| Lignocellulosic biomass is a kind of abundant renewable resource,while cellulose is wrapped by hemicellulose and lignin tightly,which led to a big challenge in effective utilization of biomass.At present,the promising method is enzymatic hydrolysis.So this paper mainly studied a potential hemicellulase p-mannanase on its structure functions and hydrolysis ability assisting cellulase.Endo-1,4-β-mannanase from Trichoderma reesei(EC3.2.1.78)is a member of the glycoside hydrolase family 5 and consists of a catalytic domain,a linker region,and a fungal-type cellulose-binding module(CBM).The full-length gene(man1)and two truncated genes(man1△CBM and manl△LCBM)were obtained using RT-PCR and expressed in Pichia pastoris GS115.The recombinant strains expressing Manl and Manl △CBM produced 34.5 IU/mL and 42.9 IU/mL of mannanase activity in the culture supernatant respectively,while only 0.36 IU/mL of mannanase activity was produced by the recombinant strain expressing Man1△LCBM due to the lack of Linker and CBM module,which might change obviously in the protein structure.The recombinant Man1 and Man1 △CBM had similar enzyme properties,while showed obvious differences in temperature stability.Man1 retained 72.7%of its activity after incubation at 60 ℃ for 120 h,while Manl △CBM retained only 47.9%.But when dealing with at 70 ℃ for 60 min,the retained activity of Manl ACBM increased by 13.3%than Manl.Compared with Man1△CBM,Man1 showed relatively low specific activities on soluble locust bean gum and galactomannan;However,When in association with cellulose ECⅡ for cellulose and pretreated sugarcane bagasse hydrolysis,the reducing sugar yields were significantly improved 28.54%和 20.31%when adding Man1.Neutral glutamine Gln substituted for Asn in β-mannanase N-glycosylation sites by site-directed mutagenesis.This stuy removed an N-glycosylated site(N131,N158 or N329)through site-directed mutagenesis and constructed three kinds of Pichia mutants.The results showed that the mannanase activities were all decreased in mutants.So N-glycosylation were required for efficient expression of β-mannase,and substitution at N131 and N158 decreased mannase production by 85.43%and 79.48%comparing the recombinant Man1.But there was a slight decrease of 16.3%for N-329 mutant,and the thermostability increased by 15.37%. |
