Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
J Microbiol Biotechnol. 2013 Nov 28;23(11):1519-28. doi: 10.4014/jmb.1301.01048.
Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.
短小芽孢杆菌 SG2 是一种耐盐菌株，可表达两种主要的几丁质酶 ChiS 和 ChiL，这两种酶受几丁质诱导并分泌到上清液中。本研究旨在通过紫外线照射和亚硝酸处理组合对短小芽孢杆菌 SG2 进行诱变，获得具有更高几丁质酶活性的突变体。经过几丁质琼脂上的诱变和筛选以及随后的 halo 形成，在不同条件下检查突变株对几丁质的降解情况。由于其更高的几丁质酶活性，选择了一株突变株命名为 AV2-9。为了寻找 ChiS 和 ChiL 整个操纵子中可能的突变，对整个几丁质酶操纵子，包括基因间区、启动子和 ChiS 和 ChiL ORF 对应的两个区域，进行了测序。对 SG2 和 AV2-9 菌株的完整几丁质酶操纵子的核苷酸序列分析表明，几丁质酶（ChiL）的催化结构域（GH18）存在突变。结果表明，AV2-9 中 ChiL 序列发生了单个碱基变化。野生型几丁质酶 ChiL 和突变体（命名为 ChiLm）在大肠杆菌中克隆、表达和纯化。两种酶在不同的 pH、NaCl 浓度和温度范围下显示出相似的活性谱，但在所有测试条件下，突变酶的催化活性约提高了 30%。本研究结果表明，突变菌株中几丁质酶的热稳定性增加。进行了生物信息学分析以预测突变引起的蛋白质稳定性变化。
