Research Of Breeding And Fermentation Conditions Of Cellulase High Production Strain-Trichoderma Reesei

Cellulase is a complex enzyme system to hydrolyze cellulose, which is mainly composed of exoglucanase, endoglucanase and P-glucosidase. Cellulase has a huge potential market in the fields of feed, food, textile and bio-energy and other fields. In the process of industrial production of cellulase, the activity level and cost of cellulase are important factors that restrict its practical application.In this paper, traditional mutation and genome shuffling were used to achieve cellulase high production mutants of Trichoderma reesei T306. And the fermentation conditions of high production mutants in 5 L bioreactor were studied. The results were as follows:1、The screening medium of Trichoderma reesei mutants was optimized. The results showed that addition of 0.1%(w/v) lactose, peptone and sodium deoxycholate was beneficial for the screening of mutants. The optimum carbon source and nitrogen source of culture medium and fermentation conditions were as follows:lactose 1.50%, ammonium sulfate 0.14%, urea 0.05%, peptone 0.10%, tween-800.6%(v/v), initial pH natural, fermentation time 7d, medium volume 30mL/150mL, rotation spead 180 r/min, inoculation volume of medium 50μL/30mL. Six mutant strains with high-production endoglucanase were screened through UV mutation and microwave mutation which were named mutant 0409、0505、0506、 0516、W0103 and W0608.2、The optimum conditions of preparation and regeneration of protoplast of Trichoderma reesei were as follows:lywallzyme concentration 3.5%, mycelium age 22h, enzymolysis time 4h, enzymolysis temperature 30℃, the stablizer buffer system was pH 5.0 sodium hydrogen phosphate-citric acid buffer, regeneration medium was hypertonic screening medium contaning 0.8 mol/L NaCl. Under the optimum conditions, the protoplast yields and regeneration rate were up to 1.44×107/mL and 2.64%.3、The protoplast fusion conditions of Trichoderma reesei were studied. The protoplasts were mixed together after two inactivation methods of UV irradiation for 90s or microwave irradiation for 200s. The protoplast fusion rate reached 0.5% when treated at 30℃ for 12min in the system composed of 30%PEG and 30mmol/L Ca2+. A fusant G2-0832 with higher production of endoglucanase and genetic stability was obtained after two rounds of genome shuffling from parent strains 0409、0516、 W0103 and W0608. The CMCase activity of fusant G2-0832 reached 86.40 U/mL, which was 57.08% higher than original strain. The expression of extracellular protein of fusant G2-0832 enhanced obviously compared with original strain.4、The expanded fermentation experiment of Trichoderma reesei in 5 L fermentor was studied to search for the suitable fermentation conditions of cellulase production. The results were as follows:the main nitrogen source of culture medium was corn steep liquor powder; the feed rate of carbon source should be controlled about 12 mL/h when cultured for 24-40h and 18 mL/h after 40h; the inoculation volume was 10%; and the initial agitation speed was about 400r/min. Under these fermentation conditions, the CMCase activity of original strain and fusant G2-0832 reached about 450 U/mL and 600 U/mL. And the enzyme activity of G2-0832 was about 30% higher than original strain in 5L fermentor.