CN103333874A - Actinomycete fibrinolytic enzyme and preparation method thereof - Google Patents

Abstract

The invention discloses actinomycete fibrinolytic enzyme and a preparation method thereof. According to the actinomycete fibrinolytic enzyme, a actinomycete strain YY21 is adopted as a strain, and culture fermentation, 30-60% W/V saturation degree ammonium sulfate salting-out, Octyl-SepharoseFF hydrophobic interaction chromatography, and Phenyl-Sepharose HP hydrophobic interaction chromatography are performed to prepare the actinomycete fibrinolytic enzyme, wherein a molecular weight of the actinomycete fibrinolytic enzyme is about 32000 daltons, and the actinomycete fibrinolytic enzyme has a fibrin dissolving function. According to the present invention, the used strain has characteristics of short enzyme production period and excellent performances, and can produce other substances having bacterial inhibition activity while producing the enzyme; the actinomycete fibrinolytic enzyme is an extracellular enzyme, such that subsequent separation and purification during preparation are easily performed; and only two step hydrophobic interaction chromatography with advantages of rapid flow rate, high capacity and good sample activity storage are required to prepare the high purity product.

Description

A kind of actinomycetes plasmin and preparation method thereof

Technical field

The present invention relates to a kind of actinomycetes plasmin, the invention still further relates to a kind of training method processed of this plasmin.

Background technology

Thromboembolism class disease serious harm human life and health, the thrombolysis method is the main means for the treatment of and prevention thrombotic diseases at present, development is efficient, fast, the novel thrombolytic drug that prevents embolism again and can reduce untoward reaction such as hemorrhage is pressing for of modern medicine.

Plasmin has fibrinolytic function, and fibrinogen is the main component that constitutes thrombus, and it is with short production cycle to utilize microbial fermentation production plasmin to have, and floor space is little, product content advantages of higher.Therefore, development microorganism plasmin efficient, high specificity is the main mode of producing novel thrombolytic drug.

In the time in nearly ten years, the applicant is exploring the application potential of microorganism in modern biological thrombolytic agent preparation always, respectively at 2006,2010 successfully training to have made number of patent application be two kinds of microorganism plasmins of 200610163497.6 and 200810137564.4, it all is fungi that these two kinds of plasmins are cultivated employed bacterial classification, the fungi fermentation meta-bolites is more, cause the later separation purification step of plasmin more, too much purification procedures can reduce the vigor of plasmin, cause the fibrinolytic effect to descend, therefore, it is less that the applicant is striving to find the fermentating metabolism product kind always in recent years, be easy to the good zymogenic bacteria kind of product separation and purification, the present invention is exactly the partial content of the research progress.

Summary of the invention

Goal of the invention of the present invention is to provide a kind of actinomycetes plasmin, and this plasmin not only has good thrombolytic performance, also intestinal bacteria is had biocidal property preferably, and relative molecular weight is little, is easy to absorption of human body.Another goal of the invention of the present invention also is to provide a kind of training method processed of this plasmin.

Product of the present invention is to be CGMCC No.5816 with the preserving number, its taxonomy called after actinomycetes Actinomycete sp.Actinomycetes YY21 bacterial strain be bacterial classification, through slant culture, dull and stereotyped cultivation, liquid fermentation and culture, separation and purification, plasmin product of the present invention is made in training at last.

Identify that through the Q-TOF2 tandem mass spectrum this actinomycetes plasmin is novel protein, recording wherein, five peptide section sequences are respectively: A-Q-S-V-P-Y-G-L-S-Q-L-K, its molecular weight are 1289.6644; V-A-V-L-D-S-G-L-D-S-S-H-P-D-L-K, its molecular weight are 1651.8243; N-S-L-E-S-T-A-T-N-L-G-N-P-A-G-A-T-Y-G-K, its molecular weight are 1964.8844; H-p-n-w-T-n-t-n-v-r, its molecular weight are 1237.5844; Y-P-S-V-L-A-V-G-A-V-D-N-G-V-S-N-K, its molecular weight are 1688.8043.

The concrete steps of product preparation of the present invention:

1, is bacterial classification with actinomycetes strain YY21, produces the actinomycetes plasmin through slant culture, liquid fermentation and culture;

2, cultural method: slant culture → seed culture → fermentation culture;

Slant medium: glucose 0.4-0.5%, yeast extract paste 0.3-0.6%, malt extract 0.5-1.5%, calcium carbonate 0.1-0.2%, agar 1.5%, pH6-7 cultivates 4-7d for 28 ℃.

Seed culture medium: glucose 0.4-0.5%, yeast extract paste 0.3-0.6%, malt extract 0.5-1.5%, calcium carbonate 0.1-0.2%, adjusting pH is 6-7, inoculation back 170-190r/min cultivates 28-32h for 28 ℃.

