High-yield Cellulase Strain Breeding,enzyme Production Optimization And Preliminary Application Research

Cellulose is an important and abundant renewable resources,so high-efficient development and usage of cellulose is of great importance for solving the problems of resources and environment.Cellulase is able to hydrolyze cellulose into glucose,which can be converted into varieties of beneficial substances via biological fermentation.Thus,cellulase exhibits important prospect in feed,textile,food,medicine and other fields.Cellulase is widely found in microorganisms,protozoa,and plants.At present,a large number of microorganisms having the cellulase producing capacity have been found,such as Trichoderma,Aspergillus,Penicillium and so on.In this study,we obtained microorganisms with highly cellulase producing capacity via screening and selection from nature.Then,compound mutation,medium component and fermention condition optimization were carried out to improve the cellulase producing capacity of the selected microorganism in liquid and solid fermentation conditions.Finally,the cellulase producing microorganism was initially used in the fermentation of rape straw feed.The main conclusions are as follows:?1?Cellulase producing strains were obtained from natural humus soil and rotten wood,and a cellulase high-producing strain?Cladosporium sp.B03?was obtained via screening through Congo red staining and rescreening by the measurement of enzyme activity.Its CMC enzyme activity was 2.37±0.02IU/mL and FPA enzyme activity was0.26±0.01IU/mL.The strain B03 was identified as Cladosporium by morphological and molecular biological identification,and was named as Cladosporium sp.B03.?2?Cladosporium sp.B03 was induced by ultraviolet ray?UV?and ARTP aiming at further improve its cellulase producing capability,then the mutants were screened by Congo red staining and enzyme activity.After UV mutagenesis,the CMCase activity of the optimal mutant?UV-03?increased 18.79%compared with the original strain.After ARTP mutagenesis,CMC activity and FPA activity of the optimal mutant?Cladosporium sp.AY-42?were detected to be 3.23±0.01IU/mL and 0.51±0.02IU/mL,which increased by 36.14%and 97.03%,compared with UV-03,respectively.?3?In order to improve the cellulase producing capacity of Cladosporium sp.AY-42 under liquor environment,rape straw powder and yeast powder were the optimized carbon source and nitrogen source,respectively.Then,six components of the medium were analyzed using PB design,and three significant influencing factors including rape straw powder,MgSO₄?7H₂O,and KH₂PO₄ were obtained.Response surface design analysis was carried out for those three significant factors,and the final optimal medium composition?for 1 L?was 12.5g rape straw powder,2g yeast powder,0.75g MgSO4?7H2O,0.5g NaCl,5g KH2PO4,and 0.01g FeSO4?7H2O.Under the optimal medium composition,fermentation conditions were further optimized.The most suitable situations for Cladosporium sp.AY-42 were:3 days for cultivation,5%of inoculation quantity,70mL of fluid volume,200r of speed,and 28?.In this case,the CMC enzyme activity of Cladosporium sp.AY-42 increased from 3.23±0.01IU to4.20±0.06IU,which inreased by 30.03%compared with the previous result.?4?In order to improve the cellulase production of Cladosporium sp.AY-42 under solid-state,single factor optimization was performed,and the following conditions were confirmed:rape straw powder and wheat bran was optimum carbon?the radio was 3:2?,optimum nitrogen source was?NH₄?₂SO₄?2%?.In addition,the radio of material and water was 1:2;the optimum fermentation temperature is 30^oC and the optimum fermentation time is 4 days.Under these conditions,the CMC enzyme activity of Cladosporium sp.AY-42 reached 8.17±0.05IU increasing by 57.72%.To ferment rape staw producing mycoprotein,when co-culture Cladosporium sp.AY-42 and Anqi yeast,the final true protein content was increased from 5.75%to 8.30%,and increased by44.34%compared with the initial value,18.45%and 17.41%of degradation rates of cellulose and hemicellulose,respectively.