Molecular Modification Of Cellobiohydrolase (CBHⅠ, CBH Ⅱ) And Endo-β-glucanase (EGⅠ) From Thermophilic Fungi

Many researches have been focused on cellulases since Seilliere found the enzyme in the digestive juice of snails in 1906. It has been shown that cellulases unpollutedly degrade cellulose waste, the most abundant renewable resources on earth, into a variety of useful products. Cellulases can also be used in industries including textile, paper pulp, food, feed and detergent. Recently, lignocellulose is degradated by cellulases to produce the fuel ethanol. Therefore, many researchers have begun to find a stain that can produce cellulases with the higher activity, the enhanced thermal stability and the increased pH stability to meet the needs of industrial production.In our laboratory, thermostable cellulases have been isolated from the thermophilic fungi and thermophilic fungi Thermoascus aurantiacus var. Levisporus and Chaetomium thermophilum CT2. In this study, the cellobiohydrolase genes cbh2 and cbh1 were isolated from C. thermophilum CT2 and were expressed in Pichia pastoris. Then, two expression strains were gotten through screening and were named as WTCBHⅡand WTCBHⅠ. After that, methods of direct evolution and high throughput screening for higher activity in P. pastoris were used to enhance the activity and stability of the cellobiohydrolase from C. thermophilum CT2. As a result, CBHⅡof two transformants showed 2 fold higher activities than that expressed from a wide-type cbh2 gene. The mutant CBHⅡin the two selected transformants were designed CBHⅡX16 and CBHⅡX305, respectively. CBHⅡX16 had five mutant amino acids: R1S, A29T, L203Y, Q204K and E252G. Meantime, A29T, T115I, I195V, L203Y, Q204K and E252G were exhibited in CBHⅡX305. In contrast, CBHⅠof two transformants exihibited 1 fold higher activities than that expressed from a wide-type cbh1 gene. The mutant CBHⅠin the two selected transformants were designed CBHⅠX88 and CBHⅠX260. We found 10 mutant amino acids (C13Y, S15P, S84P, N86D, N179D, D212E, C225R, M348K, D383G and S412G) in CBHⅠX88 and 7 mutant amino acids (C13Y, S15P, S101Y, M208T, D212E, N290T and Q473R) in CBHⅠX260. After the mutant and wild-type cellobiohydrolases were purified by using methods of the fractional ammonium sulphate precipitation and ion exchange chromatography on DEAE-Sepharose, their natures were compared.To understand how mutant amino acids affect CBH property, 3-dimentional structures of the wide-type and mutant CBH proteins from C. thermophilum in the study were predicted by the method of homology modeling. In CBHⅡX16 or CBHⅡX305, L203Y and A29T suggested that the activity was improved, and K204Q, E252G and T115I indicated the increasing of the thermal stability, while Q204K and E252G might be the reasons that the optimum reaction pH was enhanced. In CBHⅠX88 or CBHⅠX260, C225R, M208T and Q473R might lead to the increase in activity. In C13Y, S15P, S84P and C225R, the side chain of Y, P and R could improve the packing efficiency which might cause the thermal stability to be increased. Meantime, C225R, M348K and Q473R might be the reasons that the optimum reaction pH was enhanced.The method of site-directed mutagenesis was used to modify endo-β-glucanases encoded by eg1 gene from T. aurantiacus var. Levisporus. The enzymes with five mutant non-conservative amino acids (N41D, L52M, Y129H, W165Y and H193A) were expressed in P. pastoris. Endo-β-glucanase activities were significantly reduced by two amino acid mutation N41D and H193A. The enzymes with L52M, Y129H and W165Y were separately expressed in P. pastoris, and the constructed engineering stains were named as L52MEG1, Y129HEG1 and W165YEG1, respectively. After the mutant and wild-type endo-β-glucanases were purified by using fractional ammonium sulphate precipitation and ion exchange chromatography on DEAE-Sepharose, their characterizations were compared. The three-dimensional structure of enzymes produced from L52MEG1, Y129HEG1 and W165YEG1 were predicted by using the method of homology modeling, and the possible mechanisms of the changes in the natures of the mutant enzymes were discussed.