Laboratory 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.
Laboratory 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.
Int J Biol Macromol. 2019 Mar 15;125:876-891. doi: 10.1016/j.ijbiomac.2018.12.103. Epub 2018 Dec 14.
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)-tag enzyme (His-rSAPRH) was purified in a single affinity chromatography step and its biochemical properties were determined and compared to those of SAPRH and rSAPB. Interestingly, His-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 His-rSAPRH has a t 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.
从巴氏芽孢杆菌 RH12 中分离出编码丝氨酸碱性蛋白酶 SAPRH 的 sapRH 基因，并测定了其 DNA 序列。推导的氨基酸序列与其他芽孢杆菌蛋白酶具有很强的同源性。与来自解淀粉芽孢杆菌 CBS 的 SAPB 的最高序列同一性值（97%）仅相差 9 个氨基酸。使用 GATEWAY™pDEST™17 表达载体在大肠杆菌 BL21-AI™细胞中异源表达编码 SAPRH 的区域。重组（His）-标签酶（His-rSAPRH）通过单一亲和层析步骤纯化，并测定其生化性质并与 SAPRH 和 rSAPB 进行比较。有趣的是，与 SAPRH 和 rSAPB 相比，His-rSAPRH 表现出更好的热稳定性。与 SAPB 相比，SAPRH 的分子动力学研究表明其结构更耐热，这证实了体外结果表明 His-rSAPRH 在 70°C 下对合成肽的 t1/2 分别为 120 min、90 min 和 30 min，以及不同的动力学参数。对接模拟数据可以深入了解一些关键氨基酸在底物结合中的参与情况，并解释其选择性。总的来说，这是首次从巴氏芽孢杆菌中克隆出 sapRH 基因，它可能是未来在洗涤剂配方中应用的有前途的潜在候选物。
