Gene Cloning And Heterologous Expression Of The Cellulase And Hemicellulase From Phialophora Spp

Due to the shortage of fossil fuels and the increasing concern for the environment, people worldwide are striving to find an alternative energy. Lignocellulose is the most abundant renewable material in nature, which conversion into biofuel is attracting much attention. During the bioethanol production process, lignocellulosic biomass is generally pre-treated with acid at high temperatures for higher conversion efficacy, followed by enzymatic hydrolysis to obtain the final product glucose. Therefore thermoacidphilic and stable cellulolytic enzymes are favorable for bioconversion.In this study, two fungal strains G5and P13with cellulolytic abilities were isolated from the acidic wastewater of a tin mine. Morphology and ITS sequence analysis indicated that these two strains belong to genus Phialophora. Strain G5had ability to produce high levels of various cellulases, including endo-glucanase, cellobiohydrolase, β-glucosidase and xylanase, when using Avicel or corn cob as the single carbon source. These enzymes had pH optima of4.0-5.0and temperature optima of above60℃except for xylanase (optimal temperature at50℃), and were highly stable at50℃and below. Compared with the commercial cellulase Accellerase1500(Genencor), the enzyme filtrate under study showed60and80%of the capacity to hydrolyze pure cellulose and natural substrate, respectively.By using degenerate PCR and TAIL-PCR techniques, a GH5endo-glucanase encoding gene (egG5), a GH45endo-glucanase gene (egGH45), a GH6exo-cellulase gene (cbh6A), and a GH1β-glucosidase gene (egGH45) were cloned from Phialophora sp. G5. These genes were relatively novel, which deduced amino acid sequences shared67-85%identities with known proteins. After heterologous expression in Pichia pastoris, the recombinant proteins were purified to electrophorestic homogeneity and subjected to enzyme characterization. Recombinant EgG5showed optimal activity at pH4.0-5.0and70℃, retained more than70%of the activity at80℃and was highly thermostable at60℃(retaining80%of the activity after24-h incubation). Putative tertiary structure analysis indicated that EgG5had negatively charged residues in close proximity to the catalytic sites and a special β-sheet. These structure characters of EgG5may attribute to its acidophilicity and thermostability. To verify its function, two mutants, EgG5-Mut (replacing the β-sheet with a loop by four amino acid substitutions) and EgG5-CBM (removing the CBM) were constructed, expressed in P. pastoris and characterized. Both mutants had similar pH optima (pH4.0) and temperature optima (70℃) as wide-type EgG5, but varied in pH stabilities (pH2.0-10.0and pH2.0-7.0, respectively) and thermostabilities. The thermolability of EgG5-Mut (13.4%activity v.s.52.5%of EgG5at65℃for12h) confirmed the effect of β-sheet on enzyme thermostability. EgG5-CBM was more thermostable (94.9%activity at65℃for12h and15.5%. activity at80℃for30min) and had higher specific activity (711.6U/mg v.s.60.3U/mg of EgG5). Recombinant EgGH45and CBH6A showed maximum activity at pH6.0and60℃and pH7.0and65℃, respectively. At the range of pH5.0-8.0and5.5-8.0, both cellulases retained80%of maximal activity and remained stable at pH2.0-11.0and70φ (retaining more than80%of the activity). Recombinant BglGH1had optimal pH and temperature at5.5-6.0and50℃and had activity towards aryl-β-glucoside and cellooligosaccharides. An acidic β-1,4-glucanase (BglG5) was isolated from the culture filtrate of strain G5when induced with Avicel. BglG5had pH optimum at4.5-5.0and temperature optimum at55-60℃, even remained active even at pH1.5-2.0towards barley P-glucan and was stable at pH2.0-9.0and55℃. The enzyme had strong resistance against pepsin and trypsin and was stable under simulated gastric conditions. The predominant hydrolysis products were glucose and cellobiose. Besides, this enzyme could decrease the viscosity of barley-soyben feed and mash and improve the filtration rate of mash. The full-length BglG5-encoding gene was obtained by the combination of degenerate PCR with primers designed based on the internal peptide sequences obtained from mass spectrum,5’-TAIL-PCR and3’-RACE techniques. Deduced BglG5belonged to GH7and shared highest identity of69%with known proteins.Compared with strain G5, Phialophora sp. P13had higher hemicellulase activities. A GH5mannanase gene man5AP13and a GH11xylanase gene xylllA were cloned from strain P13. The deducted amino acid sequences of man5AP13and xylllA had53%and81%identity with known sequences, respectively. Recombinant MAN5AP13produced in P. pastoris showed maximal activity at pH1.5and60℃. This enzyme retained more than70%activity at pH1.0-7.0. After incubation at55℃for2h,83%of the activity was remained. Moreover, MAN5AP13had ability to hydrolyze locust bean gum mannan under simulated gastric fluid. The underlying mechanism of acidophilicity and acid-stability were analyzed based on primary and tertiary structure analysis. Recombinant XYL11A in P. pastoris had pH optimum at5.0-5.5and temperature optimum at65℃.In this study seven novel cellulases/hemicellulases were identified in two strains of Phialophora. These enzymes include thermostable cellulases, an acidophilic mannanase, a purified acidic β-1,4-glucanase and a (3-glucosidase with broad substrate specificity. Moreover, enzymes either directly purified or heterologously expressed with special properties provide us good materials for basic study of acidophilicity, thermophilicity and enzyme stability. Strain G5could be directly used for cellulase production after proper mutagenesis thus has potential for application in a rapid, efficient and low-cost bioconversion process.