几丁质酶基因的克隆表达及酶学性质  

Cloning,expression and characterization of the chitinase gene from Vibrio sp. GR52

作　　者：郑家敏 梁燕辉[1] 朱凡[1] 叶秀云[1] 林娟[1] ZHENG Jia-Min, LIANG Yan-Hui, ZHU Fan, YE Xiu-Yun, LIN Juan(Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fuzhou, Fujian 350116, Chin)

机构地区：[1]福建省海洋酶工程重点实验室福州大学,福建福州350116

出　　处：《微生物学通报》2018年第5期1027-1034,共8页Microbiology China

基　　金：福建省海洋经济创新发展区域示范项目(2014FJPT02);福建省中青年教师教育科研项目(JAT160054)~~

摘　　要：【背景】几丁质是自然界中储藏量仅次于纤维素的有机物,几丁质酶能降解几丁质生成几丁寡糖,实现废弃物的高值化利用,目前菌株产几丁质酶能力低限制了它的生产应用。【目的】克隆弧菌(Vibrio sp.)GR52的几丁质酶基因,实现其在大肠杆菌中的异源表达,对分离纯化的重组几丁质酶进行酶学性质研究。【方法】以弧菌GR52菌株基因组DNA为模板,克隆得到几丁质酶基因GR52-1,构建重组基因工程菌BL21(DE3)/p ET22b-chi GR52-1,诱导表达的产物通过Ni-NTA树脂纯化后进行酶学性质研究。【结果】重组酶的最适反应pH为6.0,在pH5.0-10.0范围内37°C保温1 h仍能保持85%以上的相对酶活力,具有较好的pH稳定性;最适反应温度为50°C,在45°C保温1 h其酶活力基本没有损失,在50°C保温1 h其残余酶活力仍达60%;在1 mmol/L浓度下,Cu^(2+)、Ca2+对该酶具有促进作用,Hg+对该酶具有明显的抑制作用;在5 mmol/L浓度下,Ni+对该酶具有一定的促进作用,Mn^(2+)、Co^(2+)、Li^+、Fe^(2+)、Hg^+、SDS(十二烷基硫酸钠)对该酶具有明显的抑制作用。以胶体几丁质为底物时,动力学参数Km、Vmax、kcat分别为0.85 mg/m L、0.19μmol/(m L·min)和7.02 s-1。底物特异性分析表明该重组酶能特异性降解几丁质。【结论】重组几丁质酶具有良好的酶学性质,为几丁质酶的开发应用奠定基础。[Background] Chitin is the second most abundant natural resource next to cellulose. Chitinase can catalyze chitin into chitosan oligosaccharide which achieved high value utilization of waste. [Objective] The chitinase gene of Vibrio sp. GR52 was cloned and expressed in Escherichia coli, and the properties of recombinase were characterized. [Methods] The chitinase gene chi GR52-1 was cloned from the genomic DNA of Vibrio sp. GR52 and the recombinant strain BL21（DE3）/p ET22 b-chi GR52-1 was constructed. The recombinase rchi GR52-1 was purified by Ni-NTA affinity chromatography and the enzymatic properties were studied. [Results] The optimum catalytic pH of rchi GR52-1 was 6.0. It was stabled in the pH range of 5.0 to 10.0 and could maintain more than 85% of its relative activity after incubation at 37 ℃ for 1 hour. The optimum catalytic temperature of the enzyme was 50 ℃ and 60% of enzyme activity was remained after incubation at 50 ℃ for 1 hour. At the concentration of 1 mmol/L, Cu^2＋ and Ca2＋ had stimulation on rchi GR52-1, while Hg＋ had significant inhibition. At the concentration of 5 mmol/L, Ni＋ stimulated the chitinase, while Mn^2＋, Co^2＋, Li^＋, Fe^2＋, Hg^＋ and SDS inhibited the enzyme. The kinetic parameters Km, Vmax and kcat of rchi GR52-1 were 0.85 mg/m L, 0.19 μmol/（m L·min） and 7.02 s^-1, respectively. Substrate specificity analysis showed that rchi GR52-1 could catalyze chitin specifically. [Conclusion] Recombinant chitinase has good enzymatic properties which provide a theoretical foundation for chitinase applications.

关 键 词：几丁质酶 弧菌 克隆表达 酶学性质 

分 类 号：Q55[生物学—生物化学] Q78