Endo-β-1,4-glucanase Gene Homologous Expressed In Aspergillus Niger

Cellulose is the most widely distributed on Earth, the most abundant carbohydrate, but also exists in nature is the cheapest, most abundant renewable resources, a wealth of research value and significance.Endo-β-1,4-glucanase in cellulase is the most extensive and in-depth an enzyme,an endo-glucanase can truncate long chains of cellulose in cellulose non-crystalline region through random hydrolysis of (3-1,4-glycosidic linkage, which plays an important role in the overall degradation of cellulose, forming cellooligosaccharides. Cellooligosaccharides is functional oligosaccharides, bifidobacterium proliferation factor, reduce serum cholesterol and triglyceride levels, promote the absorption of minerals such as calcium, magnesium, iron, low heat value. But the production of fiber oligosaccharides rely mainly on cellulose by acid or enzyme hydrolysis, Therefore, in order to obtain high purity of a single degree of polymerization of cellooligosaccharides, requires a single component, high purity endo-2-1,4-glucanase. Nowadays, the production of endo-glucanase rely on microbial fermentation, mostly the microbial strains of fungi, such as Trichoderma, Penicillium, Aspergillus and Humicola, is widely used in the production of Endo-glucanase. Single strain of endo-glucanase production have a few factors such as low stability, high costs and unsatisfactory still restricts the Cellulose production and application of oligosaccharide. Therefore, to carry out cutting endo-glucanase high-production recombinant strains and fermentation technology research of work is very necessary. Endo-glucanase are widely used in beer, feed, paper and other industries.This research is used the fungus Aspergillus niger CICC2462as receptors, although Aspergillus niger secrete endo-β-glucanase, But the expression of wild-type strains low quantity, and at the same time has exo-(3-glucanase,β-glucosidase, can’t satisfy the special requirements such as oligosaccharide production. Using Aspergillus niger high expression of the glaA gene loci gene, This result showed that the endo-glucanase gene is successfully transformed and homologously expressed in Aspergillus niger, which lays the foundation for the construction of an engineered strain to obtain high yield, high purity of endo-glucanase.The main results in this study are as follows:1. Cloning of endo-β-1,4-glucanase gene (engl) and construction of endo-β-1,4-glucanase gene (Engl) of Aspergillus niger expression vector. Using the highly expressed elment of glaA encoding a glucoamylase, the recombinant expression vector pSZHG-engl was constructed. 2. Agrobacterium-mediated transformation of Aspergillus niger and screening of transformants and identification of engl gene homologous transformantsTaking of freeze-thaw method make Aspergillus niger expression vector pSZHG-engl transformed into Agrobacterium AGL1. Transformed Aspergillus niger in the use of agrobacterium-mediated method. In the end,2positive homologous transformants were screened out by the method of PCR. Identification of2positive transformants with homologous recombination primers by PCR. As a result,2positive strains can amplify1701bp target fragments. It showed that they were homologous recombinants. The homologous recombinantion rate of transformation pSZHG-engl was100%.3. Endo-β-1,4-glucanase expression analysis in the recombinant strainsUnder the shaking flask fermentation condition, the endo-β-1,4-glucanase activity of the A.niger transformant reached272U· mL-1in the fermentation solution, which was5.2-fold as high as that of the original strain. Recombinant strains the optimum pH is6.0, the optimal temperature of60℃, temperature stability is good. By used of SDS-PAGE analysis of engineering strain culture supernatant, the result showed that all of2engineering strains had been secreted and expressed successfully, significantly higher than the original strain. By the ALPHAIMAGER SYSTEMS software analysis, the expressed enzyme was quantitated to be165～193μg·mL-1.This result showed that the endo-β-1,4-glucanase gene is successfully transformed and homologously expressed in Aspergillus niger, which lays the foundation for the construction of an engineered strain to produce a food-grade endo-β-1,4-glucanase in large-scale industrial production.