Wang Chuan, Yang Yuze, Ma Chunjuan, Sunkang Yongjie, Tang Shaoqing, Zhang Zhao, Wan Xuerui, Wei Yaqin
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China.
Center for Anaerobic Microbes, Institute of Biology, Gansu Academy of Sciences, Lanzhou 730000, China.
Microorganisms. 2023 May 25;11(6):1387. doi: 10.3390/microorganisms11061387.
β-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency β-glucosidase, in this study two subunits ( and ) of β-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria ( NZ9000). The engineered strains NZ9000/pMG36e-usp45-, NZ9000/pMG36e-usp45-, NZ9000/pMG36e-usp45--usp45- were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher ( < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (max, m, cat, and cat/m) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K and Fe concentrations, the Bgl enzyme activity was significantly higher ( < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn, Hg, and Tween20 concentrations, the Bgl enzyme activity was significantly lower ( < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of β-glucosidase.
源自微生物的β-葡萄糖苷酶具有广泛的工业应用。为了构建具有高效β-葡萄糖苷酶的基因工程菌，本研究将从牦牛瘤胃中获得的β-葡萄糖苷酶的两个亚基（和）在乳酸菌（NZ9000）中分别表达为独立蛋白和融合蛋白。成功构建了工程菌株NZ9000/pMG36e-usp45-、NZ9000/pMG36e-usp45-、NZ9000/pMG36e-usp45--usp45-。这些细菌分别显示出BglA、BglB和Bgl的分泌表达。BglA、BglB和Bgl的分子量分别约为55 kDa、55 kDa和75 kDa。对于再生无定形纤维素（RAC）、羧甲基纤维素钠（CMC-Na）、脱脂棉、微晶纤维素、滤纸和1%水杨苷等底物，Bgl的酶活性显著高于BglA和BglB（<0.05）。此外，1%水杨苷似乎是这三种重组蛋白最合适的底物。这三种重组酶的最佳反应温度和pH值分别为50℃和7.0。在随后以1%水杨苷为底物的研究中，发现BglA、BglB和Bgl的酶活性分别为2.09 U/mL、2.36 U/mL和9.4 U/mL。分别在50℃和pH 7.0条件下，以1%水杨苷为底物分析了这三种重组菌株的酶动力学参数（max、m、cat和cat/m）。在K和Fe浓度增加的条件下，Bgl酶活性显著高于BglA和BglB酶活性（<0.05）。然而，在Zn、Hg和吐温20浓度增加的条件下，Bgl酶活性显著低于BglA和BglB酶活性（<0.05）。总体而言，本研究中构建的工程乳酸菌菌株能够高效水解纤维素，为β-葡萄糖苷酶的工业应用奠定了基础。
