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

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.

Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability

“…Compared with that of the wild-type Tl PGA (58.5 °C), the T m value of the T316C/G344C mutant increased by 10 °C (68.5 °C). Kinetic and thermodynamic stabilities should be distinguished when determining enzyme stability . The thermodynamic stability of the T316C/G344C mutant was significantly higher than that of the wild-type Tl PGA, which further confirmed that introduction of both cysteines significantly contributed to the thermostability of the T316C/G344C mutant.…”

Creatinase: Using Increased Entropy to Improve the Activity and Thermostability

“…A previous study has shown that the creatinase from Alcaligenes is suitable for diagnostic use because of its superior substrate-binding affinity . The present work compared the wild type of creatinase from Alcaligenes with a four-point mutant (L6P, D17V, F108Y, and T199S) derived from nonbiased phylogenetic consensus-guided mutagenesis . As demonstrated in ref , this four-point mutant has a higher melting temperature (5 °C increment), whose lifetime is more than 1000 times longer than the wild type.…”

“…The present work compared the wild type of creatinase from Alcaligenes with a four-point mutant (L6P, D17V, F108Y, and T199S) derived from nonbiased phylogenetic consensus-guided mutagenesis . As demonstrated in ref , this four-point mutant has a higher melting temperature (5 °C increment), whose lifetime is more than 1000 times longer than the wild type. Furthermore, by performing the catalytic experiments, we found that the mutant has a broader catalytic temperature zone with a lower optimum working temperature and shows higher activity at ambient conditions (25–37 °C).…”

“…Scheme for the principle of the creatinine detection kit and biosensor . Creatinine is converted to creatine by creatininase, then creatine is hydrolyzed to sarcosine by creatinase, and sarcosine is oxidized to detectable hydrogen peroxide by sarcosine oxidase.…”

“…Scheme for the principle of the creatinine detection kit and biosensor. 24 Creatinine is converted to creatine by creatininase, then creatine is hydrolyzed to sarcosine by creatinase, and sarcosine is oxidized to detectable hydrogen peroxide by sarcosine oxidase. Under the catalysis of horseradish peroxidase, hydrogen peroxide produces a purple signal with 4-AA (4-aminoantipyrine) and TOOS (N-ethyl-N-(2-hydroxy-3sulfopropyl)-3-methylaniline).…”