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Abstract：
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.
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.
摘要：
背景：肌酐的酶定量已成为临床评估肾功能的重要方法。虽然肌酸酶（CR）经常用于此目的，其差的热稳定性严重限制了工业应用。在这里，我们报道了一种来自粪产碱杆菌（afCR）的新型肌酸酶，具有较高的催化活性和较低的KM值，比目前使用的肌酸酶。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。
结果：我们应用了一种无偏倚的系统发育共有方法，从24个肌酸酶家族同源物中鉴定了59个候选共有残基，用于筛选具有改善的热稳定性的afCR突变体。选择afCR的21个氨基酸进行诱变，与亲本酶（afCR-M0）相比，其中11个显示出改善的热稳定性。连续筛选中的单点突变组合产生了四重突变体D17V/T199S/L6P/T251C（M4-2），其在57°C下的半衰期增强了约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，因此显示出在肌酐检测中应用的强大前景。结构同源性建模揭示了与单个突变相关的广泛的潜在分子相互作用，这些突变有助于改善afCR热稳定性。
结论：这项研究的结果清楚地表明，用于改善afCR中热稳定性的非偏向系统发育共识设计在改善更多酶的热稳定性方面是有效且有希望的。
结果：我们应用了一种无偏倚的系统发育共有方法，从24个肌酸酶家族同源物中鉴定了59个候选共有残基，用于筛选具有改善的热稳定性的afCR突变体。选择afCR的21个氨基酸进行诱变，与亲本酶（afCR-M0）相比，其中11个显示出改善的热稳定性。连续筛选中的单点突变组合产生了四重突变体D17V/T199S/L6P/T251C（M4-2），其在57°C下的半衰期增强了约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，因此显示出在肌酐检测中应用的强大前景。结构同源性建模揭示了与单个突变相关的广泛的潜在分子相互作用，这些突变有助于改善afCR热稳定性。
结论：这项研究的结果清楚地表明，用于改善afCR中热稳定性的非偏向系统发育共识设计在改善更多酶的热稳定性方面是有效且有希望的。
