CN101235367B - Method for preparing ganoderma sinensis streptokinase and application thereof - Google Patents

Abstract

The invention relates to a process for preparing purple chi thrombolytic enzyme, which comprises solid state fermentation process of purple chi, firstly, activating bacteria, evenly coating purple chimycelium on inclined medium surface to culture, secondly, culturing seeds, utilizing soya bean to be seed culture medium, inoculating activated strain which is obtained from the step one to seed culture medium shake bottle to culture, thirdly, preparing soya bean to be solid fermentation culture medium, fourthly, fermenting to produce enzyme, inoculating bacterial liquid which is obtained from the step two to solid fermentation culture medium which is obtained from the step three to ferment and culture to obtain solid fermentation material, separation and purification process of enzyme comprises firstly, preparing crude enzyme preparation, adding solid fermentate material which is obtained from the above solid state fermentation process into sodium phosphate buffer solution homogenate, stirring to prepare and centrifuging, getting supernatant fluid to be crude enzyme solution, second, purifying enzyme, doing Sephadex G-50 pillar layer chromatography for crude enzyme solution which is obtained from step one, gathering eluent portion which has enzymatic activity, utilizing CMC ion to change pillar layer chromatography, gathering eluent portion of enzymatic activity peak, and obtaining purified purple chi thrombolytic enzyme.

Description

The preparation method of ganoderma sinensis streptokinase and application thereof

Technical field

The present invention relates to a kind of preparation method of streptokinase and the purposes of this streptokinase, relate in particular to a kind of from purple sesame, the prepare method of streptokinase and the prepared application of streptokinase in preparation treatment thrombotic disease medicine.

Background technology

Thrombotic disease is one of principal disease of harm health of people and life, at present because the blood embolism disease that a variety of causes causes, sickness rate as cerebral thrombosis, myocardial infarction, coronary heart disease, extremity vascular embolism etc. is in rising trend, wherein, the whole world has 1,500 ten thousand thrombotic disease patients approximately, its sickness rate, disability rate and mortality ratio are all very high, serious threat human beings'health and life.The one of the main reasons that causes thrombotic disease is that clinical manifestation is Acute Myocardial Infarction, cerebral thrombosis apoplexy and vascular embolization owing to form thrombus in the blood vessel.

The method of treatment thrombotic disease mainly contains following three kinds: the first, and surgical operation is promptly removed the blood vessel at thrombus or excision embolism position with physical method and is replaced with plastics tubing; Complicated condition, expense height that this method requires, and easy embolism again after the operation.The second, conservative treatment allows the patient take antithrombotics for a long time, lowers the blood coagulation tendency; This method combines as assisting therapy with other method usually.The 3rd, thrombolytic therapy is promptly injected thrombolytics and is made the blood vessel mediation.Thrombolytic therapy is one of effective way for the treatment of at present thrombotic disease.

At present, listing both at home and abroad or the thrombolysis medicine of studying are a lot, can be divided into 3 classes from raw material sources:

1, gene recombination product is as recombined streptokinase, lepirudin 023 ludon etc.;

2, Bio-engineering Products are as grape enzyme, Nattokinase etc.;

3, biochemical drug is as urokinase, fiber eliminating enzyme, fibrolase, Lumbrukinase etc.

Mainly streptokinase (SK) and urokinase (UK) in existing clinical used thrombolytics, but these two kinds of medicines all have its limitation: streptokinase is owing to easily produce streptokinase antibody in the human body, therefore there is antagonistic action, the bad grasp of consumption, and have significant side effects; Though urokinase does not have tangible antigenicity, very unstable because of urokinase, activity is difficult to control, causes profuse bleeding easily or does not have thrombolytic effect because of inactivation.Simultaneously, the complicated process of preparation of these thrombolytics costs an arm and a leg, and is difficult to wide popularization and application.

Source difficulty in view of present commercial thrombolysis medicament, factors such as complex manufacturing, the output of this series products is restricted, thereby directly have influence on treatment of diseases, for example urokinase by collect urine, accumulating, smelly supervisor disappears, environmental health is caused bad consequence, influence peripheral resident's life; Simultaneously, because the variation of weather, urine is apt to deteriorate, thereby directly influences the output of producing; And the Nattokinase that utilizes natto to produce is not high owing to the Nattokinase output of wild-type bacillus natto (Bacillus natto), and the production method of the Nattokinase that obtains thus is also not ideal enough.All all factors like this are that the output and the quality of thrombolysis preparation accepted influence, thereby have limited applying of they.

