Using Shuffling Technology Improve Enzyme Activity Of Cellulose Endogiucanase(Eg Ⅰ)

Cellulose is a macromolecule widely exits in the nature and it also has been widely used in real life. However, the technique and utilization efficiencies limited the value and benefit of the cellulose. In the past decades, many scholars did many researches through the technique of traditional methods, protoplast fusion and genetic engineering and so on. All the researches are focuses on attempting to convert the mass, renewable cellulose resources of the earth to replace fossil energy which cause damage to the environment of increasing depletion. But the high cost of cellulose production severely restrict the development of bioconversion of cellulose industry, the key to resolve the problem is to search new high yields strains or through innovative technology to reform the gene of cellulase, therefore, to improve the efficiency of enzymatic biotransformation. Herein, we hope relieving these conflicts on a certain extent through improve the activity of cellulase.In recent years, the development of vitro directional evolution technique can be used to obtain the proteins which possess the improved function through random mutation, recombination and directed screening of the encoding gene in the condition of3D structure information and action mechanism of the protein is unknown. This technique is available for the improvement of protein which has any appropriate screening or selection methods. In this study the mould Trichoderma viride cellulose as the female parent, we screened four high yield strains respectively. Through the definition we found most of these belong to heat resistance and isopropyl-beta-D-thiosulfate galactosidase (IPTG) induced strains, and then the endoglucanase gene was cloned from it. The results of sequence alignment shows the size of the sequence is stability and has certain diversity and the comparison of the results is51.96%, we also found directed evolution in vitro has higher filtering rate than random evolution. Meanwhile, we can confirm EGI may have higher evolutionary space.Methods:1. Extraction of Trichoderma DNA genomic, we obtained the wild EGI gene sequence through PCR cloning, transformation of competent Escherichia coli and the identification of recombinants, which was used to design the sequence primer and sequencing. Finally we got accuracy EGI sequence of99.68%similarity.2. Using shuffling reorganization technology carried out mutation and reorganization of cellulose enzyme gene through DNasel degradation, Primerless PCR, Touchdown PCR, and then the organized cellulose enzyme gene was inserted into prokaryotic expression vector pET-28a (+) to construct a recombinant mutant library. Then the library was selected through sodium carboxymethyl cellulose culture medium, we obtained higher active cellulose glucanase strains.3. Enzyme active assays were carried out to these strains through3,5-two nitro salicylic acid method (DNS), we calculated the activity of the enzyme according to the standard curve of glucose concentration and the absorption value of550nm after enzymatic reaction.4. In the conditions of a temperature gradient (20,30,40,50,60) and the setting of IPTG concentration gradient (0.01,0.05,0.1,0.2,0.5,1.0,2.0mmol/L), the induced expression conditions were optimized.Results:1. We screened Trichoderma viride from the samples of Changbai Mountain and the surrounding farmland and the genomic was extracted and identified of Trichoderma viride. Used this as a template, the endoglucanase cellulose was cloned through PCR reaction.2. This research adopted vitro directional evolution technology of DNA shuffling, we established EGI mutant library from BL21(DE3). We used Congo red staining to screen1×103clonal strains of the mutant library through the prokaryotic expression vector of pET-28a (+) and obtained four high activity mutant strains. The enzyme activity among F1, F2, F3and F4was increased to5.75,4.78,3.19and3.61times to the wild-type, respectively. The highest enzyme activity value is3. Optimization of induction conditions on F1generation strains. When the concentration of IPTG was0.011mmol/L, enzyme production capability was the best. And along the increasing of the temperature, both the reaction rate and the inactivation of enzyme are increased. In the effect of two kinds factors, when the temperature was50℃, enzyme production capability was the best.The research of bioinformatics detection and enzymatic properties found that the recombinant protein had not transmembrane region, the relative molecular mass was54517.4and the isoelectric point was9.51, with formula of C2419H3753N715O675S27. We found the similarity was51.96%contrast to the original gene, and also there had55%similarity between the recombination protein and the original protein.This study provide value technical support for adopting the method of DNA shuffling reorganization to organize cellulose endoglucanase, and also provide referenced experience for directed revolution of other proteins.