Establishment Of Expression System Of Humicola Insolens And Exploiting Novel ?-1,4-glucanase From It

Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase,?-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%,440%,320%,41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.We cloned a new glycoside hydrolase family 6 gene, Hicel6C, from the thermophilic fungus Humicola insolens Y1 and expressed it in Pichia pastoris. Using barley ?-glucan as a substrate, recombinant HiCel6C protein exhibited neutral pH (6.5) and high temperature (70?) optima. Distinct from most reported acidic fungal endo-?-1,4-glucanases, HiCel6C was alkali-tolerant, retaining greater than 98.0,61.2, and 27.6% of peak activity at pH 8.0,9.0, and 10.0, respectively, and exhibited good stability over a wide pH range (pH 5.0-11.0) and at temperatures up to 60?. The Km and Vmax values of HiCel6C for barley ?-glucan were 1.29 mg/mL and 752 ?mol/min-mg, respectively. HiCel6C was strictly specific for the ?-1,4-glucoside linkage exhibiting activity toward barley ?-glucan, lichenan, and carboxy methylcellulose sodium salt (CMC-Na), but not toward laminarin (1,3-?-glucan). HiCel6C cleaved the internal glycosidic linkages of cellooligosaccharides randomly and thus represents an endo-cleaving enzyme. The predominant product of polysaccharide hydrolysis by HiCel6C was cellobiose, suggesting that it functions by an endo-processive mechanism. The favorable properties of HiCel6C make it a good candidate for basic research and for applications in the textile and brewing industries.A novel endo-?-1,3(4)-glucanase gene, HiCel16A, was cloned from the fungus Humicola insolens Y1. The 988-bp full-length gene encoded a 286-residue polypeptide consisting of a putative signal peptide of 20 residues and a catalytic domain belonging to glycosyl hydrolase family 16. It was successfully overexpressed in Pichia pastoris GS115. The purified recombinant HiCel16A exhibited highest specific activity toward barley ?-glucan, followed by lichenan and laminarin, but not toward CMC-Na, birchwood xylan, Avicel and filter paper, indicating that HiCel16A is an endo-?-1,3(4)-glucanases. Recombinant HiCel16A had a pH optimum at 5.5 and a temperature optimum at 55? with a specific activity of 693 U/mg toward barley ?-glucan. It exhibited good stability over pH 5.0-9.0 and at temperatures up to 50?, retaining over 80% maximum activity. The Km and Vmax. values of HiCel16A for barley ?-glucan were 0.91 mg·ml-1 and 1530 ?mol·min-1·mg-1, respectively. All these favorable enzymatic properties of HiCel16A make it a good candidate for applications in various industries.