Cloning,Expression and Characterization of Endo-beta-1,4-Mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris

摘要

To be utilized in biomass conversion,including ethanol production and galactosylated oligosaccharide synthesis,namely prebiotics,the gene of extracellular endo-beta-1,4-mannanase(EC 3.2.1.78)of Aspergillus fumigatus IMI 385708(formerly known as Thermomyces lanu-ginosus IMI 158749)was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase(gpdA)and the alcohol oxidase(AOX1)promoters,respectively.The highest production of mannanase(352 U mL~(-1))in A.sojae was observed after 6 days of cultivation.In P.pastoris,the highest mannanase production was observed 10 h after induction with methanol(61 U mL~(-1)).The fold increase in mannanase production was estimated as ~12-fold and ~2-fold in A.sojae and P.pastoris,respectively,when compared with A.fumigatus.Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities(~350 U mg~(-1)protein).Temperature optima were at 60°C and 45°C,and maximum activity was at pH 4.5 and 5.2 for A.sojae and P.pastoris,respectively.The enzyme from P.pastoris was more stable retaining most of the activity up to 50°C,whereas the enzyme from A.sojae rapidly lost activity above 40°C.