贵州木霉NJAU4742几丁质酶基因chi 8的克隆、表达和酶学特性研究

Cloning,expression and enzymology characteristics of an endo-chitinase gene chi 8 from Trichoderma guizhouense NJAU4742

[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chi 8并深入研究其酶学特性,分析Chi8水解胶体几丁质的产物,为其在农业生产中的实际应用提供理论依据。[方法]基于序列比对获取chi 8与其他几丁质酶基因的同源关系,并设计引物扩增得到完整的chi 8序列,利用pET-29a质粒和大肠杆菌BL21进行异源表达;经镍柱纯化后并研究Chi8酶学特性;利用MALDI-TOF/MS检测Chi8水解胶体几丁质的最终产物;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。[结果]本试验克隆、表达得到的Chi8与哈茨木霉几丁质酶(ACM47359)的序列相似度为93.38%;Chi8在30~40℃酶活性较高且能保持较好的热稳定性,在pH6.0时酶活性为0.50 U·μmol^-1·h^-1;Ca^2+可以显著提高Chi8的酶活性,可达到原酶活性的110.62%,而Co^2+、Cu^2+、Fe^3+均有抑制作用,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,Chi8水解胶体几丁质的最终产物主要为几丁二糖[(GlcNAc)2]。Chi8同源建模结果表明,其含有18个螺旋和15个β-折叠,末端的IMD残基可形成1个咪唑环;底物对接结果表明,Chi8中Asn197、Trp110、Tyr218、Arg274、Arg31可以与几丁六糖形成氢键,可能为该蛋白的活性中心。[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,能够高效水解胶体几丁质并生成壳寡糖,产物主要为几丁二糖。

[Objectives]The purpose of this study was to clone chi 8 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 chi 8 gene with other chitinase genes was obtained by sequence alignment,and the open reading flank of chi 8 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℃and 40℃,meanwhile the enzyme activity was the highest when the pH value reached 6.0.Ca^2+could increase the activity of Chi8 significantly,and the activity reached to 110.62%of that in CK,while the co-reaction with Co^2+,Cu^2+and Fe^3+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)2.The protein homologous structure model of Chi8 indicated that it contained 18 helices and 15β-barrel,with an IMD residue as the imidazole ring at the end.The substrate docking results showed that Asn197,Trp110,Tyr218,Arg274 and Arg31 in Chi8 could form hydrogen bond with chitohexaose hydrochloride,which might be 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)2 and chitin monosaccharide.