Cloning And Expression Of ?-mannanase From Trichoderma Reesei And Research On Its Structural Domain

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-?-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 EC? for cellulose and pretreated sugarcane bagasse hydrolysis,the reducing sugar yields were significantly improved 28.54%? 20.31%when adding Man1.Neutral glutamine Gln substituted for Asn in ?-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 ?-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%.