Cloning, Expression And Anylysis Of Enzymic Properties Of β-glucosidase From The Thermophilic Fungus Thermoascus Aurantiacus Var. Levisporus

So far,β-glycosidase gene has been cloned from over a hundred microorganisms, and heterogenously expressed in many microorganisms. At present time, the research forβ-glycosidase in fungus is focused on yeast, Trichoderma, Aspergillus and so on, and found few in the thermophilic fungus, only reported in Humicola grisea, Talaromyces emersonii and Thermoascus aurantiacus (stock). The thermostableβ-glycosidases are superior to mesophileβ-glycosidase in application, because the thermostable beta-glycosidases expressed by thermophilic fungus have high stability and activity in high temperature .Thermoascus aurantiacus var. levisporus is a widespread thermophilic fungus with higher growth temperature,β-glycosidase can be produced in the culture medium containing lactose as the only carbon source under condition of 50℃. The thermostableβ-glycosidases were expressed fast and effectively in the normal temperature after the gene was transformed into Pichia pastoris using molecular biology, which was expected reduceing energy consumption, raiseing economic efficiency and laying a foundation for the large-scale fermentation and the industrial application.Theβ-glycosidase gene bglⅠand bglⅡwere cloned through RT-PCR from Thermoascus aurantiacus var. levisporus RNA. EU269025 and EU263993 are the Genbank accession numbers of the two genes respectively. The bglⅠand bglⅡwere inserted into the expression vector pPIC9K of the Pichia pastoris separately, then the reorganized plasmid pPIC9K/ bglⅠand pPIC9K/ bglⅡ, were transformed into Pichia pastoris GS115 by tip-and-run method after linearization, the yeast project strains were obtained , which could expressβ-glycosidase effectively under the function of the promotor AOX1 gene.The recombinantβ-glucosidase was purified by DEAE-Sepharose chromatography separately , whose molecular mass is 120kDa and 118kDa respectively by SDS-PAGE.β-glycosidase's activity is 1.2U/mg and 0.23U/mg respectively. The fermentation quantity of the two yeast project strains both are 0.45mg/mL in small scale fermentation. The formerβ-glycosidase exhibited optimum catalytic activity at pH5.0 and 60℃, and remained 80% of its original activity after 30min at 70℃. The enzyme has high thermostability. Optimum activity was obtained in the range of beffer from pH3.0~9.0, which means it was a high acid and alkali tolerancant enzyme. The latterβ-glycosidase exhibited optimum catalytic activity at pH5.0 and 50℃, but its thermostability is worse than the former's.