CN101701191B

CN101701191B - N.sitophila and method for breeding plasmin by same

Info

Publication number: CN101701191B
Application number: CN2009100726309A
Authority: CN
Inventors: 刘晓兰; 郑喜群; 郑宏臣; 邓永平
Original assignee: Qiqihar University
Current assignee: Qiqihar University
Priority date: 2009-07-30
Filing date: 2009-07-30
Publication date: 2012-03-14
Anticipated expiration: 2029-07-30
Also published as: CN101701191A

Abstract

The invention discloses an N.sitophila and a method for breeding plasmin by the same. The method comprises the following steps of: producing plasmin by using a No.17 bacterial strain of N.sitophila as a strain through solid fermentation and culture in a shallow tray; extracting fermentation products with physiological saline for 6h, filtering thalli and solids, collecting the filtered solution, and salting out the filtered solution with ammonium sulfate in grades; and then, purifying by using the modern chromatographic separation technology to enable the plasmin purity to reach the chromatographically pure level. The invention has the advantages that the plasmin molecules are small, and the molecular weight is 34000+/-3000, thus the plasmin can be absorbed easily by human bodies; and the optimal action temperature is 41 DEG C which is more close to the temperature of human bodies. The activity of the plasmin produced by the invention is stable at 23 DEG C-42 DEG C, the requirement of the optimal temperature is wider, and the plasmin can be stored at room temperature. In the original scheme, only Fe<2+> and Fe<3+> have the activation on the plasmin, but in the technical scheme of the invention, Ca<2+>, Mg<2+>, Na<+>, Mn<2+>, K<+> and Zn<2+> have the activation on the plasmin of the invention in different degrees.

Description

Eat mould the reaching of arteries and veins spore well and eat the method for the mould training system plasmin of arteries and veins spore well by this

Technical field

The present invention relates to that a kind of to eat the arteries and veins spore well mould, the invention still further relates to and a kind ofly eat the method for the mould training system plasmin of arteries and veins spore well by this.

Background technology

The life and health that is threatening the mankind that thrombus disease is serious be to cause one of dead main reason of mankind nowadays, and the sickness rate of this disease is still rising at present year by year.Thrombolytic therapy is a kind of the most frequently used treatment means of current thrombus disease, and employed thrombolytics has developed into the third generation now, and exploitation thrombolysis material is the important channel of this generation thrombolytics research and development of products from the various Biological resources of nature.The applicant has screened a kind of good food arteries and veins spore trichoderma strain with good thrombolytic performance in March, 1999; This bacterial strain provides preservation on October 19th, 2006 to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) by the applicant; The taxonomy called after is eaten arteries and veins spore mould (N.sitophila) bacterial strain well No. 17, and preserving number is CGMCC No.1836.Utilize this bacterial strain the applicant to turn out a kind of plasmin, and to have submitted application number on December 14th, 2006 to State Intellectual Property Office be 200610163479.6 application for a patent for invention with good thrombolytic performance.Plasmin in this invention application has following characteristic: " through the assay determination of SDS-PAGE method and gel-filtration chromatography; be proved this plasmin enzyme molecule and be made up of two subunits; relative molecular weight is respectively 30000 and 15500, it is 7.9 ± 0.2 that IEF measures iso-electric point.This plasmin enzyme molecule has direct solution fibrin and the indirect fibrinolytic effect of plasminogen activation, the former α of fibrin degradation, β and γ chain in order.All right hydrolysis chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-pNA, the Michaelis-Menton constant of the enzymatic reaction that records is 0.24mmol/L; This plasmin human serum protein that do not degrade, the righttest action pH is 7.4, suits under Human Physiology pH, to play a role, enzyme activity remains on more than 80% behind 37 ℃ of insulation 24h in the pH4.8-9.8 scope; Optimum temperature is 50 ℃, remains on more than 75% at 32 ℃-42 ℃ insulation 4h enzyme activities; Fe ²⁺And Fe ³⁺Activity to enzyme when ionic concn is 0.5mmol/L has activation, and this fibrinolytic receives the inhibition of serpin." eat well No. 17 bacterial strains of arteries and veins spore mould (N.sitophila) be a kind of be difficult to excellent species, in this plasmin process of preparation, also be separated to a kind of physico-chemical property and the diverse plasmin of this plasmin, content of the present invention belongs to be studied the continuation of this project.

