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Gene cloning, expression, molecular modeling and docking study of the protease SAPRH from Bacillus safensis strain RH12
- Rekik, Hatem, Frikha, Fakher, Zaraî Jaouadi, Nadia, Gargouri, Fares, Jmal, Najah, Bejar, Samir, Jaouadi, Bassem
- International journal of biological macromolecules 2019 v.125 pp. 876-891
- Bacillus pumilus, DNA, Escherichia coli, affinity chromatography, amino acid sequences, detergents, genes, molecular cloning, molecular dynamics, molecular models, nucleotide sequences, sequence analysis, serine proteinases, synthetic peptides, thermal stability
- 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.