High Expression Of Endocellulase And Sterol Esterase In Pichia Pastoris

With the rapid development of paper industry,such problems as shortage of raw materials,lacking of energy and pollution have arisen.Recycling and utilization of waste paper represents the new trend,enzymatic deinking which has been developed in recent years,with the advantages of energy saving,environmental protection and pertinence compared with traditional chemical deinking.Besides the common lipase,endoglucanase and sterol esterase also showed better performance in deinks,like endoglucanase can reduce cellulose content of polymerization which is good for ink detachment and sterol esterase can remove stickies,effectively.At present,the price these enzymes is on the high side,but the large-scale use is still hurdlest.Therefore,to improve the expression of endoglucanase and sterol esterase,reduce the cost of production and application has special meaning for practice.First of all,this research selected a endoglucanase EG1?GenBank:AF329732?from the fungus Volvariella volvacea has been optimized expression in P.pastoris,which has a preliminary application and good effect in papermaking deinking.In this research,the target gene is obtained from the constructed recombinant P.pastoris x33/pPICZ?A-EG1 and cloned to the plasmid pPICZ?A.Then,build recombinants plasmids pPICZ?A-EG1_?2c?,pPICZ?A-EG1_?4c?and pPICHKA-EG1_?8c?,by using isocaudamers to construct multiple plasmids in vitro,and transform it into the competent cells of P.pastoris to get recombinant P.pastoris strains of different copies,combining with the Real-time PCR analysis.The fermentation results show,after 144 h of induction with methanol,that the two-copy strain's endoglucanase activity reached to 24.83 U/mL,67.20%higher than the single-copy strain,and activity of the strain contain four copy EG1 has reached 39.58 U/mL,59.71%and166.55%higher than the two and single copy strain.After changing the expression plasmid and strain,the activity of recombinant P.pastoris GS115-pPICHKA-EG1_?8C?contain eight copy EG1 has reached 55.65 U/mL,274.75%higher than the single-copy strain and 40.59%higher than the four-copy strain,after 144 h of induction with methanol.With the improving of copy number,the increment of enzyme activity in gradually reduce.So we over-express transcription factors HAC1 or P180 to further improve the enzyme activity of EG1 in the high copy strain.The results show that the highest expression strain is overexpressing HAC1 with eight copies of EG1,the activity of which reached 91.96U/mL,519.27%higher than the single-copy strain.EG1 activity of 650.1 U/mL was achievedina3-Lscaled-upfed-batchfermenterofhigh-expressedstrain GS115/pPICHKA-EG1_?8c?-HAC1 after inducing 144 h,which has been increased by 7.07times compare to the single-copy strain.The protein yield had been reached 4.05 g/L.At the same time,the characterization of recombinant EG1has been detected,the optimal pH temperature is 5.0 and 60°C,when incubated in lower 60°C for 60 min,the residual activity is still retain more than 80.00%,after treated with pH5.0-6.0 buffer at room temperature for 24 h,about more than 90.00%is maintained.Meanwhile,the activity of recombinant EG1 was promoted by metal ions and reagents of K⁺,Mn²⁺,Ba²⁺,and DTT.The kinetic parameters of recombinant EG1 against CMC shows a K_M of 14.29mg·mL^-1,a K_catat of4470.37 min^-1,a V_maxax of 0.10?M·min^-1·mL^-1and a K_cat/K_M of 312.85 mL·mg^-1·min^-11 with pH5.0 and the temperature 60°C.Secondly,this study constructed the recombinant CHE expression strain by P.pastoris expression system.The secretion capacity of recombinant CHE about alpha signal peptide is higher than the auto-signal peptide of CHE and secretion with short peptide.The highest activity strain of eight recombinant strain by conventional construct building is x33/pPICZ?A-CHE_???.After induced 120 h by methanol,the activity of recombinant CHE has reached 0.38 U/mL,666.73%higher than the highest activity 0.05 U/mL from the original CHE after 140 h cultivation in Streptomyces.Meanwhile,the activity of mutant strain?the first amino of acid-alanine amino in CHE gene was mutation into aspartic acid?x33/pPICZ?A-CHE_??T?is 0.95 U/mL,2.51 times higher than recombinant strain x33/pPICZ?A-CHE_???.It may be that codon bias of aspartic acid GAU 35.7?2899?is better than alanine GCU 28.9?2351?in P.pastoris,so the expression of recombinant CHE has been improved.The optimum reaction temperature and pH of recombinant esterase CHE is 50°C and 7.0,and the optimum substrates is 4-Nitrophenyl butyrate?4 c?.When incubated in lower 45°C for 60 min,the residual activity is still retained more than 76.78%.After treated with pH6.0-8.0 buffer at room temperature for 24 h,about more than 80.12%is maintained.The temperature and acid alkaline of recombinant esterase CHE meet the needs of paper making.