Zhou Hai-Yan, Zhou Jian-Bao, Yi Xiao-Nan, Wang Yan-Mei, Xue Ya-Ping, Chen De-Shui, Cheng Xin-Ping, Li Mian, Wang Hong-Yan, Chen Kai-Qian, Liu Zhi-Qiang, Zheng Yu-Guo
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No. 18 Chaowang Road, Hangzhou, 310014, Zhejiang, People's Republic of China.
The National and Local, Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Bioprocess Biosyst Eng. 2021 Jan;44(1):67-79. doi: 10.1007/s00449-020-02420-7. Epub 2020 Aug 9.
To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55-75 °C and high thermostability (about 85% residual activity) at the temperature of 55 °C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0-5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0-6.0) at 4 °C for 24 h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and β-D-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters V, K, and K/K values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against β-D-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.
为开发用于高效利用木质纤维素的新型纤维素酶，合成了来自嗜兰炭疽菌的一种内切葡聚糖酶（CoCel5A）基因，并构建了重组毕赤酵母GS115/pPIC9K/cocel5A用于CoCel5A的分泌表达。纯化后，对蛋白CoCel5A进行了生化特性分析。内切葡聚糖酶CoCel5A在55 - 75℃表现出最佳活性，在55℃孵育3小时后具有高热稳定性（约85%的残留活性）。当使用100 mM柠檬酸缓冲液（pH 4.0 - 5.0）时检测到CoCel5A的最高活性，并且在4℃于100 mM柠檬酸缓冲液（pH 3.0 - 6.0）中孵育24小时后观察到优异的pH稳定性（高达95%的残留活性）。在测试的底物中，羧甲基纤维素钠盐（n≈500）（CMC）和β - D - 葡聚糖是CoCel5A的最佳底物。针对CMC的动力学参数V、K和K/K值分别为290.70 U/mg、2.65 mg/mL和75.67 mL/mg/s；针对β - D - 葡聚糖的分别为228.31 U/mg、2.06 mg/mL和76.45 mL/mg/s，表明CoCel5A具有高亲和力和催化效率。这些特性支持了CoCel5A在生物技术和环境领域的潜在应用。
