Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutage
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Microbial Cell Factories Open Access
RESEARCH
Improved thermostability of creatinase from Alcaligenes Faecalis through non‑biased phylogenetic consensus‑guided mutagenesis Xue Bai1†, Daixi Li1*†, Fuqiang Ma2†, Xi Deng3, Manjie Luo4, Yan Feng3 and Guangyu Yang3*
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. Keywords: Creatinase, Thermostability, Consensus approach, Multiple sequence alignment, Phylogenetic analysis Background Creatinine is the final product of phosphocreatine metabolism in humans [1], and has been established as a reliable clinical marker for the determination of renal and muscular dysfunction. One of the most commonly used methods for the detection of creatinine is based *Correspondence: [email protected]; [email protected] † Xue Bai, Daixi Li and Fuqiang Ma joint first authors 1 Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology, Shanghai 200093, People’s Republic of China 3 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai 200240, People’s Republic of China Full list of author information is available at the end of the article
on an enzymatic cascade [2] that includes creatininase (E.C.3.5.2.10), creatinase (E.C.3.5.3.3), and sarcosine oxidase (E.C.1.5.3.1) (Fig. 1). In this way, creatinine is eventually converted into H2O2, and thus the concentration of creatinine can be determined by conv
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