Summary of the invention

Technical problem to be solved by this invention provides a kind of new method for preparing streptokinase from purple sesame, and its raw material sources are extensive, preparation is easy.

The present invention also aims to simultaneously above-mentioned ganoderma sinensis streptokinase is applied to prepare in the medicine for the treatment of thrombotic disease.

To achieve these goals, the preparation method of ganoderma sinensis streptokinase provided by the invention is characterized in that may further comprise the steps: the separation and purification process of solid ferment process of purple sesame and enzyme, wherein:

The solid ferment process of purple sesame comprises: 1. actication of culture is uniformly coated on the slant culture primary surface with purple camphorata mycelium and cultivates; 2. seed culture is a seed culture medium with the soya bean, and the activated spawn that 1. step is obtained is seeded to the seed culture medium shake-flask culture; 3. soya bean is made the solid fermentation substratum; 4. enzymatic production, the kind daughter bacteria liquid that 2. step is obtained are seeded to that fermentation culture obtains the solid fermentation material in the solid fermentation substratum that 3. step obtain;

The separation and purification process of enzyme comprises: the preparation of i crude zyme preparation, and get the solid fermentation material that obtains in the aforementioned solid ferment process and add sodium phosphate buffer homogenate, stir preparation, centrifugal, getting supernatant liquor is crude enzyme liquid; The purifying of ii enzyme, the crude enzyme liquid that step I is obtained carries out Sephadex G-50 column chromatography and CMC ion-exchange chromatography, with ionic strength pH linear gradient elution, collects the active elutriant part of tool streptokinase, obtains the ganoderma sinensis streptokinase of purifying.

The strain activation and culture base of described purple sesame solid ferment process step in 1. is made of by weight following material: potato 180-220 weight part, Radix Dauci Sativae 180-220 weight part, agar 18-22 weight part, peptone 0.9-1.1 weight part, glucose 18-22 weight part, potassium primary phosphate 0.4-0.6 weight part, sal epsom 0.2-0.3 weight part.

The seed culture medium of described purple sesame solid ferment process step in 2. is made of by weight following material: soya bean 45-55 weight part, corn 8-12 weight part, sucrose 18-22 weight part, sal epsom 0.5-0.6 weight part, peptone 3.5-4.5 weight part, glucose 8-12 weight part, potassium primary phosphate 1.4-1.6 weight part, calcium chloride 0.2-0.3 weight part.

Described purple sesame solid ferment process step fermention medium 3. is made of by weight following material: soya bean 90-110 weight part, corn 27-33 weight part, sucrose 2.5-3 weight part, glucose 2.5-3 weight part, calcium chloride 0.5-0.6 weight part, potassium primary phosphate 0.8-1.2 weight part, sal epsom 0.5-0.6 weight part, peptone 0.3-0.4 weight part, yeast extract paste 0.5-0.6 weight part.

The solid fermentation material is 1: 3～5 with the ratio of the sodium phosphate buffer that adds homogenate among the separation and purification process steps i of described enzyme, and churning time is 2-3 hour.

The 4. middle fermentation culture conditions of described purple sesame solid ferment process step is to cultivate 13～20 days under ℃ temperature of relative humidity≤70%, 24～28.

1. middle inoculation back strain activation and culture time of described purple sesame solid ferment process step and 2. middle shake-flask culture time of step are 8～9 days.

The soya bean of adopting in the described purple sesame solid ferment process obtains through the process that bubble rises, removes the peel, drains.

The bubble condition of rising of described soya bean is: soaked 7～9 hours under 20～25 ℃ of conditions; Or under 30～35 ℃ condition, soaked 5～7 hours; Or under condition more than 35 ℃, soaked 4～6 hours.

