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Study on Cloning, Expression and Evolution of Douchi Fibrinolytic Enzyme

Author: DiaoMiao
Tutor: WangShiHua
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: Tempeh fibrinolytic enzyme Bacillus subtilis Error-prone PCR Staggered extension technology
CLC: Q786
Type: Master's thesis
Year: 2009
Downloads: 78
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Abstract


Thrombosis is a serious hazard to human health diseases, heart disease, cerebrovascular disease caused by thrombosis ranking second and third in the rankings of mortality, which makes thrombolytic drugs and health food with thrombolytic function trituration rapid development. As a new microbial fibrinolytic enzyme plasmin, in theory and clinical have some value, and of positive significance to the development of China's pharmaceutical biotechnology. The laboratory has successfully isolated from tempeh screening to the strains of a thrombolytic enzyme, and named as Bacillus subtilis LD-8547. The test by PCR screened out from tempeh production tempeh fibrinolytic enzyme of Bacillus subtilis tempeh fibrinolytic enzyme-encoding gene fragment was amplified, connected to the pET-28a () vector, and then transformed into E. coli BL21 (DE3 ) host bacteria. Positive clones were induced with IPTG. The results show that: successfully constructed the recombinant bacteria E. coli BL21/pET28a-fe. Induced expression, the expression good broth sonication, after purification on a nickel column for activity detection, and the results show that protein the E. coli BL21/pET28a-fe Expression thrombolytic activity of the enzyme. E. coli BL21/pET28a-fe to SDS-PAGE analysis showed two protein bands in the E. coli BL21/pET28a-fe the size of 60 kDa and 28 kDa, respectively, compared to cells after crushing precipitate and supernatant the strips can be found, the 60 kDa protein exist only precipitate and after crushing of the cells, while not found in the supernatant, while the 28 kDa protein in the precipitate and the supernatant have. In order to improve the vitality of the recombinant thrombolytic enzymes, the trial by error-prone PCR and staggered extension technology tempeh thrombolytic genes encoding enzymes transformation. And the resultant mutated gene is connected to the pET-28a () vector, and then transformed into E. coli BL21 (DE3) host strain, the recombinant bacteria obtained in a two-step screening results filter 4 with respect to the starting strain enzyme Activity significantly improved recombinant bacteria. Be compared to the mutant enzyme and the wild enzymes, a Bacillus subtilis fermentation broth as the material, tempeh fibrinolytic enzyme isolated and purified, and the resulting wild enzyme and mutant enzymes were carried out kinetic analysis. Kinetic analysis showed that obtained four mutant enzyme The Km values ??and wild Km of the enzyme is similar, but Mut 1 Km, Km values ??of less than wild enzymes described with respect to the affinity of the wild enzymes, Mut 1 on a substrate with slightly improved. Compared with wild enzymes Kcat, Kcat value of the four mutant enzyme has increased, indicating that the catalytic ability of the substrate to obtain improved. Compared with the wild enzyme Kcat / Km Mut 1, Mut 2, Mut 3, three mutant enzyme Kcat / Km values ??have improved catalytic substrate relative to the wild enzyme a certain increase. Mut 4 Kcat / Km with respect to the wild enzymes, but decreased to some extent, mainly due to its Km is too high, may be due to a decreased affinity of the enzyme substrates.

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