Construction Of Neurospora Crassa CBH 60 Degrading Cellulose System

Straw is a renewable resources which is abundant and cheap,but its fiber refractory characteristics become the bottleneck limiting their effective utilization.Cellulose secreted by microorganism can degrade straw with low energy consumption,low cost,and less pollution production.We have successfully constructed an CBH?cellobiohydrolase?gene over-expressed strain of N.crassa.The preliminary work has proved that CBH60 strains have stronger ability to secrete cellulase.In order to improve the utilization of straw,achieve the resource utilization and solve pollution problems caused by burning straw,we use solid-state fermentation to investigate the degradation of strain CBH60 on corn stalks,wheat straw,rice straw,and rice husk.Also the degradation system would be optimized and established by considering the factors of straw pretreatment,inducers for cellulase production and cellulose hydrolysis conditions.Firstly,4 kinds of straws were meshed into different particle sizes and then pretreated with acid.The results showed that the particle size had no effect on the contents of cellulose,hemicellulose and lignin,but had greatly effect on cellulase activity when fermented by strain CBH 60.The smaller the particle size,the higher the cellulase activity.when it reached to 60 mesh,further griding would not effectively increace cellulase activity.The sulfuric acid treatment of straw would change the content ratio of cellulose,hemicellulose and lignin.Cellulose content of Corn stalks,wheat straw,rice straw,rice husk respectively increased by 21.13%,23.7%,17.8%,9.3%.It's good for the following fermentation degradation.Meanwhile the acid treatment would partially hydrolyzed the cellulose components of straws.with no more than 33%of corn stalk,22%of wheat straw,24%of rice stalks and 10%of rice shell.The optimum pretreatment is:pulverized particle size of 60 mesh,2%dilute sulfuric acidand 90?treatment for 2h.After pretreament,the response surface methodology was used to optimize the degradation condition of straws.The factors considered included carbon sources,surfactant,tempreature,solid-liquid ratio,and the pH.We obtained 3 major factors which influenced the activity of cellulase,they were solid-liquid ratio,pH and CaCl₂concentration.The optimized results indicated that the optimal degradation system of strain CBH60 was:Corn stalks as single carbon resources?or wheat straw,rice straw,rice husk?,NH₄NO₃ 10g/L,Solid-liquid ratio1:4.5,pH5.90,0.03%PEG 6000,5.06g/L CaCl₂,fermented for 7d at 30?,the cellulase activity reached up to 56.02U/mL.Based on the pretreatment and optimized degradating condition,we finally drawed out a degrading system for 4 kinds of straws,and investigated the degradation effect during the process of degradating.The result showed that after 7days of degrading,the degradation rate of corn stalks,wheat straw,rice straw,rice husk respectively reached to 44.9%,40.46%,22.07%and 16.54%.SEM electron microscope can clearly see the missing of waxy in the surface of straw after acid treatment,and cellulose fibers were broken and degraded,causing a loose strcture.Enzyme activity analysis also demonstrated the improvement of cellulase activity.