单个突变相关的多种潜在分子相互作用，这些相互作用有助于改善afCR热稳定性。这项研究的结果清楚地表明，用于改善afCR的热稳定性的无偏差系统进化共识设计是有效的，并且有望改善更多酶的热稳定性。