Cloning,Sequence Analysis,Analysis,Structure Model And Function Study Of Marine Low-temperature Alkaline Protease

Marine Low-temperature Alkaline Protease (MLAP) is a kind of extracellular enzyme from a kind of marine bacterium of QD80.The study mainly concerned about gene engineering bioinformatics and homology modeling of MLAP.The gene libraries constructed by the partially filled methods. This method has several strong points: (1) Eliminating the possibility of ringing self of vector. (2)The inserting fragments can't ligate each other. (3) The translating rate with the partially filled in method is equal to phosphatase method.One positive clone was obtained from the libraries with casein flat and electrophoresis comparison method. Analyzed the sequencing result, the positive clone include the Open Read Frame of MLAP gene and upper regulatory sequence. The Fragment codes the enzyme of 459 amide acids, the molecule weight of which was 49903.92Dal. The constructed expression vector expressed in E coli.The MLAP was analyzed with bioinformatics software. It has two domains: Zincins catalytic domain and C-terminal domain of Metalloprotease. The similarity indicates that it has a "HExxHxUGUxH" motif.The MLAP was modeled with different methods, then analyses the model with software. The result indicates that the Geno3d model is good in stereochemistry and biology function. The modeling indicates MLAP has a slot to catalyze the reaction. The hydrogen bond is important to the cold-adaptation of MLAP.Studied the pL molecular weight Histidine of catalytic group of MLAP and the influence of Zn2+ Ca2+ and EDTA to enzyme activity for validation the results of bioinformatics analysis and homology modeling. The cold-adaptation is validated by the catalysis of MLAP. The results indicate that model accords with the enzyme character and function.These results have some advance and innovation, and filled up some blankness in the domain of marine low-temperature enzyme in our country. Otherwise, this study has important theoretically and practical meaning to accelerating to exploit marine bioactivity resource of our country.