A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations

et al. 2020

Microb Cell Fact

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.

Section: Kinetic and Thermodynamic Stability Of The Improved Afcr Commentioning

confidence: 93%

“…The af CR gene (BAA88830.1) was synthesized at GenScript Crop (Nanjing, China) and cloned into a pANY1 expression vector (gift from College of Biosciences and Biotechnology, Shenyang Agricultural University) [ 50 ]. Restriction enzymes and T4 ligase were purchased from New England Biolabs (Ipswich, MA).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

Microb Cell Fact

“…The afCR gene (BAA88830.1) was synthesized at GenScript Crop (Nanjing, China) and cloned into a pANY1 expression vector (gift from College of Biosciences and Biotechnology, Shenyang Agricultural University) [31]. Restriction enzymes and T4 ligase were purchased from New England Biolabs (Ipswich, MA).…”

Section: Experimental Operations 411 Media and Reagentsmentioning

confidence: 99%

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

et al. 2020

Preprint

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. 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 ºC and a 4.2 ºC 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.

“…The afCR gene (BAA88830.1) was synthesized at GenScript Crop (Nanjing, China) and cloned into a pANY1 expression vector (gift from College of Biosciences and Biotechnology, Shenyang Agricultural University) [50]. Restriction enzymes and T4 ligase were purchased from New England Biolabs (Ipswich, MA).…”

Section: Media and Reagentsmentioning

confidence: 99%

“…T50 15 values were determined by incubating the puri ed enzyme over a range of temperatures from 35 ºC to 70 ºC for 15 min. After incubation, the sample solution was then cooled immediately in an ice bath and the remaining afCR activity was measured as described above.…”

mentioning

confidence: 99%

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

et al. 2020

Preprint

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 ºC and a 4.2 ºC 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.