Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis

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 K_M 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.

Consensus approach; Creatinase; Multiple sequence alignment; Phylogenetic analysis; Thermostability.