Through zymologic property research and hemolytic experiment checking, ganoderma sinensis streptokinase of the present invention has the intensive thrombus dissolving activity, and there is serine residue in the active centre, is serine protease, belongs to the Trypsin enzyme.This enzyme is constant substantially 40 ℃ of 15min pH6-8 activity, optimum temperuture 40-45 ℃, optimal pH 6-8, this enzymic activity is suppressed by divalent manganesetion, cupric ion, and ferrous ion has activation to enzymic activity, and divalent calcium ion, magnesium ion do not have obvious influence to enzymic activity.

Description of drawings

Fig. 1 is that the present invention is through Sephadex G-50 gel-filtration elution curve;

Fig. 2 is that the present invention is through CMC ion-exchange chromatography elution curve;

Fig. 3 is separation and purification the results list of the present invention;

Fig. 4 measures the active figure of ganoderma sinensis streptokinase on the fine albumen flat board;

Fig. 5 is the thermostability tabulation of enzyme among the present invention;

Fig. 6 is the optimum temperuture graphic representation of enzyme among the present invention;

Fig. 7 is the ph stability graphic representation of enzyme among the present invention;

Fig. 8 is the optimal pH graphic representation of enzyme among the present invention;

Fig. 9-1 is restraining effect (the no substrate protection) graphic representation of Serine (Ser) to ganoderma sinensis streptokinase;

Fig. 9-2 is restraining effect (the substrate protection arranged) graphic representation of Serine (Ser) to ganoderma sinensis streptokinase;

Figure 10 is ganoderma sinensis streptokinase hemolytic experiment the results list;

Figure 11-1 is crude enzyme liquid of the present invention hemolytic experiment contrast figure under different time;

Figure 11-2 concentrates preparation liquid hemolytic experiment contrast figure under different time for the present invention;

Figure 11-3 crosses Sephadex G-50 crude enzyme liquid hemolytic experiment contrast figure under different time for the present invention;

Figure 11-4 crosses the pure enzyme liquid of CMC hemolytic experiment contrast figure under different time for the present invention;

Figure 11-5 is pH7.5, and the 0.1mol sodium phosphate buffer is hemolytic experiment contrast figure under different time.

Embodiment

The preparation method of ganoderma sinensis streptokinase of the present invention serves as to produce bacterial strain with purple sesame, is substratum with the soya bean, adopts the method for solid state fermentation, prepares streptokinase from purple sesame.

Bacterial classification of the present invention is purple sesame (Ganoderma Sinensis), by driving to such an extent that health-preserving food research institute provides (Chinese invention patent application number: 200710027289.6).

The preparation method of ganoderma sinensis streptokinase of the present invention comprises the separation and purification process of solid ferment process and the enzyme of purple sesame.

Embodiment 1

The solid ferment process of purple sesame

1. actication of culture

Strain activation and culture base (g/L): potato 180-220, Radix Dauci Sativae 180-220, agar 18-22, peptone 0.9-1.1, glucose 18-22, potassium primary phosphate 0.4-0.6, sal epsom 0.2-0.3.

Bevel behind the sterilization 30min under 121 ℃ of temperature is used inoculation hook picking 0.5～1cm then under aseptic condition ²Mycelium is inoculated in slant medium, and mycelia is uniformly coated on the slant culture primary surface, cultivates under 25 ℃ of temperature.

2. seed culture

Seed culture medium (g/L): soya bean 45-55, corn 8-12, sucrose 18-22, sal epsom 0.5-0.6, peptone 3.5-4.5, glucose 8-12, potassium primary phosphate 1.4-1.6, calcium chloride 0.2-0.3.

With soya bean, corn adds little water homogenate, is equipped with other material again and adds water to 1000mL, boils.Capacity is the bottled liquid of the triangle of 500ml, and loading amount is 300mL.

30min sterilizes under 121 ℃ of temperature.The activated spawn that 1. step obtains is used inoculation hook picking 3～4cm under aseptic condition ²Be seeded to seed culture medium, under 24～26 ℃ of temperature, the 120r/min shake-flask culture.

3. fermentation culture

Fermention medium (g): soya bean 90-110, corn 27-33, sucrose 2.5-3, glucose 2.5-3, calcium chloride 0.5-0.6, potassium primary phosphate 0.8-1.2, sal epsom 0.5-0.6, peptone 0.3-0.4, yeast extract paste 0.5-0.6.

