Chitin, a long-chain polymer of N-acetylglucosamine, is a major structural component of the insect exoskeleton and the peritrophic membrane (PM). Chitinases are able to effectively break down glycosidic bonds of chitin polymer thus can be used in agriculture to control plant pathogen insects. These enzymes can be synthesized by higher plants, animals, protista, bacteria, and viruses. In this study, viral and bacterial chitinases were compared for their potential activity on a laboratory test insect. The genes encoding chitinases of Autographa californica nucleopolyhedrovirus (AcNPV) and Cydia pomonella granulovirus (CpGV) were amplified from genomic DNAs by PCR and cloned into the pET-28a (+) expression vector. The chitinase proteins of these 2 viruses (AcNPV-Chi, CpGV-Chi) and Serratia marcescens chitinase C (ChiC) protein which was previously cloned were overexpressed in Escherichia coli. Expressed proteins were purified and confirmed by western blot analysis as 50, 63, and 68 kDa for AcNPV, CpGV, and S. marcescens chitinases, respectively. Enzyme activities of the chitinases were confirmed. Chitinases were also compared to each other in silico. The insecticidal effects of these proteins were evaluated on Galleria mellonella L. larvae. Bioassays were performed on the 3rd instar larvae for each chitinase protein in triplicate. The results showed that although there were differences in enzymatic activities and domain organizations, all 3 microbial chitinases produced almost the same level of insecticidal activity on the test insect. LC50 and LT50 values were compatible with the mortality results. These results were a preanalysis for comparing the effects of microbial chitinases. Potential activity experiments should be carried out on more insects to provide detailed information on the insecticidal effects of bacterial and viral chitinases.

中文翻译：

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病毒和细菌几丁质酶潜在活性的比较

几丁质是 N-乙酰氨基葡萄糖的长链聚合物，是昆虫外骨骼和围营养膜 (PM) 的主要结构成分。几丁质酶能够有效地分解几丁质聚合物的糖苷键，因此可用于农业防治植物病原昆虫。这些酶可以由高等植物、动物、原生生物、细菌和病毒合成。在这项研究中，病毒和细菌几丁质酶对实验室测试昆虫的潜在活性进行了比较。通过 PCR 从基因组 DNA 中扩增了编码 Autographa californica 核多角体病毒 (AcNPV) 和 Cydia pomonella 颗粒病毒 (CpGV) 几丁质酶的基因，并将其克隆到 pET-28a (+) 表达载体中。这两种病毒的几丁质酶蛋白 (AcNPV-Chi, CpGV-Chi) 和粘质沙雷氏菌几丁质酶 C (ChiC) 蛋白（先前克隆）在大肠杆菌中过表达。表达的蛋白质被纯化并通过蛋白质印迹分析确认为 AcNPV、CpGV 和粘质链球菌几丁质酶的 50、63 和 68 kDa。几丁质酶的酶活性得到证实。几丁质酶也在计算机上进行了相互比较。这些蛋白质对大蜡螟幼虫的杀虫效果进行了评估。对每种几丁质酶蛋白的第 3 龄幼虫进行生物测定，一式三份。结果表明，虽然酶活性和结构域组织存在差异，但所有3种微生物几丁质酶对受试昆虫的杀虫活性水平几乎相同。LC50 和 LT50 值与死亡率结果一致。这些结果是用于比较微生物几丁质酶作用的预分析。应该对更多的昆虫进行潜在活性实验，以提供有关细菌和病毒几丁质酶杀虫作用的详细信息。