Chitinases are enzymes that catalyze the degradation of chitin, a major component of the cell walls of pathogenic fungi and cuticles of insects, gaining increasing attention for the control of fungal pathogens and insect pests. Production of recombinant chitinase in a suitable host can result in a more pure product with less processing time and a significantly larger yield than that produced by native microorganisms. The present study aimed to express the synthetic chi42 gene (syncodChi42), which was optimized from the chi42 gene of Trichoderma asperellum SH16, in Escherichia coli to produce 42 kDa chitinase (Ta-CHI42); then determined the activity of this enzyme, characterizations and in vitro antifungal activity as well as its immunogenicity in mice. The results showed that Ta-CHI42 was overexpressed in E. coli. Analysis of the colloidal chitin hydrolytic activity of purified Ta-CHI42 on an agar plate revealed that this enzyme was in a highly active form. This is a neutral chitinase with pH stability in a range of 6–8 and has an optimum temperature of 45°C with thermal stability in a range of 25–35°C. The chitinolytic activity of Ta-CHI42 was almost completely abolished by 5 mM Zn²⁺ or 1% SDS, whereas it remained about haft under the effect of 1 M urea, 1% Triton X-100 or 5 mM Cu²⁺. Except for ions such as Mn²⁺ and Ca²⁺ at 5 mM that have enhanced chitinolytic activity; 5 mM of Na⁺, Fe²⁺ or Mg²⁺ ions or 1 mM EDTA negatively impacted the enzyme. Ta-CHI42 at 60 U/mL concentration strongly inhibited the growth of the pathogenic fungus Aspergillus niger. Analysis of western blot indicated that the polyclonal antibody against Ta-CHI42 was greatly produced in mice. It can be used to analyze the expression of the syncodChi42 gene in transgenic plants, through immunoblotting assays, for resistance to pathogenic fungi.

中文翻译：

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在大肠杆菌中从合成基因中表达曲霉木霉 (Ta-CHI42) 的 42 kDa 几丁质酶

几丁质酶是催化几丁质降解的酶，几丁质是病原真菌细胞壁和昆虫角质层的主要成分，在真菌病原体和害虫的防治中越来越受到关注。在合适的宿主中生产重组几丁质酶可以得到更纯的产品，与天然微生物生产的产品相比，加工时间更短，产量明显更高。本研究的目的是表达合成chi42基因（syncodChi42），这是从所述优化chi42的基因棘孢木霉SH16，在大肠杆菌中以产生42kDa的几丁质酶（TA-CHI42）; 然后确定该酶的活性、表征和体外抗真菌活性及其在小鼠中的免疫原性。结果表明Ta-CHI42在大肠杆菌中过表达。在琼脂板上对纯化的 Ta-CHI42 的胶体几丁质水解活性进行分析表明，该酶处于高活性形式。这是一种中性几丁质酶，其 pH 稳定性范围为 6–8，最适温度为 45°C，热稳定性范围为 25–35°C。Ta-CHI42 的几丁质分解活性几乎被 5 mM Zn ²⁺或 1% SDS完全消除，而在 1 M 尿素、1% Triton X-100 或 5 mM Cu ²⁺的作用下它仍然保持大约一半。5mM的Mn ²⁺和Ca ^{2+ 等}具有增强几丁质分解活性的离子除外；5 mM Na ⁺、Fe ²⁺或Mg ²⁺离子或1 mM EDTA 对酶产生负面影响。60 U/mL 浓度的 Ta-CHI42 强烈抑制病原真菌黑曲霉的生长。蛋白质印迹分析表明，抗Ta-CHI42的多克隆抗体在小鼠中大量产生。它可用于通过免疫印迹分析分析转基因植物中syncodChi42基因的表达，以了解对病原真菌的抗性。