Identification Of Psychrobacter And Reaserch Of Genetic Engineering And Bioinformatics Of Marine Low-temperature Alkaline Lipase

Marine Low-temperature Alkaline Lipase (MLAL) is a kind of extracellular enzyme from a kind of marine bacterium of EastSeaGS-1415 isolated from the deep sea silt of East China Sea. The study mainly concerns about species identification of EastSeaG5-1415, gene engineering, bioinformatics and homology of MLAL. The mostly innovative points as following.On the basis of physiology, morphology, structural characteristics and phylogenetic analysis by comparing the sequence of 16 sRNA, the strain EastSeaG5-1415 was identified as Psychrobacter g/acmc0/a,We called it P. glacincola EastSeaG5-1415Using rarefied MLAL, immunized rabbit by multi-point injection in butt and tissue injection in partes subcutanea methods and obtained the polyclone antibody. Subsequently, we purified the polyclone antibody. After detecting it's titration by ELISA, the titration is 64000. The western blotting test demonstrated that the antigen and the antibody have specific.For the preparation of gene librarie, Genomic DNAfrom P. glacincola EastSeaG5-1415 was digested partially with SauSA I ,and the Fragments from 2kb to 10kb were recovered.. The cohesive ends partially filled in by klenow fragment of DNA polymerase I (Pol I k). DNA of plasmid pUC19 was cleaved with sal I and subsequently partially filled in with Pol I k. The plasmid and the genomic DNAs were mixed and ligated. Then the recombinant plasmid transformed ?co//JM109.This method was called partially filled in technology, which has several strongpoints: (1) Eliminating the possibility of ringing self of vector. (2)The inserting fragments which were partially filled in also can't ligated each other. (3) Compared with alkaline phosphatase method, the translating rate with the partially filled in method is equal to phosphatase method.One positive clone was obtained from the libraries with the tributyrin flat and ELISA sieve method. Analyzed the sequencing result, the positive clone include the Open Read Frame of low-temperature alkaline lipase gene. The Fragment codes the enzyme of 317 amide acid, the molecule weight of which was 35001.83Dal. Through Southern hybridization, we confirmed the Fragment coming from P. glacincola EastSeaG5-1415.The sequence of MLAL was analyzed with bioinformatics software. The result of the homology analysis showed that MLAL contained a conservative catalyzing sequence which including a pentapeptide G-X1-S-X2-G. The model obtained by CPHmodels method indicates MLAL has 10 a-helixes and 7 p-sheets .The sequence of three amino acid residues, which make up active-site is Seri42-Aspi66-His292.The active-site, which is embed in the inside of lipase molecule, is covered with a lid of a-helixes. The quantity of hydrogen bond of PGLip lipase is apparently less than that of the protein Iglh as moulding board. The mol ratio of alkaline amino acid (Arg/Arg + Lys) (nearly 0.27) is much less than that of medium-temperature lipase(0.4) and of high- temperature lipase(0.48).These two characters make the structure of PGLip lipase more unwound, which help to form more flexible tertiary structure.And all of these do good to change of conformation of the lipase when it catalyses at low temperature.These result have some advance and innovation, and filled up some blankness in the domain of marine low-temperature enzyme in my country. Otherwise, this study has important theoretically and practical meaning to accelerating to exploit marine bioactivity resource of my country.