Screening For Cellulase And Hemicellulase High-yielding Strains

Cellulase produced by microorganism is wildely used in a number of ways, such aswashing industry, animal husbandry, fermentation industry and environmental protection, etc.And hemicellulase is universally applied in animal feed, food, wine and so on. But thecurrent industrial enzymes have obvious weakness of poor specificity, low productionefficiency and high cost, sudy on cellulase and hemicellulase of good specificity, highefficiency, suitable for industrialization production is still very important. Therefore breedingof high-yielding strains for cellulase and hemicellulase, and research on the fermentationconditions has important significance.Cellulase and hemicellulase-producing strains were isolated from rotten wood andfarmland composts in the countryside of Baoding of Hebei Province, and deciduous forst ofpine needles piled years collected from Chinese Academy of Qingyuan Experimental Stationof Forest Ecology and other5samples. After enrichment cultured,450strainsof producing cellulase were separated by CMC as the sole carbon source in the medium.85strains had decolorization circle through of Congo red staining and gram’s iodine staining,screened30strains of high cellulase yield through the rescreening method of determining thefilter paper activity (FPA) by DNS, including18strains of bacteria and12strains of fungi.Strain X9-202with the highest FPA activity of was obtained, of which the FPA productivitywas191.68U/mL after shaking for4d at28℃,180r/min. By enriched in hemicellulose assole carbon source in the culture medium,87strains that can use hemicellulose as a solecarbon source were obtained.11high-yielding strains of hemicellulase were selected afterfermented at28℃,180r/min, for4d, of which strain B-090has the highest hemicellulaseproductivity of145.18U/mL. The strains combination X10-279+B-212that can simultaneoushydrolyse cellulose and hemicellulose of the corn straw was screened out by antagonistic tests.The activity of cellulase producing strain X10-279was138.25U/mL, and the activity ofhemicellulase producing strain B-212was114.85U/mL. So the artificial construction ofcomplex microbial system was of practical maneuverability. After cultivatd with fermentationmedium containing acid aeration treated corn stover at28℃,180r/min for5d, and post-hydrolyzed at50for2h, the total saccharification rate of the corn stover reached35%.High cellulase producing strains were screened by mutagenesis treatment of strainX10-279using UV-Lithium chloride and ARTP, and the FPA activity of the best strain Y-006reached143.96U/mL after subcultured for5generations，which increased4.13%comparedwith the original strain. The fermentation medium components of strain X10-279wereoptimized after the pH of medium, fermentation time and culture temperature were optimizedby single factors experiment. PB experiment was carried out and wheat bran, CMC-Na andMgSO47H2O were screened as the major factors influenced cellulase production. Andthe most appropriate concentrations of three factors bran, CMC-Na, MgSO47H2O wereclose to the area of1.05%,1.7%and0.0685%through steepest ascent experiment. Furtherly,Box-Behnken method was used in response surface experimental design, and experimentaldata were analysised by using Minitab software. As a resut, the response surface mode wasconstructed, and the optimal culture conditions for cellulase production of strain X10-279wascalculated as following：wheat bran1.24%, CMC-Na1.73%, MgSO47H2O0.0678%,beef extract0.3%, peptone0.6%,(NH4)2SO40.5%, KH2PO40.2%, CaCl20.04%,33℃, pH7.0, cultured for5d. The verification test got a productivity of180.34U/mL, which is veryclose to the predictive value and can confirm the model well In general, the cellulaseproduction of X10-279increased30.44%than the original level after fermentationoptimization.Based on the morphological characteristics and phlogenetic analysis result of18S-ITS1-5.8S-ITS2-28S rDNA D1/D2sequence, cellulase producing strains X10-279,X10-280, X10-281and hemicellulase producing strain B-212were premliminary identified astaxa most closely related to Penicillum oxalicum CCF2315. Hemicellulase producing strainB-090was identified as taxa most closely related to Penicillium chrysogenum NRRL820.Cellulase producing strains X9-202was identified as taxa most closely related to Aspergillufumigatus NRRL163.