Study On High Activity Endo-β-glucanase Production By Recombinant Trichoderma Reesei Strains

Cellulase is a class of enzymes that catalyze hydrolysis of cellulose to glucose. The cellulase system is composed of exo-β-glucanase, endo-p-glucanase and β-glucosidase. The major cellulase component of Trichoderma reesei is exo-p-glucanase, which accounts for 60% of the total secreted protein of T. reesei. The endo-p-glucanase activity is low, which decrease the efficiency of synergetic degradation of cellulose. In this study, we used recombinant DNA technology to achieve the directed evolution of T. reesei. The endo-p-glucanase gene was highly expressed. The results suggest that cellulase production by recombinant T. reesei at large scale had industrial application potential. This study is meaningful in biomass energy and cotton fabric biofinishing.Homologous expression of endo-β-glucanase gene egl2 was carried out in T. reesei. The endo-p-glucanase gene egl2 was cloned from T. reesei by the method of RT-PCR. The gene was placed between cellobiohydrolase I gene promoter including signal peptide sequence (Pcbhl-ss) and cellobiohydrolase I gene terminator sequence (Tcbh1). A new recombinant plasmid pCB-PHT containing the egl2 gene expression cassette and the hygromycin B resistance marker for transformants screening was constructed. The plasmid pCB-PHT was introduced into T. reesei conidia using Agrobacterium tumefaciens mediated transformation method (AMT). The influencing factors for transformation were studied. The density of T. reesei conidia was 107/ml. The density of A. tumefaciens cells was OD660 of 0.8. The effect of pH was also assessed. It was found that the optimal pH of induction media was 5.3. The optimal co-cultivation temperature leading to the highest transformation frequency was 25℃. In AMT experiment,241 transformants were obtained and then cultured on screening plates with CMC as sole carbon source. Six fast-growing ones, as indicated by their larger colonies, were selected for further analysis. After shaking-flask fermentation T. reesei E5 was obtained. After 120 h shaking-flask fermentation, the endo-p-glucanase activity from recombinant T. reesei E5 reached 3472 U/ml, which were about 3.8 times higher than that of the original strain. Meanwhile, the filter paper activity of recombinant T. reesei was 1.32-fold of the host strain. This research is meaningful in the biological catalysis of lignocellulosic material and biomass energy industrialization.Heterologous expression of Humicola insolens endo-β-glucanase gene cel5A in T. reesei was studied. The endo-β-glucanase gene cel5A was cloned from the RNA of H. insolens by the method of RT-PCR. The length of the gene is 1116 bp, encoding 372 amino acid residues. The gene was placed between Pcbhl-ss and Tcbh1. A new recombinant plasmid pCB-HI, containing the cel5A gene expression cassette and the hygromycin B resistance marker for transformant screening, was constructed.In AMT experiment,323 transformants were obtained and then cultured on screening plates with CMC as sole carbon source. Eight fast-growing transformants, as indicated by their larger colonies, were selected for further analysis. After shaking-flask fermentation, the culture supernatants of the eight transformants were collected for enzyme activity assay. After the screening, T.reesei H3 transformant was obtained. Recombinant T. reesei H3 was used to produce endo-β-glucanase. An obvious protein band (approximately 52 kDa) of H. insolens endo-β-glucanase was detected by SDS-PAGE from fermentation broth. The enzyme from T.reesei H3 shows high catalysis in the range of pH 5.0-7.0 and the optimal pH value is 6.0.The fermentation properties of the recombinant T. reesei H3 were studied. The results show that the carbon sources play an important role in the formation of endo-β-glucanase. When using lactose and microcrystalline cellulose as composite carbon source, both enzyme activity and productivity reach high level. The suitable concentration of corn steep powder as a nitrogen source is 12 g/L. The optimum initial pH value of fermentation medium is 5.0. After 120 h shaking-flask fermentation, the endo-p-glucanase activity of recombinant T. reesei H3 reached 3068 U/ml. When endo-β-glucanase production was carried out in a 2 m3 fermenter, the neutral endo-β-glucanase activity reached 8012 U/mL after 96 h fermentation. The higher endo-β-glucanase activity in the fermenter was considered to have resulted from the higher level of dissolved oxygen and the more efficient mass transfer as compared to shake-flask cultivation.The molecular structure of the cellulase can be divided into cellulose binding domain (CBD), linker and catalytic domain. To study the impact of CBD fragment from T. reesei endo-β-glucanase gene egl2 on H. insolens endo-β-glucanase gene cel5A, the egl2 CBD sequence was cloned and connected to H. insolens cel5A gene. A new recombinant plasmid pCB-CEG, containing cellobiohydrolase I gene promoter and the hygromycin B resistance marker for transformants screening, was constructed. The plasmid pCB-CEG was introduced into T. reesei using AMT method.260 transformants were obtained and then cultured on screening plates with CMC as sole carbon source. Seven fast-growing transformants, as indicated by their larger colonies, were selected for further analysis. After shaking-flask fermentation T. reesei HC4 was obtained. After 120 h shaking-flask fermentation, the endo-β-glucanase activity from recombinant T. reesei HC4 reached 3254 U/ml. The enzymatic properties of cellulase from T.reesei HC4 were investigated. The enzyme from T.reesei HC4 shows high activity in the range of pH 5.0-7.5 and the optimal pH value is 6.5. It indicated that the enzymatic property of H. insolens endo-β-glucanase Cel5A containing the egl2 CBD is more close to neutral pH and has wider pH range than H. insolens endo-β-glucanase Cel5A. This study provides a new way for the directed improvement of catalytic property of cellulases.According to the characteristics of enzyme preparation from recombinant T. reesei, basic tests for application were carried out. Experiments of denim biofinishing indicated that neutral enzymes from recombinant T. reesei H3 and HC4 present better appearance for the denim garments with little backstaining on the fabrics than acidic enzyme from T. reesei. The results showed a good application prospect of neutral cellulase from T. reesei transformants. The hydrolysis yield of corncob cellulose can reach 84.9% using enzyme from T. reesei E5, which was 10.7% higher than that of the host strain.