Fermention medium group: millet powder 6-8%, glucose 2-4%, calcium carbonate 0.1-0.3%, sodium-chlor 0.4-0.6%, peptone 0.6-0.7%, pH7.0-7.4,5% inoculum size, 150-170r/min, 22 ℃ of fermentation 90-100h.

3, the actinomycetes plasmin of slightly purifying: with liquid fermentation and culture thing 10000rpm/min, 4 ℃, centrifugal 20 min, centrifuged supernatant is crude enzyme liquid behind the grade ammonium sulfate salting-out of 30%-60%W/V saturation ratio.

4, smart purification actinomycetes plasmin: adopt modern chromatographic technique that crude enzyme liquid is carried out essence and purify,

Adopt following purification step to carry out successively:

A, Octyl-Sepharose FF hydrophobic interaction chromatography are transferred to 30% with sample salt loading degree, and with 30～0%W/V ammoniumsulphate soln gradient elution, collecting has the fibrinolytic component behind the last sample; With

B, Phenyl-Sepharose HP hydrophobic interaction chromatography are transferred to 15% with sample salt loading degree, and with 15～0%W/V ammoniumsulphate soln gradient elution, collecting has the fibrinolytic component behind the last sample.

Advantage of the present invention: experimental results show that this actinomycetes plasmin has good dissolving fibrinogen performance, and there is not tangible acute toxicity, thereby have the prospect of carrying out test, Application and Development clinically, also increased a new member for thin plasmin family.It is short that bacterial strain uses therefor of the present invention produces the enzyme cycle, produces the enzyme activity height, is the good bacterial classification of a strain production performance.Actinomycetes plasmin of the present invention is a kind of extracellular enzyme, is particularly conducive to follow-up separation and purifying in preparation.

Description of drawings

Fig. 1 measures plasmin to Cryodesiccant Human Fibrinogen's degraded picture for the SDS-PAGE method

Fig. 2 utilizes fibrin plate method to confirm the picture of product plasmin fibrinolytic effect of the present invention

Actinomycetes strain YY21 used in the present invention provides culture presevation on February 27th, 2012 to the China Committee for Culture Collection of Microorganisms common micro-organisms center that is positioned at No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City by the applicant, and preserving number is CGMCC N _O .5816, its taxonomy called after actinomycetes Actinomycete sp.

Specific embodiment

Embodiment 1

1, bacterial classification: actinomycetes strain YY21, preserving number are CGMCC N _O .5816

2, substratum and culture condition:

(1) slant medium: glucose 0.4-0.5%, yeast extract paste 0.3-0.6%, malt extract 0.5-1.5%, calcium carbonate 0.1-0.2%, agar 1.5%, pH6-7.

Slant culture condition: cultivate 4-7d for 28 ℃.

(2) seed culture medium: glucose 0.4-0.5%, yeast extract paste 0.3-0.6%, malt extract 0.5-1.5%, calcium carbonate 0.1-0.2%, adjusting pH is 6-7, the sterilization back is standby.

Culture condition: rotating speed is 170-190r/min, and 28-32h is cultivated in 28 ℃ of concussions, as liquid seeds.

(3) fermention medium: millet powder 6-8%, glucose 2-4%, calcium carbonate 0.1-0.3%, sodium-chlor 0.4-0.6%, peptone 0.6-0.7%, pH7.0-7.4,5% inoculum size.

Culture condition: 150-170r/min, 22 ℃ of fermentation 90-100h.

Embodiment 2

1, slant culture is with embodiment 1

2, seed culture is with embodiment 1

3, fermentation culture is with embodiment 1

4, the separation and purification of actinomycetes plasmin:

With liquid fermentation and culture thing 10000rpm/min, 4 ℃, centrifugal 20 min, centrifuged supernatant is crude enzyme liquid behind the grade ammonium sulfate salting-out of 30%-60%W/V saturation ratio.

A, Octyl-Sepharose FF hydrophobic interaction chromatography are transferred to 30% with sample salt loading degree, and with 30～0%W/V ammoniumsulphate soln gradient elution, collecting has the fibrinolytic component behind the last sample; With

B, Phenyl-Sepharose HP hydrophobic interaction chromatography are transferred to 15% with sample salt loading degree, and with 15～0%W/V ammoniumsulphate soln gradient elution, collecting has the fibrinolytic component behind the last sample.

Embodiment 3

1, slant culture is with embodiment 1

2, seed culture is with embodiment 1

3, fermentation culture is with embodiment 1

4, the separation purification method of actinomycetes plasmin is with embodiment 2

5, the aminoacid sequence of the plasmin after the mensuration separation and purification: Recording wherein through the Q-TOF2 tandem mass spectrum, the aminoacid sequence of five peptide sections is respectively: A-Q-S-V-P-Y-G-L-S-Q-L-K, its molecular weight are 1289.6644; V-A-V-L-D-S-G-L-D-S-S-H-P-D-L-K, its molecular weight are 1651.8243; N-S-L-E-S-T-A-T-N-L-G-N-P-A-G-A-T-Y-G-K, its molecular weight are 1964.8844; H-p-n-w-T-n-t-n-v-r, its molecular weight are 1237.5844; Y-P-S-V-L-A-V-G-A-V-D-N-G-V-S-N-K, its molecular weight are 1688.8043.

