Key Laboratory of Animal Epidemic Etiology and Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou 310029, People's Republic of China.
Appl Biochem Biotechnol. 2011 Sep;165(2):728-36. doi: 10.1007/s12010-011-9291-2. Epub 2011 May 31.
Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and β-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.
高效降解纤维素需要纤维素酶的协同反应，其中包括外切葡聚糖酶、内切葡聚糖酶和β-1,4-葡萄糖苷酶。在本研究中，我们使用了一种改良的 Bac-to-Bac/BmNPV 杆状病毒表达系统，该系统缺乏家蚕核型多角体病毒（BmNPV）编码的几丁质酶组织蛋白酶（v-cath）基因，在家蚕 BmN 细胞和幼虫中表达绿色木霉内切葡聚糖酶 V（EG V）基因，并分析了重组酶在家蚕幼虫中的特性。结果表明，在第二代重组 mBacmid/BmNPV/EG V 杆状病毒感染后 48 小时，BmN 细胞中可见约 36 kDa 的蛋白。从重组杆状病毒感染的家蚕粗酶提取物在环境条件 pH 5.0 和温度 50°C 时表现出显著的最大活性，与空白 mBacmid/BmNPV 杆状病毒感染的家蚕和正常家蚕相比，分别提高了 39.86%和 37.76%。它在 pH 5.0 到 10.0 范围内以及在 40 到 60°C 的温度范围内稳定。家蚕提供的大量 EG V 的可用性可能极大地促进未来的研究和潜在的工业应用。
