Gene Cloning, Expression And Characterization Of Novel Thermostable Cellulases From Thermophilic Fungi

As the exhausting of energy and the destruction of the environment, it has been one of the major barriers of the human development in the 21 st century. Therefore, it is imminent to look for renewable resources instead of oil. Lignocellulosic biomass is the largest source of the renewable energy on earth and the most important structural component of all plants. It is the only renewable source of carbon and covers more than 50% of the botanic body. Its enzymatic degradation to the constituent monosaccharaides has attracted considerable attention for biofuel production, which is crucial for satisfying global energy demand and cutting greenhouse gases. In the course of lignocellulose biomass enzymatic conversion, high enzymatic activity and strong stability of cellulases has been the key to reduce the cost, and improve enzymatic hydrolysis efficiency. therefore, novel cellulose hydrolysis enzymes have gained wide attention of domestic and foreign experts.Myceliophthora thermophile is a soil-borne thermophilic fungus that grows well at 50°C. As the first filamentous fungi which finished sequenced of the genome, it is a widely thermophilic fungus and it has been used to identify novel cellulolytic activity, including endo-1,4-β-glucanases(EC 3.1.1.4), exo-1,4-β-glucanases(EC 3.2.1.91; EC 3.2.1.74) and β-glucosidases(EC 3.2.1.21).In this research, M. thermophile was cultured on a medium with Avicel as carbon source, and total RN A was isolated from the mycelia to sequencing using RNA-seq. Two unknown functional genes were screened and definited, named cel1 and cel2, respectively. The obtained nucleotide sequences were deposited in GenBank database, und er the Gene Accession No. KM099282 and KM099283, respectively. The encoded proteins(Cel1 and Cel2) comprised 211 and 203 amino acid residues, and the predicted the molecular weight was 20.7 and 19.8 kDa, respectively. Blast showed that Cel1 and Cel2 belonged to DUF4360 super family. Each of the two proteins has a predicted signal peptide, it is suggested that all the proteins were the extracellular secreted proteins. Using ExPASY Position to analysis the N-linked glycosylation sites and using NetOGlyc 4.0 Server to analysis the O-linked glycosylation sites, all the results demonstrated that the two proteins might be glycosylated.The recombinant plasmids pPIC9k/cel1 and pPIC9k/cel2 were constructed and electroporated into P. pastoris cells for heterologous expression of the two genes under control of the AOX1 promoter. Hundreds of transformants were obtained by electroporation on MD/MM plates. Multicopy transformants were detected by PC R and screened out on the YPD plates with the highest concentration of antibiotic G418. The clones with the highest yield of two proteins designated GS-MT-Cel1 and GS-MT-Cel2, were chosen for proteins purification and characterization respectively. After one percent methanol induction, proteins Cel1 and Cel2 were secreted to the culture medium. The hydrolytic activity of two recombinant proteins in the culture medium reached 4.4 and 3.8 mg/ml after induced expressionfor 144 h, respectively. After purification using His TrapT M FF crude, each of the two recombinant proteins displayed a single band in the electrophoresis gel image, corresponding to a molecular mass of 73.8 and 77.5kDa, respectively. Glycoprotein staining showed that the purified recombinant Cel1 and Cel2 were glycoproteins, especially Cel1 and Cel2 were highly glycosylation.Characteristics of those proteins showed that the two proteins could efficiently hydrolyze cellulose and Cel2 could also efficiently cleave xylan. TLC and HPAEC-PAD showed the hydrolysis products were cello-oligosaccharides meaning the two proteins conformed to the characteristics of endoglucanases. The recombinant enzymes showed the highest activity at 60 °C, the activity dropped sharply above 80 oC. These results indicated that those enzymes were thermostable. The highest activity of the two proteins were pH 5.0. The deglycosylated Cel1 and Cel2 proteins prepared with the Enzymatic DeGlycoMx K it were used for determination of their melting temperature(Tm) using Q-PCR, the results suggested that there was great disparities between the temperatures of the two deglycosylated enzymes and the two glycosylated enzymes, suggesting that glycosylation affects the thermostability. Mutation of two conserved Asp residues D59 and D84 of Cel1 and Cel2 caused loss of their cellulase activity, and mutation of two conserved Glu residues E66 and E95 of Cel2 caused loss of its xylanase activity. Furthermore, specific activity of each Cel2 mutant exhibiting either the cellulase activity or the xylanase activity did not vary markedly from that o f the wild-type Cel2.These data provide direct evidence that Cel1 and Cel2 are new type of cellulolytic enzymes and Cel2 is a bifunctional enzyme. To our knowledge, Cel1 and Cel2 are two proteins to be identified in DUF 4360 superfamily for the first time. All these characterisations suggest they are promising for industrial applications.