Cloning And Expression Of Endo-β-glucanase Gene

Endo-P-glucanase (EG) is one of the important cellulases components, which is widely used in the fields of cellulose hydrolysis, cotton biofinishing, feed industry, paper and pulp, etc. This research focused on the construction, screening and EG enzyme production of high EG-producing strains based on gene engineering technology.An endo-β-glucanase gene celE from the anaerobic fungus was inserted into expression vector pET-28a(+), and transformed into Escherichia coli BL21(DE3). SDS-PAGE analysis of its expression product showed a clear protein band about 42 kDa, according with the theoretic size of catalytic domain of the protein. The enzyme exhibited maximum EG activity at pH 6.0,45℃. The enzyme production could reach 39.1 IU ml-1 in shaking flasks.The celE gene was then placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast Pichia pastoris GS115 by electroporation. Three strains with highest resistant to G418 was selected, and assayed by PCR analysis to confirm the gene of interest had been integrated into the genome DNA. This feature was good for genetic stability. Further research was carried out in shaking flasks. The favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 74.1 IU mL-1.The technique of Agrobacterium tumefaciens-mediated transformation (ATMT) of Trichoderma reesei ZU-02 was then optimized. A new recombinant plasmid pCB-hPsT, containing the hygromycin B resistance marker for transformants screening and the strong promoter Pcel7a(including secreting signal peptide sequence) and terminator Tce17a for gene expression, was constructed. This plasmid could be used to transform target gene into T.reesei and realized expression and secretion, which also provided a new technology platform for foreign gene cloning and high expression in filamentous fungi. The imfluencing factors for transformation were studied. It was noted that the pre-germination of conidia was important for improvement of transformation efficiency:the transformation efficiency could boost by 25-fold when the pre-germinated time was prolonged from Oh to 3h. This result was extraordinary for further study of filamentous fungi transformation technique.The codon used frequency differences between anaerobic fungus gene and T.reesei gene was analysed via bioinformatics technique. The catalytic domain of celE was named as celE*, it was optimized accoding to codon bias of T.reesei, obtaing the new sequence celEn. The recombiant plasmids were constructed as pCB-hE* and pCB-hEn, harboring celE* or celEn as target gene, and transformed into T.reesei ZU-02 respectively.300 T.reesei/pCB-hE* and 293 Treesei/pCB-hEn were obtained, and the recombinants with pCB-hEn grow quickly on the screening plates with CMC as sole carbon source, and eight strains T.reesei/pCB-hEn with high-CelEn yields were selected. After fermentation for 84h in shaking flasks, the EG production reached 193.6 IU ml-1 (pH6.0), about 6.0-fold as high as that of ZU-02 at corresponding period. This research achieved extracellular secretion of anaerobic fungus endo-β-glucanase in aerobic fungus T.reesei successfully, and the expression cellulases is of great importance in denim biofinishing. This study filled in the gap of the domestic field in neutral cellulases production.Furthermore, the cel5a coding for endo-β-glucanase from T.reesei was inserted between T.reesei strong promoter Pcel7a and terminator of pCB-hPsT, and then transformed into the genome DNA of T.reesei ZU-02 by ATMT technique. Four recombinants were selected by screened on the plates with CMC as the sole carbon source from 263 transformants. The highest EG activity could reach 297.1IU ml-1 after 120h cultured in shaking flasks, about 3.9-fold as high as that of ZU-02, and the filter paper activity(FPA) was increased by 30%-40%. This study can promote the development both for direction evolution of cellulases and construction of cellulase producing strain with high hydrolysis activity...