4. enzymatic production

To be seeded to by the kind daughter bacteria liquid that 2. step obtains under aseptic condition in the solid fermentation substratum that 3. step obtain, inoculum size is 4～6%.Relative humidity≤70%, 24～28 ℃ temperature bottom fermentation is cultivated and was obtained the solid fermentation material in 13～20 days.

Preferably, 2. the time of middle shake-flask culture is 8～9 days for 1. middle inoculation back actication of culture time of step and step.

The separation and purification process of enzyme

I, crude zyme preparation preparation

Get the solid fermentation material that obtains in the aforementioned purple sesame solid ferment process with 1: 3～5 ratios add sodium phosphate buffer (pH7～8,0.1～0.2mol/L) homogenate stirs preparation 2～3 hours, the centrifugal 10～20min of 4000r/min, getting supernatant liquor is crude enzyme liquid.

The purifying of ii, enzyme

(1) Sephadex G-50 (dextrane gel) column chromatography

Take by weighing 15-18gSephadex G-50, with the distilled water swelling, boil, clean, sodium phosphate buffer (pH7～8,0.1～0.2mol/L) balances, dress post (2.6 * 30cm) with tilt-pour process.Get crude enzyme liquid 8～10ml upper prop that step I obtains, with above-mentioned buffer solution elution, flow velocity is 0.5ml/min, manages with 5ml/, collects elutriant.Detect protein and enzymic activity, collect the elutriant part that enzymic activity is arranged.Result such as Fig. 1 (be protein peak, O-O is the enzymic activity peak).

(2) CMC (Xylo-Mucine) ion-exchange chromatography

Take by weighing 18-20gCMC, the distilled water swelling is soaked 30min with 0.5mol/L HCl, drains, and distilled water is washed till neutrality; Soak 30min with 0.5mol/L NaOH then, drain, distilled water is washed till neutrality; Soak 30min with 0.5mol/LHCl again, drain, distilled water is washed till neutrality; Use the phosphoric acid buffer balance of pH5.8 at last, dress post (2.6 * 30cm).The rapid enzyme liquid that obtains of previous step is concentrated into 1/3 of original volume through the ultra-filtration method, get and concentrate enzyme liquid 8～10ml upper prop, behind the upper prop with sodium phosphate buffer ionic strength, the pH linear gradient elution (containing 0.5mol/LNaCl in the pH8.0 damping fluid) of pH5.8～8.0, flow velocity is 5mL/9min, a 5ml/ pipe, detect the protein and the enzymic activity of elutriant, collect the elutriant part of tool enzymic activity.Result such as Fig. 2.

(3) purification result

Purification result such as Fig. 3.Obtain ganoderma sinensis streptokinase through purification step, this streptokinase is purified 10 times, and vigor reclaims 47%.

Enzymic activity and protein content determination method are as follows respectively in the purification step of enzyme (1), (2):

Activity determination method:

The preparation of I, fibrin plate

Getting the 2.5g agarose adds in the 50ml physiological saline, be incubated 30min in the rearmounted 45 ℃ of water-baths of dissolving, the zymoplasm 1ml that adds 200U/ml, mixing, getting the 0.2g Parenogen is dissolved in the 50ml barbitol buffer solution (pH7～8), put 45 ℃ of water bath heat preservation 5min, get respectively 5ml thrombin solution and 5ml fibrinogen original solution mix the back rapidly to diameter be in the plate of 9cm, it is standby to put into refrigerator behind the horizontal positioned 30min.

On the flat board for preparing, make a call to three holes with glue head dropper, use micro sample adding appliance application of sample 10 μ l enzyme liquid then, put 37 ℃ of thermostat containers insulation 18h, take pictures and measure the length of solusphere two perpendicular diameter, average, calculate the solusphere area, obtain unit of enzyme activity again.Unit of enzyme activity definition: the enzyme amount with 1 square millimeter of the every increase of solusphere area under these conditions is a unit of enzyme activity.

Experimental result is with reference to Fig. 4.

