Patel Dharti Keyur, Dave Gayatri
PD Patel Institute of Applied Sciences, CHARUSAT, Anand, Changa, 388421 Gujarat India.
3 Biotech. 2022 Oct;12(10):245. doi: 10.1007/s13205-022-03315-y. Epub 2022 Aug 25.
The biotechnological application of bacterial xylanases requires a high thermostability, a catalytically active state for a broad pH range. The (MTCC 1270) A gene was amplified and cloned into the pCold vector and was expressed in to evaluate the expressed proteins' thermostability The pCold, compared to other similar vectors, has unique properties-including pH and temperature tolerance due to the presence of the cspA promoter. The recombinant A-pCold (rxynApC) showed the expression of A gene with a molecular weight of ~ 27 kDa, confirmed on SDS-PAGE. The rxynApC exhibits optimal activity at 70 °C and pH 8.0. The residual activity of the recombinant enzyme was 90% at pH 8.0. The thermal decomposition temperature ( ) value for the rxynApC enzyme was 93.33 °C obtained from the thermogravimetric analysis, indicating the potent stability of the cloned enzyme. The specific activity of native xylanase and rxynApC under optimal conditions was 32.35 and 105.5 U/mg, respectively. The structural model of the A gene was predicted using the in silico tool along with the active site (containing four important Tyr-166, Gly-7, Try-69 and Arg-112 amino acids). The predicted biophysical parameters of the in silico model were similar to the experimental results. The unique feature of the A promoter is that it gave a high expression of rxynApC enzyme having alkali and thermostable properties with high yield in surrogate host Thus, the recombinant A gene can potentially be applied to different industrial needs by looking at its thermostability and enhanced enzyme activity.
The online version contains supplementary material available at 10.1007/s13205-022-03315-y.
细菌木聚糖酶的生物技术应用需要高耐热性，即在较宽的pH范围内具有催化活性状态。（MTCC 1270）A基因被扩增并克隆到pCold载体中，并在[具体宿主菌名称缺失]中表达，以评估表达蛋白的耐热性。与其他类似载体相比，pCold具有独特的特性，由于存在cspA启动子，包括耐pH和温度。重组A-pCold（rxynApC）在SDS-PAGE上证实显示分子量约为27 kDa的A基因表达。rxynApC在70°C和pH 8.0时表现出最佳活性。重组酶在pH 8.0时的残余活性为90%。通过热重分析获得的rxynApC酶的热分解温度（[具体温度单位缺失]）值为93.33°C，表明克隆酶具有强大的稳定性。天然木聚糖酶和rxynApC在最佳条件下的比活性分别为32.35和105.5 U/mg。使用计算机工具预测了A基因的结构模型以及活性位点（包含四个重要的酪氨酸-166、甘氨酸-7、色氨酸-69和精氨酸-112氨基酸）。计算机模型预测的生物物理参数与实验结果相似。A启动子的独特之处在于它在替代宿主[具体宿主菌名称缺失]中高产表达具有碱性和耐热性的rxynApC酶。因此，考虑到其耐热性和增强的酶活性，重组A基因有可能应用于不同的工业需求。
在线版本包含可在10.1007/s13205-022-03315-y获取的补充材料。
