Heterologous Expression Of Cellulase In Pichia Pastoris And Its Synergistic Effect

Cellulase hydrolysis of lignocellulose biomass to obtain renewable energy is an important way to solve the energy shortage in recent years.Therefore,in view of the high cost of enzymatic hydrolysis and the low efficiency of enzymatic hydrolysis,we constructed recombinant strains of Trichoderma reesei EG ?,EG ? EGV,CBH ? and CBH? to make up for the deficiency of natural secretion,and discussed their properties and synergistic effects in enzymatic hydrolysis.The expression vectors pPIC9K-eg2,pPIC9K-eg4,pPIC9K-eg5,pPIC9K-cbh2 and pPIC9K-cbh1 were successfully constructed by digestion homologous recombination.The recombinant strains were transformed into Pichia pastoris GS115 to construct an inducible recombinant of Pichia pastoris.Five recombinant strains were screened by resistance plate screening and CMC-Congo red plate screening.Through shaking flask fermentation optimization,the activity of EG ? enzyme of strain P.pastoris-eg2-3 could reach 18.5 IU/mL after being induced by methanol for 168 hours,28 degrees and 260 rpm;the activity of EG ? enzyme of strain P.pastoris-eg4 could reach 4.87 IU/mL after being induced by methanol for 120 hours,28 degrees and 260 rpm;and that of strain P.pastoris-eg5-6 could reach 2.95 IU/mL after being induced by methanol for 168 hours,28 degrees and 260 rpm;and that of recombinant strain P.pastoris-eg5-6 could reach 2.95 IU/mL after being induced by methanol for 168 hours,CBH ? activity of P.pastoris-cbhl-12 was 3.559 IU/mL after 168 h,8 C and 240 RPM induced by methanol,and 6.07 IU/mL of CBH ? activity of recombinant strain P.pastoris-cbh2-8 after 168 h,26 C and 260 RPM induced by methanol.The analysis of enzymatic properties after purification showed that the optimum pH of EG ? was 4.5,the optimum temperature was 60?,the Km value was about 3.6 mg/mL,and the Vmax value was about 241.8 IU/mg;the optimum pH of EG ? was 5.0,the optimum temperature was 60?,the Km value was about 2.4 mg/mL,and the Vmax value was about 156.6 IU/mg;the optimum pH of EG V was 4.5,the optimum temperature was 55?,the Km value was about 1.6 mg/mL,and the Vmax value was about 99.7 IU/mg;The optimum pH of CBH ? is 5.5,the optimum temperature is 60 C,the Km value is about 1.8 mg/mL,and the Vmak value is about 116.3 IU/mg.The optimum pH,temperature,Km and Vmax of CBH ? were 5.5,60?,3.1 mg/mL and 214.3 IU/mg respectively.The molecular weight of recombinant enzymes EG II,EGIV,EGV is about 50 KDa,which specifically acts on non-crystalline cellulose.The molecular weight of CBH ? and CBH ? are about 65 KDa and 55 KDa respectively,which can act on the long chain of crystalline cellulose and non-crystalline cellulose.Its enzymatic properties are very close to those of the natural Trichoderma reesei secretase.Response surface methodology(RSM)was used to study the effects of CBH ?,CBH ?and EG ? on the enzymatic hydrolysis of five recombinant enzymes.There is obvious synergism between CBH ? and CBH ?,but there is no obvious synergism between CBH ?and EG ?,CBH ? and CBH ?.When the amount of recombinant enzymes CBH ?,CBH ?and EG ? were 89.6%,36.6%and 20.2%of the total cellulose,the glucose content in the hydrolysate increased by 9.5%compared with commercial cellulase.