Cloning And Expression Of Chitosanase Gene From Bacillus Subtilis And Its Enzyme Analysis

At present,the research on chitosanase mainly focused on the screening of chitosanase-producing strains,the isolation and purification of enzymes and the optimization of enzyme-producing conditions domestic and overseas.But Chitosan mostly due to the lower activity of microorganisms,low production,it is difficult to realize industrialized production.With the development of molecular biology,Chitosanase gene cloning and expression,recombinant,mutation and other technologies in the enzyme engineering applications more widely.Mainly used in enzyme gene site-directed mutagenesis,chemical modification to improve the enzyme properties,increase the enzyme activity and expression.For chitosanase enzyme of fine structure,the structure of the catalytic mechanism and no in-depth research.using genetic engineering technology research enzyme structure,build production chitosan enzyme expression vector,to obtain a higher and more stable than natural enzyme activity of industrial enzymes.Combination of microbiology and molecular biological technology,reduce the production cost of chitosan enzyme,and no pollution to the environment,can realize large-scale commercial production method,enzyme engineering is misused to solve the problem.This report with bacillus subtilis genomic DNA as a template,successfully cloned a chitosan enzyme genes.The gene sequence of size 831 bp,encoding 277 amino acids,31.3kDa theoretical molecular weight,isoelectric point theory of 9.51.gene,cloning and fixed point mutation technique,bacillus subtilis genome as a template,chitosan enzyme gene cloning in table to host to e.coli bacteria,recombinant expression to obtain chitosan enzyme bacteria.The first successful build in pHsh heat shock expression vector.Recombinant expression vector into a host bacterium escherichia coli,to induce into the positive clone of culture identification.Based on the research of enzymatic properties of chitosan enzyme.Chitosan enzyme that the optimal reaction temperature is 50 ℃,the optimum reaction pH 5.0,Thermal stability through the study on enzymology properties of chitosan enzyme performance of 35 ℃ relative enzyme activity is 100%,50 ℃ when the relative enzyme activity is half the original,good thermal stability.SDS-PAGE was carried out on the host after induction of purification,get the size of molecular weight of 28 kda protein bands.Mie equation to calculate the Kinetic parameter Km is 2.952 mg/ml min,Vmax = 0.111 mg/ml.Because of the low amount of chitosan enzyme clone of expression,in order to improve the expression quantity,most of the use of molecular biological fixed point mutation technique.This paper attempts to use of microbial cultures means to not induction and induction of microbial carbon and nitrogen source analysis and antibiotics,utilization of carbon and nitrogen source and antibiotics sensitivity analysis.Using PYMO homology modeling analysis,general molecular structure shows: 14 alpha helix and five beta chain,the enzyme has two globular upper and lower structure domain,which used for the substrate in combination with the active site of crack.Molecular folding similar to chitnanse.Study on the Utilization of Carbon and Nitrogen Sources by Recombinant Escherichia coli Using Biolog Automatic Identification System.The results showed that 26 kinds of carbon sources such as D-fructose could be used as the reducing agent,and four kinds of nitrogen sources could be used to sensitize 8 kinds of antibiotics.