| Cellulose is the most abundant organic compound on Earth,widely spreading as one of the cheapest renewal resources, but most of it can't be used directly and becomes cellulose waste.Almost all of the cellulose waste, such as crop residual, used paper, forest waste and so on, is merely burned or deserted without treatment in China, which causes many environmental, ecological problems and several billion yuan economic losses in one year.The cellulose waste is difficult to be used because it is a tough molecule to break down, but it can be converted into sugars, which can be easily used in industry, by cellulases.Cellulases can also used in textile industry, paper and pulp industry, food industry, feed industry, detergent industry and so on.The cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyzes the hydrolysis of cellulose by Hydrolyzing 1,4-beta-D-glycosidic linkages in cellulose.Although the cellulase has been widely used as an important industrial enzyme, it should be improved in enzyme activity, thermostability, pH stability to meet the industry need.Thermophiles are main sources of cellulases with high thermostability.Cellulases from the thermophilic fungi have been reported to be stable and highly active at high temperature and, like mescophilic fungi, thermophilic fungi can produce multiple forms of the cellulase components, mostly.Thermoascus aurantiacus var.levisporus and chaetomium thermophilum CT2 are both widespread thermophilic fungi.They can thrive at temperature between 45℃and 50℃, while most fungi would die above 40℃.Several thermostable enzymes have been isolated from the thermophilic fungi.Current study shows that both the thermophilic fungi's cellulases have multiple components and have high activity and thermostability, which makes the thermophilic fungi's cellulases have great research and industry value.In this study, an endo-β-glucanase encoding gene, eg1 (GenBank accession number: AY 847014), has been isolated from T.aurantiacus var.levisporus and expressed in pichia pastoris.A high expression efficiency strain named GpN24 was gotten through screening, and this strain can express recombinant endo-β-glucanase with excellent thermostability.The molecular of the enzyme was 33kDa.The optimum pH and temperature of the recombinant enzyme activity were 5.0 and 55℃respectively.The recombinant enzyme remained 50% of its original activity after 30 min at 90℃and the activity of the recombinant enzyme can remain stable in pH between 3.0 and 5.0.Direct evolution based on eg1's error-prone PCR and high throughput screening for higher activity in pichia pastoris was used to enhance activity and stability of the endo--β-glucanase from T.aurantiacus var.levisporus and the expression efficiency of its encoding gene eg1.A mutant library of eg1 was constructed by error-prone PCR.In this research, small amount ferment in 1.5ml centrifuge tube after two-layer plate was used to screen for higher enzyme activity.Through mutagenesis and screening, a mutant showed 3.5 fold enzyme activity and optimum pH changing from 5.0 to 4.0.The result of alignment of the mutant's and the wild type amino acid sequences showed that the wild type's 44 amino acid N was replaced with D,and the wild type's 129 amino acid Y changed to H, and wild type's 235 amino acid G mutate to E.The mutant site 229 and 235 belong to conserved sites.Both the mutant endo-β-glucanase and wild type endo-β-glucanase were purified by fractional ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose hydrophobic interaction chromatography.The preliminary mechanism was studied by comparing the two purified endo-β-glucanases.Cbh1 of C.thermophilum was linked to 5'end of eg1 by fusion PCR.After sequencing to make sure a correct ORF, an expression vector was constructed to transform into pichia pastoris, and then a high expression strain of fusion gene eg1cbh1 was screened, and named GPNI3.The molecular of the enzyme was 88kDa .The fusion protein expressed by this strain exhibited maximal activity at pH 4.0 and 60oC and showed higher enzyme activity on all the different substrates than endo-β-glucanase eg1Most cellulases have catalysis domain (CD) and binding domain (BD) .The CD has active sites of catalyzingβ-1,4 glycosidic bond , while the CBD have specificity to the supramolecular structure of cellulose, and increase the absortion to cellulose, and accelerates the hydrolysis of cellulose.As the CBD has key role in the hydrolysis of cellulose, lots of researchs were done by deletion,change or site-directed mutagenesis to the CBD.Cbh1 gene and cbh3 gene were cloned from C.thermophilum, and cbh1 has CD CBD and a link, while cbh3 don't have CBD.We linked the CBD of cbh1 to the C- terminal through fusion PCR.The expression plasmid was constructed and transformed into Pichia pastoris.A strain with high expression was selected and named as GPB103.After DEAE-Sepharose chromatography, the fusion enzyme homogeneously by SDS-PAGE was purified and the characterization of the purified enzyme was carried out.The molecular of the fusion protein expressed was 51kDa.The optimum temperature and pH for enzyme activity were 60℃and 4.0 respectively, and the activity increased 1.2 times on filter paper.
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