Screening Of Cellulose-degrading Fungi, Cloning And Expression Of Cellulase Gene In Pichia Pastoris

Enzymatic hydrolysis of cellulose using cellulase is attractive, however, high cost in liberating sugars from cellulosic biomass is still the bottleneck of the commercialization of biofuels and bio-based chemicals, and thus screening of cellulose-degrading microorganisms which produced more efficient cellulosic enzymes are expected to be developed.In this article, a new strain of high efficiency cellulose-degrading is isolated from soil of Shang dong province by Congo red method and analyzeing the activities of cellulosic enzymes. It is a fast-growing and strongly cellulolytic fungus that has enormous potential to produce cellulose-degrading enzymes. It was identified as Penicillium oxalicum M (CGMCC No. 4357) based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. However, information is lacking on cellulose-degrading of Penicillium species, and this is particularly the case for P. oxalicum. The screening of high efficiency cellulose-degrading P. oxalicum M enriched the microorganism resources of degrading cellulosic biomass.Little is known about cellulases of P. oxalicum, thus, to address this deficiency, we cloned two complete open reading frame of endoglucanase and cellobiohydrolases gene, egl1 (GeneBank No. HQ843505) and cbh1 (GeneBank No. HQ843504), from strain M. The egl1 gene consists of a 1434 bp open reading frame that encodes a putative protein containing 478 amino acid residues; the cbh1 gene of 1 641bp encoded 547 amino acids. The two gene contained no intron and were both identified as members of glycoside hydrolase family 7 based on highly conserved catalytic residues -Glu-X-Asp-X-X-Glu-. The successful cloning of the egl1 and cbh1 gene enriched the gene resources of cellulase and provided the fundament for their effectiveness in attacking cellulose substrate.The egl1 and cbh1 were expressed in Pichia Pastoris, and the two recombinant proteins were characterized respectively. Using sodium carboxymethyl cellulose (CMCNa) as substrate, recombinant EGL had optimal activity at 50°C and pH 6.0, and EGL was stable ranging from pH 5.0 to 7.0; the optimum pH and temperature of the recombinant enzyme CBH was 6.0 and 55℃respectively using p-nithrophenyl-β-D-cellobioside (pNPC) as substrate, and the stability of pH and temperature were both very high. Mg²⁺ modestly stimulated EGL and CBH activity, whereas Ag+ and Cu²⁺ were inhibiters. The successful expression of two cellulases in Pichia pastoris provided the fundament for the construction of gene engineering strains and elucidation of the underlying mechanism of the conformations of the enzyme molecules.