| With the increasing demand of cellulase,acquisition of efficient cellulolytic microorganisms and cellulase are becoming more and more popular in the world,especially in biofuel and organic fertilizer field.The process of cellulose decomposition is mainly accomplished by cellulase secreted by microorganisms.Cellulase is a general term for a series of enzymes which can hydrolyze cellulose and its derivatives,mainly including endo-glucanase,exo-glucanase and β-glucosidase.Those cellulase usually work together to decompose cellulose.The secretion of cellulase is affected not only by the type of microorganisms,but also by the component of culture medium,such as carbon and nitrogen sources,minerals,temperature,water and pH values especially.The purpose of this study was to understand the effect of different pH values on the decomposition of lignocellulose by Trichoderma harzianum NJAU4742.Our conclusions are as follows.1.Liquid-state fermentation was carried out with rice straw as the sole carbon resource.Trichoderma harzianum NJAU4742 could grow on straw in liquid medium with initial pH of 3.0-8.0,and could also increase the initial pH of 3.0,4.0,5.0,6.0 and 7.0 by an average of one pH unit,while the treatments with initial pH of 2.0,9.0 and 10.0 remained stable throughout the culture period.The results indicated that Trichoderma harzianum NJAU4742 grew fastest at pH value of 6.0.The concentration of extracelluar enzyme achieved the highest value(36.3mg·g-1)in the treatment of 6.0,and the activity of enzymes were the strongest(87.3 U·g-1 for endoglucanase and 244.4 U·g-1 for xylanase),which also meant the best decomposition effect for rice straw.NJAU4742 could increase the pH value of the medium to its optimum growth environment with an initial pH of 3.0.Under this condition,the strain still keeps a high concentration of extracellular protein(28.9 mg·g-1)and enzyme activity(39.0 U·g-1 for exoglucanase and 45.6 U·g-1 endoglucanase),which meant that it could decompose rice straw efficiently.The analysis of NMR indicated that solid fermentation mainly consists of alkyl-C,O-alkyl-C,aromatic C,carboxyl and carbonyl C.Among them,O-alkyl-C took the greatest percent,which was related to the carbohydrates mainly.The results showed that the degradation of carbohydrates was greatly affected by pH during solid fermentation.2.Based on the results above,the quantity and abundance of cellulase secreted by strain NJAU4742 under different pH conditions as well as the expression differences of various proteins were analyzed by SWATH,and the results are listed as follows.The proteomic analysis was compared between SSF3.0 and SSF6.0 treatment,and there were 224 proteins with significantly differential expression,among which 100 proteins were significantly up-regulated and 124 were significantly down-regulated in SSF3.0.The proteomic analysis result of SSF9.0 and SSF6.0 treatment was also compared,and there were 251 proteins with significant differential expression.Among them,103 proteins were significantly up-regulated and 148 were significantly down-regulated in SSF3.0.The classification of all the identified proteins indicated that the abundance of glycoside hydrolase and protease was above 70%,and they might play a key role in the degradation of rice straw.The results of carbohydrate enzymes analysis showed that the secretion level of carbohydrate enzymes was significantly different under different pH conditions.In SSF 6.0 treatment,the cellulase and hemicellulase enzymes secreated by NJAU472 reached the highest level.However,in SSF3.0 treatment,NJAU4742 could also secret enough enzymes to decompose the rice straw efficiently.Distribution analysis of extracellular protein abundance showed that there was no significant difference in the types of extracellular proteases secreted by Trichoderma NJAU4742 under different pH conditions.However,in order to decompose rice straw and obtain necessary nutrients rapidly,there were significant differences in the proportion of protein secretion. |
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