Byeon Gyeong Min, Lee Kwang Sik, Gui Zhong Zheng, Kim Iksoo, Kang Pil Don, Lee Sang Mong, Sohn Hung Dae, Jin Byung Rae
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Comp Biochem Physiol B Biochem Mol Biol. 2005 Aug;141(4):418-27. doi: 10.1016/j.cbpc.2005.05.001.
A digestive beta-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori beta-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori beta-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori beta-glucosidase to be a single gene. Northern blot analysis of B. mori beta-glucosidase gene confirmed larval midgut-specific expression. The B. mori beta-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori beta-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant beta-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori beta-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant beta-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per microg of recombinant B. mori beta-glucosidase. The purified recombinant B. mori beta-glucosidase showed the highest activity at 35 degrees C and pH 6.0, and were stable at 50 degrees C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori beta-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.
从家蚕（Bombyx mori）中克隆出一种消化性β-葡萄糖苷酶cDNA。家蚕β-葡萄糖苷酶cDNA包含一个1473 bp的开放阅读框，编码491个氨基酸残基。家蚕β-葡萄糖苷酶拥有在糖基水解酶家族1中保守的参与催化和底物结合的氨基酸残基。基因组DNA的Southern印迹分析表明家蚕β-葡萄糖苷酶是一个单基因。家蚕β-葡萄糖苷酶基因的Northern印迹分析证实其在幼虫中肠特异性表达。家蚕β-葡萄糖苷酶mRNA在幼虫中肠的表达仅在取食期可检测到，而在饥饿期间其表达下调。家蚕β-葡萄糖苷酶cDNA在杆状病毒感染的昆虫Sf9细胞中表达为一种57 kDa的多肽，重组β-葡萄糖苷酶对纤维二糖和乳糖有活性，但对水杨苷无活性，表明家蚕β-葡萄糖苷酶具有2类酶的特性。在杆状病毒感染的昆虫细胞中表达的纯化重组β-葡萄糖苷酶的酶活性约为每微克重组家蚕β-葡萄糖苷酶665 U。纯化的重组家蚕β-葡萄糖苷酶在35℃和pH 6.0时活性最高，在50℃至少稳定10分钟。用N-糖基化的特异性抑制剂衣霉素处理重组病毒感染的Sf9细胞，结果显示重组家蚕β-葡萄糖苷酶是N-糖基化的，但碳水化合物部分对酶活性不是必需的。
