[Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design].

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design / 生物工程学报

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

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 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.

Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase.

BACKGROUND: Pseudomonas putida (P. putida) ATCC12633 can produce creatinase. It is a microbial enzyme which degrades creatinine in bacteria and provides source of carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of creatinine concentration for diagnosis of renal and muscles functions and diseases. Our purpose was recombinant production of creatinase for using in clinical measurement of serum or urine creatinine. METHODS: A 1209bp of open reading frame of creatinase was amplified by PCR from P. putida ATCC12633 genome and cloned into pET28a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET28a vector was transformed to Escherichia coli (E. coli) BL21 (DE3). Creatinase expression was induced in E.coli BL21 (DE3) using IPTG and confirmed by SDS-PAGE and western blotting. Purification of creatinase was performed using Ni-NTA column. The specific activity of this enzyme was also investigated. RESULTS: The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in E. coli (57.4% of total protein). SDS-PAGE and western blot analysis showed a 45 kDa creatinase protein. Purification of creatinase was done with high purity. The specific activity of recombinant enzyme is 26.54 unit/mg that is much higher than other creatinase used in the commercial kits (9 unit/mg). CONCLUSION: The P. putida ATCC12633 recombinant creatinase was expressed efficiently in E. coli BL21 and 57% of total protein was the recombinant creatinase. Also, expressed creatinase has high solubility and also the enzyme has good activity compared to enzymes used in commercial kits, so a new source of creatinase was produced for creatinine assay kit in this study.

Consecutive hydrolysis of creatinine using creatininase and creatinase encapsulated in Saccharomyces cerevisiae spores.

OBJECTIVES: To achieve consecutive conversion from creatinine to urea and sarcosine using creatininase and creatinase encapsulated in spores of Saccharomyces cerevisiae. RESULTS: Creatininase encapsulated into the spore wall was produced and its specific activity was 3.4 ± 0.4 U/mg. By deletion of OSW2 gene, which causes a mild spore wall defect, the activity was increased to 10.9 ± 0.5 U/mg. Compared with soluble enzymes, spore-encapsulated creatininase was tolerant to environmental stresses; creatininase encapsulated in osw2∆ spores retained more than 90 % of the activity after treatment by SDS or proteinase K. Creatinase capsules could also be produced through spore encapsulation. The mixture of spores containing either creatininase or creatinase could mediate a two-step reaction to produce urea from creatinine; 5 mg spores produced 19 µmol urea in 10 min. Spores co-expressing creatininase and creatinase could also mediate the reactions more efficiently than the mixture of spores individually expressing each enzyme; the yield in 10 min was 38 µmol. CONCLUSIONS: Yeast spores can hold creatininase and creatinase simultaneously and catalyze the consecutive reactions.

Production of encapsulated creatinase using yeast spores.

Yeast spores can be used as a carrier to produce enzyme capsules. In the present study, this technique was applied to a diagnostic enzyme named creatinase. We found that a secretory form of Pseudomonas putida creatinase could be entrapped in the spore wall, and such spores were used as creatinase capsules. The activity of the encapsulated creatinase was largely improved by mild spore wall defective mutations, such as DIT1 or OSW2 deletions. The advantages of this method include the following: encapsulated and freeze-dried creatinase is produced without preparing the purified enzyme, and it exhibits resistance to environmental stresses, such as high temperature and SDS treatments. Thus, yeast spores could be applied to establish quick and easy clinical diagnostic methods.

Production of novel NaN3-resistant creatine amidinohydrolase in recombinant Escherichia coli.

Creatinase (creatine amidinohydrolase), an important medical enzyme, has been used for clinical diagnosis of renal function because of its high substrate specificity. Recently, we successfully cloned a NaN3-resistant creatinase encoding gene from Arthrobacter nicotianae. By optimizing the cultivation process, we realized its high-level expression in Escherichia coli. In this addendum, production of this NaN3-resistant creatinase in E. coli and future research were further discussed.