Summary of the invention

The technology that the present invention will solve provides a kind of plasmin that the fermentation of arteries and veins spore trichoderma strain produces of eating well, and this plasmin not only has good thrombolytic performance, and uses last security good, and relative molecular weight is little, is easy to absorption of human body, and preparation cost is also very cheap.Another technical problem that the present invention will solve provides a kind ofly eats the method for the mould training system of arteries and veins spore system plasmin well by this.

Of the present invention eat well the mould the applicant of being of arteries and veins spore in March, 1999 in China's northern sauce based food fermentation intermediate product sauce piece, separate, screening obtains; Be a kind of good food arteries and veins spore trichoderma strain with good thrombolytic performance; This bacterial strain provides preservation on October 19th, 2006 to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) by the applicant; The taxonomy called after is eaten arteries and veins spore mould (N.sitophila) bacterial strain well No. 17, and preserving number is CGMCC No.1836.The arteries and veins spore is mould extensively to be distributed in the nature soil, on the grass and on the food of rich in starch.The mould aerobic microbiological that belongs to of this arteries and veins spore, when oxygen was sufficient, sporulation was rapid.The righttest growth pH is 5～7.5.25～36 ℃ down growth is the fastest, the bacterium colony initial white, Cheng Cong, become very soon faint yellow, the fine hair shape.Mycelia spreads growth, and tabula is arranged.After the bacterium colony maturation, the upper strata covers into the conidium of agglomerate.Conidium sphere or almost spherical, chaining, orange red.

The training system method of product of the present invention is that to eat arteries and veins spore mould (N.sitophila) bacterial strain well with mikrobe be for No. 17 bacterial classification; Cultivate the generation plasmin through the tray solid fermentation; With fermented product with saline water lixiviate 6h; Elimination thalline and solid substance are collected filtered solution, with the ammonium sulfate classification salt of 40%～80% (W/V) saturation ratio

In technical scheme of the present invention, Ca ²⁺, Mg ²⁺, Na ⁺, Mn ²⁺, K ⁺, Zn ²⁺Plasmin of the present invention all there is activation in various degree.

Fig. 1 one-level MS spectrogram I

Fig. 2 one-level MS spectrogram II

Description of drawings

Fig. 3 one-level MS spectrogram III

Fig. 4 secondary MS spectrogram I

Fig. 5 secondary MS spectrogram II

Fig. 6 utilizes fibrin plate method to confirm that product plasmin of the present invention dissolves the picture of fine effect

Fig. 7 measures the degraded picture of product plasmin of the present invention to the Cryodesiccant Human Fibrinogen for SDS-PAGE

Bacterial classification used in the present invention is eaten arteries and veins spore mould (N.sitophila) well and to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation was provided on October 19th, 2006 by the applicant No. 17, and preserving number is CGMCC No.1836.

Embodiment 1:

1, bacterial classification: eat arteries and veins spore mould (N.sitophila) bacterial strain well No. 17

Specific embodiment

2, substratum and culture condition:

(1) slant medium (PDA inclined-plane): glucose 2%, agar 2%, with the preparation of 20% murphy juice, pH nature, 28 ℃ of constant temperature culture 3d.

(2) nysfungin resistance primary dcreening operation plate culture medium: contain 10U/ml nystatin in the slant medium, 0.02% (w/v) goes into LiClH ₂O, 0.08 (w/v) sodium deoxycholate, 25～28 ℃ of constant temperature culture 36h;

(3) multiple sieve is used the solid fermentation substratum: bean dregs: wheat bran=4: 1 (dry weight ratio), CaCl ₂0.75%, FeSO ₄7H ₂O 0.045%, pH nature, inoculum size is 5 * 10 ⁵Individual/bottle, 28～30 ℃ of fermentation 3d.

Embodiment 2

1, slant culture is with embodiment 1

2, solid fermentation is cultivated: the composition of shake flask fermentation substratum is: bean dregs: wheat bran (4: 1)+CaCl ₂0.75%+FeSO ₄7H ₂O 0.045%.Every flask culture base inoculation 3.4 * 10 ⁵Individual spore is cultivated 48h at 28 ℃ of thermostat containers, uses saline water lixiviate 4～6h then, and the shallow tray fermentation substratum is formed and the same shake-flask culture of culture condition.

3, the separation and purification of plasmin: after the lixiviate of solid fermentation product process saline water; Through 40～80% (W/V) saturation ratio grade ammonium sulfate salting-out, Octyl-SepharoseFF hydrophobic interaction chromatography, SP-SepharoseHP ion exchange chromatography, Phenyl hydrophobic interaction chromatography and RESOURCE hydrophobic interaction chromatography, collecting has the fibrinolytic composition successively.

