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Fermentation, Purification and Characteristics of a Novel Fibrinolytic Enzyme SPFE-Ⅲ

Author: LiuZhu
Tutor: JiangBo
School: Jiangnan University
Course: Of Food Science
Keywords: Bacillus Optimization of liquid fermentation Plasminogen SPFE-III Purification Enzymatic Properties Fibrinolytic mechanism
CLC: R363
Type: Master's thesis
Year: 2009
Downloads: 57
Quote: 1
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Abstract

Thromboembolic disease including cerebral thrombosis, myocardial infarction, venous thrombosis, clinical highest mortality diseases, and the incidence is increasing every year worldwide, so people have been in the research and development of new dissolved thrombosis drugs. From traditional Asian fermented foods - shrimp paste screened to a production the plasminogen ability strains, given by the Chinese Type Culture Collection the named Bacillus sp.nov.SK006 (CCTCC NO.: M 205 071), has been proof of the strains were able to produce four different plasminogen, one based on optimization bacteria enzyme production conditions, mainly produced by Bacillus Bacillus sp.nov.SK006 plasminogen (SPFE-Ⅲ) made in-depth research. First investigated in shake flask level medium composition and culture conditions of the Bacillus sp.nov.SK006 fermentation plasminogen, the results show that the group was divided into the strains enzyme production medium: glucose 20 g / L pancreatic peptone 30g / L, Na _{, 2} HPO ₄ · 12H ₂ O 15g the / L, NaH _{, 2} PO ₄ · 2H ₂ O 1.3g / L, MgSO ₄ · 7H ₂ O 0.5g / L, CaCl ₂ 0.1g / L; optimal culture conditions: species age 18h, inoculum size of 3%, the fermentation period 24h, initial pH 7.0, shaking speed 180r/min, fermentation temperature 37 ° C, in this condition, the fermentation liquid fibrinolytic activity (plasmin standard) up to 2.63 U / mL, 3.76 times 0.70 U / mL before optimization. The fermentation broth of Bacillus Bacillus sp.nov.SK006 by ethanol precipitation, ion exchange chromatography (DEAE-Sepharose CL-6B), after gel filtration chromatography (Superdex 75) is isolated and purified from an electrophoretically pure plasminogen SPFE - Ⅲ, a molecular weight of about 42.8 kDa, the enzyme activity of 7.1 U / mg (to plasmin as standard). SPFE-Ⅲ on fibrinogen degradation process the first degradation of the α chain, followed by the γ-chain, and the degradation of the β chain is the slowest; heated fibrin plate method plasminogen SPFE-Ⅲ the mode of action of fibrin and found that the enzyme fibrin degradation, while the lower the sensitivity to plasminogen. Secondly, the enzymatic properties of the purified plasminogen SPFE-Ⅲ: optimum reaction temperature of 30-40 ° C, optimum pH range 7.0-8.0 at 40 ℃ for 1h residual enzyme activity of about 60%, at 50 ℃ the 1h remaining enzyme activity is only about 30% of the plasminogen SPFE-Ⅲ high temperature is more sensitive. Metal ion experiments showed that: In the experiment of the selected range, except Ba ^{2 outer other metal ions the plasminogen SPFE-III fibrinolytic activity have a certain degree of inhibition, wherein Ca ^{2 , Mn ^{2 the the Fe ^{3 and Zn ^{2 strong inhibition, inhibition rate was about 50%. Inhibitor experiments show different degrees of inhibition of plasminogen SPFE-Ⅲ: protease inhibitors, EDTA and β-mercaptoethanol have a strong inhibition of plasminogen SPFE-Ⅲ. Last selected for each of the three synthetic luminescent substrate D-Val-Leu-Arg-pNA and D-Val-Leu-Lys-pNA and N-Succ-Ala-Ala-Pro-Phe-pNA with plasminogen SPFE of - III reaction. The SPFE-Ⅲ substrate N-Succ-Ala-Ala-the Pro-Phe-pNA has the strongest specificity, its amidolytic activity 75.77AU, the substrate is chymotrypsin and subtilisin optimum substrate. N-Succ-Ala-Ala-Pro-Phe-pNA Michaelis constant K _m 0.239 mmol / L, the enzyme reaction conversion number of k _cat for 475.5s < sup> -1 , show that the affinity of of plasminogen SPFE-Ⅲ this substrate better reaction speed.}}}}}

Fermentation, Purification and Characteristics of a Novel Fibrinolytic Enzyme SPFE-Ⅲ

Abstract

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