贵州木霉 NJAU4742 几丁质酶基因 chi8 的克隆、 表达和酶学特性研究.

[Objectives] The purpose of this study was to clone chi8 gene from a beneficial strain Trichoderma guizhouense NJAU4742, and then to obtain the pure Chi8 protein through the heterologous expression by Escherichia coli expression system and the enzymology characteristics of Chi8 by evaluating the enzyme activity under different conditions, and to analyze the hydrolysates by using colloidal chitin as substrate, which will provide theoretical basis for its practical application in agricultural wastes treatment. [Methods] The homology of chi8 gene with other chitinase genes was obtained by sequence alignment, and the open reading flank of chi8 was amplified based on specific primers and then heterologously expressed in E.coli BL21 by using the pET-29a vector. The enzymology characteristics of Chi8 was obtained by determining the activities at different conditions with DNS method after purification by nickel gel, and the MALDI-TOF /MS method was used to detect the final product of colloidal chitin hydrolyzed by Chi8. Construction of three-dimensional model of Chi8 and docking results with chitohexaose hydrochloride was based on homologous modeling. [Results] Chi8 encoded by Trichoderma guizhouense NJAU4742 owned a highest sequence similarity ( 93. 38%) with the chitinase from Trichoderma harzianum ( ACM47359). Chi8 owned high enzyme activities and maintained a considerable thermal stability between 30 °C and 40 °C, meanwhile the enzyme activity was the highest when the pH value reached 6. 0. Ca2+ could increase the activity of Chi8 significantly,and the activity reached to 110.62% of that in CK, while the co-reaction with Co2+, Cu2+ and Fe3+ showed inhibitory effects with only 89.70%,60.17% and 5.93% of the enzyme activity by compared to CK, respectively. Under the condition of weak acid, colloidal chitin was used as the substrate for hydrolysis, and the final product was mainly chitobiose ( GlcNAc),. The protein homologous structure model of Chi8 indicated that it contained 18 helices,md 15 /3-hm-rd, with,m IMD residue as the imidazole ring at the end. The substrate docking results showed that Asn 197, Trp 110, T)T218, Arg274 and Arg3 l in Chi8 could form hydrogen bond with chitohexaose hydrochloride, which might he the active center of this protein. [ Conclusions] Chi8 owned a considerable capacity of efficient decompose colloidal chitin to generate chitosan oligosaccharides ., and the major products were ( GlcNAc), and chitin monosaccharide. [ABSTRACT FROM AUTHOR]

[目的]本研究旨在从贵州木霉 (Trichoderma guizhouense NJAU4742) 菌株中克隆、表达几丁质酶基因 Chi8 并深入研究 其酶学特性ꎬ分析 Chi8 水解胶体几丁质的产物ꎬ为其在农业生产中的实际应用提供理论依据ꎮ [方法]基于序列比对获取 Chi8 与其他几丁质酶基因的同源关系ꎬ并设计引物扩增得到完整的 Chi8 序列ꎬ利用 pET-29a 质粒和大肠杆菌 BL21 进行异 源表达ꎻ经镍柱纯化后并研究 Chi8 酶学特性ꎻ利用 MALDI-TOF/MS 检测 Chi8 水解胶体几丁质的最终产物ꎻ基于同源建模 法构建 Chi8 的三维模型并与几丁六糖进行对接获取其催化相关位点信息ꎮ [结果]本试验克隆、表达得到的 Chi8与哈茨木 霉几丁质酶 i( ACM47359) 的序列相似度为 93.38%, Chi8 在 30-40 °C酶活性较高且能保持较好的热稳定性ꎬ在 pH6.0 时酶活 性为 0.50 U·µmol-1 • h-1 ; Ca2+ 可以显著提高 Chi8 的酶活性ꎬ可达到原酶活性的 110.62% ꎬ而 Co2+, Cu2+,Fe3+均有抑制作用ꎬ 与酶作用后 Chi8 酶活性下降至原酶活性的 89.70%,60.17% 和 5.93%, 在酸性条件下ꎬChi8 水解胶体几丁质的最终产物主要 为几丁二糖[(GlcNAc) 2]ꎮ Chi8 同源建模结果表明ꎬ其含有 18 个螺旋和 15个 β－折叠ꎬ末端的 IMD 残基可形成 1 个咪唑 环ꎻ底物对接结果表明ꎬChi8 中 Asnl97, Trpl 10, Tyr218, Arg274,Arg31 可以与几丁六糖形成氢键ꎬ可能为该蛋白的活性中 心ꎮ [结论]本试验克隆、表达得到的几丁质酶 Chi8 具有良好的生化特性ꎬ能够高效水解胶体几丁质并生成壳寡糖ꎬ产物主 要为几丁二糖ꎮ [ABSTRACT FROM AUTHOR]