Solusphere 1:d=1.2cm d=1.15cm

Solusphere 2:d=1.15cm d=1.25cm

Solusphere 3:d=1.15cm d=1.05cm

Draw: the sample enzyme is lived and is 10534u/ml

The preparation of II, urokinase typical curve

Each 10 μ l point sample of urokinase sample (100,200,300,400,500U/ml) on the fibrin plate of new preparation, are placed 10min, move, take out behind the insulation 18h, measure the diameter of solusphere, calculate each solusphere area in 37 ℃ of incubators.With the solusphere area is X-coordinate, is ordinate zou with the enzyme activity, according to the vigor of typical curve calculation sample.

The protein content determination method:

By Bradford (1976) method (being the Xylene Brilliant Cyanine G method), and be standard protein with the bovine serum albumin.Join the about 100 μ g/ml testing samples of protein concn, get a test tube adding 0.1ml distilled water and do blank, one adds the 0.1ml testing sample, every the test tube in back adds 5ml Xylene Brilliant Cyanine G G-250 and shakes up, place 5min, the 595nm wavelength is surveyed down absorbancy, checks in protein content the sample according to the optical density(OD) of sample from typical curve.

Embodiment 2

Embodiment and embodiment 1 different place is: the strain activation and culture base of described purple sesame solid ferment process step in 1. also comprises vitaminB10 .07-0.1 weight part.

Embodiment 3

The fermenting process of purple sesame

1. actication of culture

Strain activation and culture base (g/L): potato 200, Radix Dauci Sativae 200, agar 20, peptone 1, glucose 20, potassium primary phosphate 0.4, sal epsom 0.2, vitaminB10 .1.

Sterilization 30min bevel under 121 ℃ of temperature.Under aseptic condition, use inoculation hook picking 1cm ²Inoculated by hypha block is uniformly coated on media surface in slant medium with mycelium, cultivates 8 days for 25 ℃.

2. seed culture

Seed culture medium (g/L): soya bean 50, corn 10, sucrose 20, sal epsom 0.5, peptone 4, glucose 10, potassium primary phosphate 1.4, calcium chloride 0.2.

With soya bean, corn adds little water homogenate, is equipped with other material again and adds water to 1000mL and boil.Capacity is the bottled liquid of the triangle of 500ml, and loading amount is 300mL.

121 ℃ of 30min that sterilize down.Under aseptic condition, use inoculation hook picking 3～4Cm ²1. the activated spawn chopping that is obtained by step is seeded to seed culture medium, and 26 ℃, 120r/min shake-flask culture 8 days.

3. fermentation culture

Fermention medium (g): soya bean 100, corn 30, sucrose 2.5, glucose 2.5, calcium chloride 0.5, potassium primary phosphate 1, sal epsom 0.5, peptone 0.3, yeast extract paste 0.5.

4. enzymatic production

The kind daughter bacteria liquid that under aseptic condition 2. step is obtained is seeded in the solid fermentation substratum that 3. step obtain, and inoculum size is 4～6%, and cultivated 13～20 days relative humidity≤70%, 24～28 ℃.

The separation and purification process of enzyme

I, crude zyme preparation preparation

(preparation 2 hours was stirred in pH7.5,0.1mol/L) homogenate, and the centrifugal 10min of 4000r/min gets supernatant liquor, is crude enzyme liquid with 1: 3 ratio adding sodium phosphate buffer to get the solid fermentation material that obtains in the aforementioned fermentation step.

The purifying of ii, enzyme

(1) Sephadex G-50 column chromatography

Take by weighing 18g Sephadex G-50, with the distilled water swelling, boil, with tilt-pour process clean, sodium phosphate buffer (pH7.5,0.1mol/L) balance, the dress post (2.6 * 30cm), get crude enzyme liquid 8ml upper prop, with above-mentioned buffer solution elution, flow velocity is 0.5ml/min, a 5ml/ pipe, collects elutriant.Detect protein and enzymic activity in the elutriant, collect the elutriant part of tool enzymic activity.

(2) CMC ion-exchange chromatography

Take by weighing 20g CMC, the distilled water swelling is soaked 30min with 0.5mol/L HCl, drains, and distilled water is washed till neutrality; Soak 30min with 0.5mo1/L NaOH then, drain, distilled water is washed till neutrality; Soak 30min with 0.5mol/LHCl again, take out in, distilled water is washed till neutrality; Use the phosphoric acid buffer balance of pH5.8 at last, dress post (2.6 * 30cm), the rapid enzyme liquid that obtains of previous step is concentrated into 1/3 of original volume through the ultra-filtration method, get and concentrate enzyme liquid 8ml upper prop, with phosphoric acid buffer ionic strength, the pH linear gradient elution (containing 0.5mo1/L NaCl in the pH8.0 damping fluid) of pH5.8～8.0, flow velocity is 5mL/9min, a 5ml/ pipe, detects protein and enzymic activity in the elutriant behind the upper prop, collect the elutriant part of tool enzymic activity, be partially purified enzyme liquid.

