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Hong, Min; Chang, Jinq Chyi; Wu, Mei; Chang, Ming

doi:10.1023/a:1018705831622

Cited by 11 publications

(3 citation statements)

References 10 publications

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“…Creatinase is used as a key enzyme for enzymatic measurement of creatinine which catalyzes the hydrolysis of creatine to sarcosine and urea. It has been found in various kinds of bacteria such as Flavobacterium , Pseudomonas , Arthrobacter and Bacillus [ 6 , 28 , 30 – 32 ]. In this work, we analyzed the creatinase family sequences base on phylogenetic tree (Additional file 1 : Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Bai

et al. 2020

Microb Cell Fact

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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.

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Section: Resultsmentioning

confidence: 99%

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

Bai

et al. 2020

Microb Cell Fact

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“…aeruginosa ’s soxBDAG , 1 h post-creatine exposure in P. putida KT2440 (, ) [5–9, 45–47]. Genes involved in the subsequent steps of sarcosine metabolism, including glyA1 encoding the serine hydroxymethyltransferase and tdcG-I encoding the l -serine dehydratase, are amongst the next most highly transcribed genes in the presence of creatine (, ), providing a more complete picture of creatine metabolism in P.…”

Section: Discussionmentioning

confidence: 99%

“…putida to grow on creatine as the sole nitrogen source. CahR binds with specificity to the promoter region of creA (), from which we conclude direct transcriptional induction of creA , which encodes a creatinase with the ability to efficiently cleave creatine into sarcosine and urea [5–10, 45–47]. Transcription of creA occurs quickly in WT P.…”

Section: Discussionmentioning

confidence: 99%

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR

et al. 2022

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Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putida ’s detection of the animal-derived amine compound creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putida’s ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small, and an electrophoretic mobility shift assay demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologues associated with cahR orthologues highlights a strain distribution and organization supporting probable horizontal gene transfer, particularly evident within the genus Acinetobacter . This study identifies and characterizes the GATR that transcriptionally controls P. putida ’s metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.

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Characterization of a Chitinase (Chit62) from Serratia marcescens B4A and Its Efficacy as a Bioshield Against Plant Fungal Pathogens

et al. 2012

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Chitinases have been suggested to be involved in pathogen-antagonist interaction during biological control progress of plant pathogenic fungi. Here, a recombinant bacterial chitinase originally from Serratia marcescens B4A was produced, purified, and assayed biochemically to ascertain the activity and determine the kinetics parameters. Active enzyme was used to determine its biocontrol features against fungal phytopathogens. The results demonstrated that the optimum pH and temperature for the enzyme activity were 6.0 and 55 °C, respectively. The K(m) and V(max) values were 3.30 mg ml(-1) and 0.92 units, respectively. The recombinant chitinase was demonstrated to be highly active in controlling fungal pathogens.

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Cited by 11 publications

References 10 publications

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

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

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR

Characterization of a Chitinase (Chit62) from Serratia marcescens B4A and Its Efficacy as a Bioshield Against Plant Fungal Pathogens

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