Embodiment 3

1, slant culture is with embodiment 1

2, solid fermentation is with embodiment 2 shallow tray fermentations;

3, the separation and purification of plasmin: behind the lixiviate of the mould solid fermentation liquid process of arteries and veins spore saline water, 40～80% (W/V) grade ammonium sulfate salting-out; Adopt Octyl-Sepharose FF hydrophobic interaction chromatography, Sephadex G-25 gel permeation chromatography, SP-SepharoseHP strong cation exchange chromatography, Superdex75 gel permeation chromatography, collecting has the fibrinolytic component.

Embodiment 4

1, slant culture is with embodiment 1

2, solid fermentation is with embodiment 2 shallow tray fermentations;

3, the plasmin separation and purification is with embodiment 3

4, the character of the plasmin after the mensuration separation and purification: the mould plasmin relative molecular weight of arteries and veins spore is 34000 ± 3000; Iso-electric point 9.3 ± 0.2; Fibrin degradation α, β and γ chain in order, recording wherein, the sequence of three peptide sections is respectively: L-A-S-T-A-N-S-G-V-L-S-G-L-L-A-G-T-V-G-G-K; A-Y-T-S-K-S-S-V-P-S-S-V-G-L-A-R; L-L-D-T-G-L-N-T-A-H-S-D-F-N-R sees Fig. 1-Fig. 5.

Embodiment 5

The plasmin fibrinolytic proves:

Adopt this enzyme of fibrin plate method and electrophoretic method check to fibrinous solvability.

The A fibrin plate method

This flat band method contains Parenogen (possibly contain scleroproein in the commercially available Parenogen) and zymoplasm; Soluble fibrin is former to form fibrin monomer in thrombin action; Fibrin monomer is spontaneous to be concluded, and multimerization forms the visible fibrin gel, enzyme liquid is added to the slab gel surface after; After cultivating after a while, be about to fibrinolysis, form transparent circle on the slab gel surface.See Fig. 6.

The B electrophoretic method

With plasmin and Cryodesiccant Human Fibrinogen's equal-volume mixing 37 ℃ of effects; Detect the mould plasmin of the arteries and veins spore Cryodesiccant Human Fibrinogen that whether degrades with SDS-PAGE, the degraded collection of illustrative plates see Fig. 7 (from right to left swimming lane be followed successively by Cryodesiccant Human Fibrinogen, the mould plasmin of arteries and veins spore mixes with Fibrinogen afterwards act on 5min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h).

As shown in Figure 7: complete behind the 5min to the α chain degradation, complete behind the 1h to the β chain degradation, and when 24h, the γ chain has been degraded fully, this degraded order is similar with the situation of former each subunit of PLI's fibrin degradation.See Fig. 6, Fig. 7.

Claims

1. eat the plasmin of the method preparation of the mould training system plasmin of arteries and veins spore well, it is characterized in that: this plasmin is that to eat arteries and veins spore mould (N.sitophila) bacterial strain well with mikrobe be for No. 17 bacterial classification, cultivates the generation plasmin through the tray solid fermentation; With fermented product with saline water lixiviate 6h; Elimination thalline and solid substance are collected filtered solution, with the grade ammonium sulfate salting-out of 40%～80% (W/V) saturation ratio; Adopt modern chromatographic separation technology to carry out purifying again; Comprise and use Sephadex G-25 gel permeation chromatography that use pH6.0 phosphate buffered saline buffer wash-out, collecting has the fibrinolytic component; The SP-Sephar ion exchange chromatography uses the phosphate buffer soln pH6.0 ladder that contains 0～0.8mol/L NaCl to wash, and collecting has the fibrinolytic component, finally makes enzyme purity reach chromatographically pure; This plasmin relative molecular weight is 34000 ± 3000, iso-electric point 9.3 ± 0.2, and fibrin degradation α, β and γ chain in order, wherein the sequence of three peptide sections is respectively: L-A-S-T-A-N-S-G-V-L-S-G-L-L-A-G-T-V-G-G-K; A-Y-T-S-K-S-S-V-P-S-S-V-G-L-A-R; L-L-D-T-G-L-N-T-A-H-S-D-F-N-R; The optimum temperature of this plasmin is 41 ℃, and the stable range of plasmin enzyme activity is 23 ℃-42 ℃, Ca ²⁺, Mg ²⁺, Na ⁺, Mn ²⁺, K ⁺, Zn ²⁺This plasmin all there is activation in various degree.