Embodiment 4

Embodiment 3 and embodiment 1, embodiment 2 different places are: the soya bean in the solid ferment process obtains through the process that following bubble rises, removes the peel, drains:

A, get selected soya bean (Glycine max Merrill), tap water is cleaned;

B, under 20～25 ℃ of conditions, soaked 7～9 hours; Or under 30～35 ℃ condition, soaked 5～7 hours; Or under condition more than 35 ℃, soaked 4～6 hours;

C, peeling were pulled draining out 3～4 hours;

With the soya bean bottling that abovementioned steps obtains, the internal diameter of packing into of the soya bean behind the draining is 6cm, and capacity is in the vial of 500ml, natural elasticity, and with the double gauze that accompanies absorbent cotton, seal with kraft paper the back, tightens with cotton cord more earlier; Above-mentioned charge bottle is put in the Autoclave, 90～100min sterilizes under 121～126 ℃ of conditions again.

Embodiment 5

Embodiment 5 and embodiment 4 different places are: soya bean is after tap water is cleaned, and 25 ℃ were soaked 8 hours, and back draining 3 hours are pulled in peeling out; With the internal diameter of packing into of the soya bean behind the draining is 6cm, and capacity is in the vial of 500ml, natural elasticity, and with the double gauze that accompanies absorbent cotton, seal with kraft paper the back, tightens with cotton cord more earlier; Above-mentioned charge bottle is put in the Autoclave 121 ℃ of sterilization 90min.

Above embodiment is only for the present invention is further illustrated, and scope of the present invention is not subjected to the limitation of illustrated embodiment.

The application of ganoderma sinensis streptokinase of the present invention on the thrombolysis capsule of preparation treatment thrombotic disease:

1, the material after will fermenting grinds in 45 ℃ of following warm air dryings, and sieve (60 order), capsule is made in can.

2, the material homogenate after will fermenting, spraying drying, 130 ℃～150 ℃ of TRANSIENT HIGH TEMPERATURE, heated time 5～7 seconds, spray pressure 0.15pa, 80～90 ℃ of temperature outs.Capsule is made in can.

Below further illustrate the characteristic of ganoderma sinensis streptokinase of the present invention by zymologic property research and hemolytic experiment.

Zymologic property research

1. the stability of enzyme

(1) thermostability and optimum temperuture

The thermostability of enzyme and optimum temperuture such as Fig. 5 and Fig. 6.From Fig. 5 as seen, the better heat stability of ganoderma sinensis streptokinase, constant substantially at vigor below 40 ℃, in the time of 50 ℃, can keep 60% enzyme activity.From the optimum temperuture of the visible enzyme of Fig. 6 is 40 ℃.

(2) ph stability of enzyme and optimal pH

The ph stability of enzyme and optimal pH such as Fig. 7 and Fig. 8.From Fig. 7 as seen, enzyme is stable at pH6～8 vigor, is 6～8 from the optimal pH of the visible enzyme of Fig. 8.

2. the chemically modified of enzyme active center

Get 2 groups of enzyme liquid, every group each 7 parts, every part of 1ml, the 1st group adds substrate casein 2ml, and the 2nd group does not add substrate, 2 groups of enzyme liquid adds respectively that final concentration is 0,0.1,0.4,0.7,1.0,1.5, the PMSF of 2.0mg/ml then, respectively add 2ml37 ℃ of accurate response 15min of substrate casein in the 2nd group, add 10% trichoroacetic acid(TCA) solution 2ml, termination reaction, leave standstill the 30min after-filtration, measure active down in the 280nm wavelength.Experimental result such as Fig. 9-1,9-2, as seen from the figure, PMSF has suppressed the activity of enzyme, illustrate that Serine is the moiety of enzyme active center, and the enzymic activity of substrate has significant protective effect.There is the chemical dose relation between inhibitor concentration and enzymic activity.