Embodiment 4

1, slant culture is with embodiment 1

2, seed culture is with embodiment 1

3, fermentation culture is with embodiment 1

4, the separation purification method of actinomycetes plasmin is with embodiment 2

5, actinomycetes plasmin fibrinolytic proves:

Adopt SDS-PAGE method and fibrin plate method to check this enzyme to the degradation property of fibrinogen.

Method

With plasmin and 37 ℃ of reactions of Cryodesiccant Human Fibrinogen's equal-volume mixing, detect actinomycetes plasmin degraded Cryodesiccant Human Fibrinogen situation with SDS--PAGE, the degraded collection of illustrative plates is seen Fig. 1 (swimming lane is followed successively by and acts on 37 ℃ of insulations 10min, 1h, 4h, 6h, 10h, 12h after Cryodesiccant Human Fibrinogen, actinomycetes plasmin mix with Fibrinogen from right to left).A α chain and B β chain are degraded fully behind the 10min, and the γ chain degradation finishes behind the 1h, and the former situation of this degraded order and Fibrinolysin (human) fibrin degradation is identical.See Fig. 1.

Fibrin plate method

Fibrin plate contains Parenogen (may 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, multimerization forms the visible fibrin gel, plasmin liquid is added to this slab gel surface, be about to fibrinolysis through this enzyme after the short period of time cultivation, form macroscopic transparent circle on the slab gel surface.See Fig. 2.

Sequence table

＜110〉Qiqihar University

＜120〉a kind of actinomycetes plasmin and preparation method thereof

<160> 5

<210> 1

<211> 12

<212> PRT

＜213〉actinomycetes (Actinomycete sp.)

<400> 1

Ala Gln Ser Val Pro Tyr Gly Leu Ser Gln Leu Lys

1 5 10

<210> 2

<211> 16

<212> PRT

＜213〉actinomycetes (Actinomycete sp.)

<400> 2

Val Ala Val Leu Asp Ser Gly Leu Asp Ser Ser His Pro Asp Leu Lys

1 5 10 15

<210> 3

<211> 20

<212> PRT

＜213〉actinomycetes (Actinomycete sp.)

<400> 3

Asn Ser Leu Glu Ser Thr Ala Thr Asn Leu Gly Asn Pro Ala Gly Ala

1 5 10 15

Thr Tyr Gly Lys

20

<210> 4

<211> 10

<212> PRT

＜213〉actinomycetes (Actinomycete sp.)

<400> 4

His Pro Asn Trp Thr Asn Thr Asn Val Arg

1 5 10

<210> 5

<211> 17

<212> PRT

＜213〉actinomycetes (Actinomycete sp.)

<400> 5

Tyr Pro Ser Val Leu Ala Val Gly Ala Val Asp Asn Gly Val Ser Asn Lys

1 5 10 15

Claims (2)

1. actinomycetes plasmin, this actinomycetes plasmin is CGMCC No.5816 with the preserving number, taxonomy called after actinomycetes Actinomycete sp.Actinomycetes strain YY21 for cultivating bacterial classification, make through fermentation, separation and purification; Recording wherein through the Q-TOF2 tandem mass spectrum, the aminoacid sequence of five peptide sections is respectively: A-Q-S-V-P-Y-G-L-S-Q-L-K, its molecular weight are 1289.6644; V-A-V-L-D-S-G-L-D-S-S-H-P-D-L-K, its molecular weight are 1651.8243; N-S-L-E-S-T-A-T-N-L-G-N-P-A-G-A-T-Y-G-K, its molecular weight are 1964.8844; H-p-n-w-T-n-t-n-v-r, its molecular weight are 1237.5844; Y-P-S-V-L-A-V-G-A-V-D-N-G-V-S-N-K, its molecular weight are 1688.8043.

2. preparation method of actinomycetes plasmin according to claim 1, be to be bacterial classification with microorganism actinomycetes strain YY21, produce plasmin through fermentation culture, with fermented product elimination thalline and solid substance, collect filtered solution, with 30%-60%W/V saturation ratio ammonium sulfate precipitation, obtain crude enzyme liquid, described crude enzyme liquid carries out purifying successively through following purification step:

A, Octyl-Sepharose FF hydrophobic interaction chromatography are transferred to 30% with sample salt loading degree, and with 30～0%W/V ammoniumsulphate soln gradient elution, collecting has the fibrinolytic component behind the last sample; With

B, Phenyl-Sepharose HP hydrophobic interaction chromatography are transferred to 15% with sample salt loading degree, and with 15～0%W/V ammoniumsulphate soln gradient elution, collecting has the fibrinolytic component behind the last sample.

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