Cloning And Expression Of Neutral Cellulase Stce1

Cellulase is the series of enzyme that able to effectively degrade cellulose into glucose,which is widely used in food, paper, textile, and many other fields. With the great progress has been made in molecular biotechnology in recent years, it has more and more breakthroughs to improve cellulase by genetic engineering. It effectively improve the activity of the cellulase enzyme, and also provides a great impetus for its industrial production.Stce1 is a new member of the family 45 endoglucanase,Which was found by Japanese scholar Jinichiro Koga, etc. through culture the supernatant of 1600 fungi, It has a high addition to micro-fiber,and also has anionic surfactants and anti-oxidant activity. Currently,At present Jinichiro Koga etc. have successfully transferred stce1 to the Humicola in a recombinant mature protein, it has high enzyme activity, per m L of culture supernatant contained 0.90 mg protein, 27% of the total protein,100 times high than in S. Coccosporum supernatants of 0.0085 mg / m L.In this study, has successfully obtained plasmid p PIC-Ppdc-Tpdc transformation from plasmid p PICZαA, referred p PIC-PT. Accrodding to PCR amplification to get target gene stce1, sequencing with NCBI, homology is 99%. By double digestion method, inserted the target gene stce1 into the plasmid p PIC-PT to constituting the expression cassette p PIC-PST.The plasmid p PIC-PCT and p AN7-1 are transformed into T.reesei QM9414 via co-transfected protoplasts method. The plasmid p AN7-1 contains resistance gene hygromycin, which can be used for screening of recombinants as a gene marker. PCR verification and SDS-PAGE verification obtain recombinant bacteria B6, Finally, flask fermentation, measure enzyme activity.After shake flask fermentation, the CMCNa activity of recombinant B6 is up to2.09 U/m L, which is 7.1-fold as high as that of the host strain, FPA activity reachs 0.44 U/ m L, which is 4.3 fold as high as that of the host strain.The study of cellulase characterization showed tha the optimum ph was about 6.0,and the optimum reaction temperature was 60 ℃The suject also used prokaryotic system to express stce1. Insert endoglucanase genestce1 stce1 into the expression vector p ET-28 a to get the prokaryotic expression cassette.Preparation competent cells of Ecoli Rosetta, transforme prokaryotic expression cassette p ET28-stce1 into Ecoli.Rosetta competent cells. Obtain recombinant bacteria A2, The study of recombinant bacteria inducing conditions show that optimum conditions is 37 ℃, 4h, 0.5m M PTG final concentration. At last, the CMCNa activity of transformant is 1.23 U / m L.In summary, this study constructed prokaryotic and eukaryotic expression box.stce1 was expressde in Ecoli Rosetta and T.reesei QM9414. Compared with original bacteria,the enzyme activity of cellulase has improved significantly, it lays the experimental foundation for the cloning and expression of neutral cellulase.