Research On Neutral Celluiase Production By Gene Recombinant Strains

Neutral cellulase has important applications on denim biofinishing. Screening the recombinant Pichia pastoris and recombinant Trichoderma reesei strains with higher neutral cellulase activity, and also study on fermentation conditions, and further study the enzymatic properties and its applications on denim biofinishing were carried out.A Pichia pastoris transformant YJ5with high activity of neutral cellulase was screened from282recombinant Pichia pastoris strains. Appropriate fermentation conditions were shown as follows:medium volume30mL in250mL Erlenmeyer flask, the initial pH4.8and uncontrolled during the fermentation process, inoculum volume10%and methanol addition1%every24h. Under the appropriate conditions, the activity of the neutral cellulase reached1144U/mL at240h. The results from the study on enzymatic properties indicate that the appropriate catalysis conditions for the neutral cellulase from the P. pastoris transformant were pH6.0and temperature50℃, and that Zn2+and Mg2+were activators of the enzyme but Fe2+, Fe3+and Cu2+were inhibitors.A Trichoderma reesei transformant HI3with high activity of neutral cellulase was screened from407recombinant Trichoderma reesei strains. Appropriate fermentation conditions were shown as follows:lactose and microcrystalline cellulose compound carbon medium, content of corn steep liquor powder0.9%, the C/N7, the initial pH uncontrolled, inoculum volume10%and the rotation speed220rpm. Under the optimal conditions, neutral cellulase activity was up to3012U/mL at240h. The fed-batch process was used in the next research, and the appropriate conditions were as follows:After fermented for120h, lml feeding solution its consistency15%and C/N5was added every24h. The conditions above allowed the activity of the neutral cellulase to reach5433U/mL at288h, and continued to rise. The results from the study on enzymatic properties indicate that the appropriate catalysis conditions for the neutral cellulase were pH6.0and temperature50℃, and it can be used for a wide range of temperature and pH value, because it reminded85%relative activity at temperature50℃-60℃and pH5.0-7.5. So it has important application value in industry.The Trichoderma reesei transformants HC6and Ce19with high activity of neutral cellulase were screened in the further study. The several different neutral cellulases production and the properties were compared, and the effect of denim biofinishing was also compared. The results were shown as follows:the neutral cellulase could significantly reduce the backstaining of indigo onto denim. Its effect on denim biofinishing was better than acid cellulases. The neutral cellulases had bright industrial propects.