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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 ( af CR) with higher catalytic activity and lower K M value, than currently used creatinases. Furthermore, we developed a non-biased phylogenetic consensus method to improve the thermostability of af CR. Results We applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening af CR mutants with improved thermostability. Twenty-one amino acids of af CR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme ( af CR-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 T 50 15 than that of af CR-M0. The mutant retained catalytic activity equivalent to af CR-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 af CR thermostability. Conclusions Results of this study clearly demonstrated that the non-biased-phylogenetic consensus design for improvement of thermostability in af CR is effective and promising in improving the thermostability of more enzymes.
Bai et al. (Sat,) studied this question.