Screening Of High-yield Cellulase Strains, Identification And Cut Inside Glucanase Build High Expression Genetic Engineering Bacteria

Cellulose are widely distributed on the earth,everywhere,our country is humongous,rich in natural resources,and cellulose resources is extremely rich,but,because of its characteristics of structure stability,not easy to hydrolysis,greatly limits its use range.If they produce can make use of a large number of microorganisms,degradation of cellulose enzyme,by converting cellulose decomposing enzyme,can not only solve the problem of environmental pollution,but also can reasonable use of fiber resources.In addition,the reasonable use of fiber resources,the development of feed resources,agricultural energy production,the development of animal husbandry resources,reduce the environmental pollution is the human is of great significance.Although people in the screening of cellulose enzyme,done a lot of research work,and has obtained certain achievements,but until now,enzyme production ability of high strain due to its own limitations,is not very good use.With the development of biotechnology,cut inside the beta glucan enzyme gene cloning and expression of research more and more deeply,but,because of various conditions such as heat resistance is poor,poor stability,really into the production practice of genetic engineering bacteria few and far between.Therefore,the method of using reasonable cut inside to build a high beta glucan enzyme gene engineering bacteria and applied to the production practice,will greatly speed up the pace of fibrous raw materials in the use of industrial and agricultural production.1.This experiment adopts the Congo red staining method(Congo red staining),choose the brush,rotten wood and straw accumulation is rich in cellulose content in the soil sampling,through the solid medium carboxymethyl cellulose sodium concentration after separation of single colony,with Congo red staining method(Congo red staining)to sieve,sieve out 6 strains of hydrolysis circle is big,high strain of CMC enzyme activity,after the number,they are 16 srdna and physiological and biochemical identification,it was found that no.2 for Waxy Waxy bacillus bacillus,5 for bacillus subtilis bacillus subtilis,9 for the great help from bacillus bacillus,10 for bacillus licheniformis bacillus licheniformis,11 for Tiny coli Tiny bacteria,12 for the Green trichoderma viride Green trichoderma viride.Using candidate strains of two different fiber material bran and rice bran by solid state fermentation,DNS method to measure the OD value,calculate the enzyme activity after the fermentation,the results showed that Bacillus licheniformis Bacillus licheniformis after fermentation of enzyme activity,the strongest bran and rice bran as a substrate after fermentation enzyme activity reached 3437 u/ml and 8541 u/ml,for the production of industry and agriculture,animal husbandry are of great application prospect and economic benefit.2.In the above candidate strains,this research choose enzyme activity of the largest 10 strains Bacillus licheniformis Bacillus licheniformis as the research object,the growth conditions including fermentation temperature,time and moisture content is optimized,the results showed that: when the temperature of 30 ° C,reach 60 h of incubation time,initial moisture content was 40%,the maximum capacity of producing enzyme.In order to further the use of the strain for industrial production and feed research laid the theoretical foundation,also pointed out the direction for the future application.3.Cut inside according to the published cellulose glucanase genes(www.ncbi.nlm.nih.gov),design specific primers,with PCR amplification for a length of 732 bp of specific product,found that the gene sequence and released after sequencing cellulase gene homology of 99%,indicating success to the gene cloning.4.On both ends of the above the cellulose enzyme gene introduced ECOR1 and Hand ?enzyme loci,to design primers for PCR products,connect the cellulose enzyme gene to PET-28 acarrier,and conversion to e.coli,gene engineering bacteria.After enzyme polyacrylamide gel electrophoresis,success for the expression of 27.1 kd product size.To show that the genetic engineering bacteria successful build and expresses the expected result.By comparing with 10 strains of enzyme activity and found that the enzyme activity of recombinant bacteria have obvious enhancement.