多粘类芽孢杆菌CP7β-1,3-1,4-葡聚糖酶基因的克隆、表达及应用

Cloning, Expression and Application of β-1,3-1,4-glucanase Gene from Paenibaccillus polymyxa CP7

【目的】克隆多粘类芽孢杆菌(Paenibaccillus polymyxa)CP7的β-1,3-1,4-葡聚糖酶基因,构建高效表达工程菌株,为CP7菌的抗菌活性组分研究和开发利用以及葡聚糖酶在农业生产中的应用提供理论依据。【方法】通过CP7菌β-1,3-1,4-葡聚糖酶基因的克隆及原核表达构建工程菌株,采用平板对峙培养和生长速率测定法研究重组酶对供试真菌的生长抑制活性,同时采用体外消化法研究该酶对麦类饲料消化率的影响。【结果】成功克隆目的基因并在原核系统获得高效表达。重组酶蛋白对4种供试真菌菌丝生长具有明显抑制作用,平均抑制率高达40%以上;添加了重组β-1,3-1,4-葡聚糖酶的处理组样品,经体外消化后黏度降低了4.69%(P﹥0.05)。【结论】β-1,3-1,4-葡聚糖酶是CP7菌发酵液中抗真菌活性组分或其中之一,对真菌生长抑制的活性作用提示其可作为农业抗病虫害新型药物进行开发研究;该酶能够有效降低麦类饲料黏度,表明其作为饲料添加剂的开发利用同样具有良好前景。

【Objective】This study was carried out to obtain high-yielding β-1,3-1,4-glucanases gene engineering strain from Paenibacillus polymyxa CP7 for the research of anti-fungi components, and provide a theoretical basis and application in agricultural production for β-1,3-1,4-glucanases.【Method】After cloning and expression of the β-1,3-1,4-glucanases gene in Escherichia. coli strain BL-21, the inhibitory effects of recombinant protein on the tested fungus were detected, and the effect of recombinant β-1,3-1,4-glucanase on animal feed digestibility in vitro using wheat as raw material of feed was studied.【Result】The bioassay results reveal that the recombinant β-1,3-1,4-glucanase has strong inhibitory effects on the testing fungus with different activities between them, and the inhibitory ratio reached up to 40% above. Compared with the control, the viscosity of wheat sample treated with the recombinant protein was reduced by 4.69% （P〉0.05）. 【Conclusion】 β-1,3-1,4-glucanase is or one of the effective components of active compounds, restrains the fungus of Paenibacillus polymyxa CP7 crude extracts, which suggest β-1,3-1,4- glucanase can be used as a new kind of agricultural chemicals for development, meanwhile, it is also a prosperous feedstuff additive.