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Gene cloning, expression, molecular modeling and docking study of the protease SAPRH from Bacillus safensis strain
Hatem Rekik1, Fakher Frikha2, Nadia Zaraî Jaouadi3
1Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour, Km 6, P.O. Box 1177, Sfax 3018, Tunisia; Biotech ECOZYM Start-up, Business Incubator, Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia; STE JMAL (EJM)-Laundry Detergent Industry, Z.I. Avenue August 13, Z.I. Poudriere 1, P.O. Box 407, Boustene, Sfax 3000, Tunisia.
Abstract:
The sapRH gene, which encodes the serine alkaline protease SAPRH, from Bacillus safensis RH12, was isolated and its DNA sequence was determined. The deduced amino-acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with SAPB from B. pumilus CBS, with only 9 amino-acids of difference. The region, encoding SAPRH was heterologously expressed in E. coli BL21-AI™ cells using GATEWAY™ pDEST™17 expression-vector. The recombinant (His)6-tag enzyme (His6-rSAPRH) was purified in a single affinity chromatography step and its biochemical properties were determined and compared to those of SAPRH and rSAPB. Interestingly, His6-rSAPRH showed improved thermostability compared to SAPRH and rSAPB. The molecular dynamics of SAPRH compared to SAPB revealed a more thermostable structure, thus confirming the in vitro results showing that His6-rSAPRH has a t1/2 of 120 min against 90 and 30 min for SAPRH and rSAPB, respectively, at 70 °C and different kinetic parameters to synthetic peptides. The docking simulations data allow in getting an insight into the involvement of some key amino-acids in substrate binding and account for the selectivity. Overall, this is the first report of a sapRH gene cloned from B. safensis which can be a promising potential candidate for future applications in detergent formulations.
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