The Composite Mutagenesis And Enzyme System Optimization Of Trichoderma Reesei

Cellulase has a wide range of uses in the bioethanol, food processing, feed, textile, traditional Chinese medicine and many other fields. As a catalyst for the degradation of cellulose and hemicellulose, Cellulose plays an important role in the process of lignocellulose into bioethanol, therefore, it has very important scientific prospect and application value to breeding out high-yield cellulase strains. And the Trichoderma reesei and Aspergillus niger have its advantages in the production of cellulose so that it has been widely studied.In order to improve on the enzyme activity of components of cellulose enzyme system, this study proceeded the repeat alternating mutation of Trichoderma reesei RUT C30, and optimized the enzyme production liquid culture medium of mutant strains; In addition, optimized the beta glycosidase enzymes production of Aspergillus niger C112, which made up for shortcomings of enzyme insufficient in the cellulase production by Trichoderma reesei; Finally, evaluated the environmental impact factors of cellulose activity and the application effect of it, the mainly results are as follows:(1) The spore suspension of start strain Trichoderma reesei Rut C30 was proceeded repeat alternating mutation by UV irradiation and ethyl methyl sulfonic acid. Via the calculation of fatality rate of strains,2 times repeated mutagenesis for UV irradiation 150s and EMS 120s was confirmed as the optimize mutation method. A high-yield cellulase mutant strain ZE 7 was finally obtained through Congo red selective plate preliminary selection and fluid fermentation re-screening respectively to the induced mutant strain, and the filter paper activity of it was 3.84 IU/mL, which has improved significantly when compared with the original strain 2.09 IU/mL. Via 8 times serial passage cultivation and the stability research, the mutant strain enzyme activity was found stable, and the average filter paper activity was 3.74 IU/mL,(2) The fermentation conditions for enzyme production of Trichoderma reesei ZE 7 were optimizid by single factor and response surface methodology. The optimal enzyme production condition were:avicel cellulose 11.15 g/L, corn steep liquor 1 g/L, ammonium sulfate 5.14 g/L,1 mol/L citrate buffer solution 75.83 mL/L. In this condition, the highest filter paper activity of the obtained Trichoderma reesei ZE 7 was 7.79 IU/mL, increasing 1.08 times than before, and the highest β-glucosidase activity was 3.45 U/mL, increasing 1.80 times than before(1.23 U/mL).(3) The fermentation conditions for enzyme production of Aspergillus niger C112 were optimizid by single factor and response surface methodology. The optimal enzyme production condition were:corn cob 27.64 g/L, rice straw 25.18 g/L, ammonium sulfate 0.76 g/L, peptone 0.76 g/L,1 mol/L citrate buffer solution 100 mL/L, culture time 6d. The β -glucosidase activity of obtained Aspergillus niger C112 was 15.67 U/mL, increasing 1.17 times than before(7.22 U/mL).(4) The environmental impact factors of cellulose enzyme activity were analyzed.The results show that the optimum reaction temperature of cellulose enzyme was 50℃, and enzyme activities were stable under the temperature, and high temperature can make the enzyme denatured inactivation. The optimum reaction pH value was 5, and enzyme activities were stable under the pH value, and overly acidic or basic can make enzyme inactivation. In addition, Metal ions like Fe2+、Mn2+、Zn2+、 Co2+ can activate the activity of cellulase, On the contrary, Fe3+、Hg2+、Al3+、Pb2+、 Ag+ can inhibit the activity of cellulase, and Na+、Mg2+、Ba2+、Cu2+、Ca2+、K+ have no significant impact on cellulase.(5) The preliminary evaluation was proceeded on the catalytic effect of cellulase. Via mixed and optimized the ratio of Trichoderma reesei enzyme liquid and Aspergillus niger enzyme liquid, and research the effect of enzyme saccharification of mixture cellulase to poplar wood,the best enzymatic hydrolysis reaction conditions was:the amount of cellulase was 15 IU/g, the ratio of β-glucosidaseand FPA was 1.5, the hydrolysis time was 48h. In this condition, the reducing sugar of the hydrolysates was 48.93 mg/mL.