| Cellulase is widely found in nature and animal bodies,and can hydrolyze ?-1,4glucopyranose to degrade cellulose into glucose.In the animal husbandry industry,the utilization of cellulose in the animal body is very low,but the cellulose degradation products has a role in promoting on the growth and development of animal body.In order to improve the utilization of cellulose in animal body,the experiment to build a strain of degrading cellulose,have reference significance to increment the source of feed raw materials.In the study,the cellulase CelKg gene was selected as the target gene,and M1,M2 were homologous arm in the genome of the wild type B.subtilis LN.The promoter P43 gene was used as a promoter to construct an integrated vector.The integrated vector was transformed into wild type B.subtilis LN,and it was hoped that a recombinant strain capable of secreting cellulase.The main research is as follows:The homologous fragments M1 and M2 were cloned by polymerase chain reaction from B.subtilis LN genome.The glucosidase gene CelKg,homologous fragment M1,M2 and strong promoter P43 were ligated to the pGEM-T vector by T4 DNA Ligase to construct the integrated vector pGEM-Kmpgmt.The integrated vector pGEM-Kmpgmt was transferred into DH5? competent cells,and recombinant E.coli Kpg was screened by blue-white screening,polymerase chain reaction(PCR)validation,and double digestion.In this study,1%,2%,3%,4% and 5% of the Tween-80,methanol and acetone were added in preparation of wild-type B.subtilis LN competent cell.The number of viable bacteria was calculated by dilution plate counting method to determine the best addition reagent and its concentration.The exogenous plasmid pGEM-kmpgt was transformed into competent cells,and the transformation results and transformation efficiency were determined by PCR using promoter gene P43 as reporter gene.The results showed that the count of viable bacterial of competent cell is 546 cell/?L and the highest transformation efficiency is 52 transformants/?g DNA when adding 4%methanol in the preparation process.The vector pGEM-kmpgmt was integrated to B.subtilis LN by double crossover homologous recombination method,based on the technology of spizizen transformation.the integration vector was successfully constructed and integrated intothe genome of B.subtilis LN by PCR detect.Congo red staining indicated that the recombinant B.subtilis could obviously degrade sodium carboxymethyl cellulose in the medium.Through the observation of the recombinant colonies and bacteria,the morphological characteristics of the recombinant and B.subtilis were similar.Using1000 times the microscope to observe,the recombinant bacteria were rod-shaped,there are spores appear,and wild-type B.subtilis LN form the same.In the experiment,cellulase activity was studied by changing the culture time of recombinant bacteria B.subtilis Kpg.Besides the optimum reaction temperature and optimum pH were determined by changing the reaction temperature and reaction pH of the cellulose.The cellulase activity in modified fermentation broth of recombinant strain was improved 115% compare with the wild type B.subtilis LN.The test results showed that the optimum temperature of cellulase reaction was 60 ° C,at this temperature cellulase activity is highest,reach 108.45 U/mL,and the most suitable pH was 6,at this pH cellulase activity is 97.72 U/m L.To study its actual ability to degrade cellulose,the recombinant strain B.subtilis Kpg was added to bran and fermented at 37 °C for 6 d.Fermentation test showed that the recombinant strain B.subtilis Kpg could decrease the content of cellulose in bran,and increase the content of true protein in bran.Through the study on enzyme activity analysis of cellulase-producing B.subtilis Kpg and effect on the cellulose content in the bran,determined the degradation ability of recombinant B.subtilis Kpg on cellulose,and the utilization value of wheat bran by B.subtilis,for the construction of engineering bacteria and application provided theoretical basis. |
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