几丁质酶基因chi6380的克隆、表达及酶学性质分析

Cloning, Expression, and Enzymatic Characterization of the Chitinase Gene chi6380

摘要:

目的 探究几丁质酶在生物降解领域的应用潜力，挖掘新型高效的几丁质酶资源。方法 利用原核表达系统对副地衣芽孢杆菌（Bacillus paralicheniformis）来源的几丁质酶基因chi6380进行克隆与异源表达；经镍柱亲和层析纯化后，对Chi6380的酶学性质、几丁质降解产物及抗真菌活性进行系统研究。结果 作者成功在大肠杆菌BL21（Escherichia coli BL21）中实现了重组几丁质酶Chi6380的异源表达。酶学性质研究结果表明，Chi6380的最适反应温度为50 ℃；最适反应pH为5.0；该酶在30~50 ℃、pH 4.0~9.0条件下具有良好的稳定性；Mg²⁺、Ca²⁺等金属离子及二硫苏糖醇（DTT）等化学试剂均对Chi6380酶活力具有抑制作用。底物特异性分析显示，Chi6380可水解多种类型的几丁质，其中胶体几丁质为最适底物，其水解产物主要为几丁二糖、几丁四糖和几丁六糖。抑菌试验结果表明，Chi6380对烟草疫霉（Phytophthora nicotianae）和新月弯孢霉（Curvularia lunata）均具有抑菌效果，抑菌率分别为（46.40±0.21）%和（30.53±0.38）%。结论 该研究获得的几丁质酶Chi6380具有良好的几丁质降解能力与植物病原菌抑制活性，在几丁质资源生物转化及植物病原菌生物防治方面具有广阔的应用前景。

Abstract:

Objective This study explored the application potential of chitinase in biodegradation, aiming to mine new efficient chitinase resources.Method This study cloned and heterologously expressed the chitinase gene chi6380 from Bacillus paralicheniformis via a prokaryotic expression system. After purification by Ni-NTA affinity chromatography, the enzymatic properties, chitin degradation products, and antifungal activity of Chi6380 were systematically investigated.Result The recombinant chitinase Chi6380 was successfully expressed in Escherichia coli BL21. Enzymatic characterization revealed that Chi6380 exhibited the optimal activity at 50 ℃ and pH 5.0, and maintained high stability within the ranges of 30~50 ℃ and pH 4.0~9.0. Common metal ions, such as Mg²⁺ and Ca²⁺, along with chemical reagents including dithiothreitol (DTT), demonstrated inhibitory effects on the activity of Chi6380. Substrate specificity analysis revealed that Chi6380 exhibited hydrolytic activity against various chitin substrates, with colloidal chitin as the optimal substrate, and the main hydrolysis products were chitobiose, chitotetraose, and chitohexaose. Antimicrobial assays indicated that Chi6380 exhibited inhibitory effects against Phytophthora nicotianae and Curvularia lunata, with inhibition rates of (46.40±0.21)% and (30.53±0.38)%, respectively.Conclusion The chitinase Chi6380 obtained in this study exhibits good capacity of degrading chitin and activity of inhibiting plant pathogenic fungi, demonstrating promising potential for applications in the bioconversion of chitin waste and the biological control of plant pathogenic fungi.