3. hemolytic experiment

Get new freshly-slaughtered poultry blood coagulation, be cut into 0.3cm * 0.3cm distilled water and clean, accurately take by weighing each five equal portions of 0.5g, do not add in five test tubes, add 4ml enzyme liquid in each pipe, wherein a test tube adding sodium phosphate buffer is contrast, and sampling is on time observed and taken pictures.The results are shown in Figure 10 and Figure 11-1,11-2,11-3,11-4,11-5 and show that crude enzyme liquid and partially purified enzyme all have tangible haemolysis effect, after enzyme-added, begin haemolysis, dissolving fully to 23 hours.

Claims (9)

1. the preparation method of ganoderma sinensis streptokinase is characterized in that may further comprise the steps:

The solid ferment process of purple sesame and the separation and purification process of enzyme, wherein

The solid ferment process of purple sesame comprises: 1. actication of culture is uniformly coated on the slant culture primary surface with purple camphorata mycelium and cultivates; 2. seed culture adds water boil with soya bean homogenate, makes seed culture medium, and the activated spawn that 1. step is obtained is seeded to the seed culture medium shake-flask culture; 3. soya bean is made the solid fermentation substratum; 4. enzymatic production, the kind daughter bacteria liquid that 2. step is obtained are seeded to that fermentation culture obtains the solid fermentation material in the solid fermentation substratum that 3. step obtain;

The separation and purification process of enzyme comprises: the preparation of i crude zyme preparation, and get the solid fermentation material that obtains in the aforementioned purple sesame solid ferment process and add sodium phosphate buffer homogenate, stir preparation, centrifugal, getting supernatant liquor is crude enzyme liquid; The purifying of ii enzyme, the crude enzyme liquid that step I is obtained carries out Sephadex G-50 column chromatography and CMC ion-exchange chromatography, with ionic strength pH linear gradient elution, collects the active elutriant part of tool streptokinase, obtains the ganoderma sinensis streptokinase of purifying.

2. the preparation method of ganoderma sinensis streptokinase according to claim 1 is characterized in that:

The strain activation and culture base of described purple sesame solid ferment process step in 1. is made of by weight following material: potato 180-220 weight part, Radix Dauci Sativae 180-220 weight part, agar 18-22 weight part, peptone 0.9-1.1 weight part, glucose 18-22 weight part, potassium primary phosphate 0.4-0.6 weight part, sal epsom 0.2-0.3 weight part.

3. the preparation method of ganoderma sinensis streptokinase according to claim 2 is characterized in that:

The strain activation and culture base of described purple sesame solid ferment process step in 1. also comprises vitaminB10 .07-0.1 weight part.

4. the preparation method of ganoderma sinensis streptokinase according to claim 1 is characterized in that:

The seed culture medium of described purple sesame solid ferment process step in 2. is made of by weight following material: soya bean 45-55 weight part, corn 8-12 weight part, sucrose 18-22 weight part, sal epsom 0.5-0.6 weight part, peptone 3.5-4.5 weight part, glucose 8-12 weight part, potassium primary phosphate 1.4-1.6 weight part, calcium chloride 0.2-0.3 weight part.

5. the preparation method of ganoderma sinensis streptokinase according to claim 1 is characterized in that:

Described purple sesame solid ferment process step fermention medium 3. is made of by weight following material: soya bean 90-110 weight part, corn 27-33 weight part, sucrose 2.5-3 weight part, glucose 2.5-3 weight part, calcium chloride 0.5-0.6 weight part, potassium primary phosphate 0.8-1.2 weight part, sal epsom 0.5-0.6 weight part, peptone 0.3-0.4 weight part, yeast extract paste 0.5-0.6 weight part.

6. the preparation method of ganoderma sinensis streptokinase according to claim 1 is characterized in that:

The solid fermentation material is 1: 3～5 with the ratio of the sodium phosphate buffer that adds homogenate among the separation and purification process steps i of described enzyme, and churning time is 2-3 hour.

7. the preparation method of ganoderma sinensis streptokinase according to claim 1 is characterized in that:

1. middle inoculation back strain activation and culture time of described purple sesame solid ferment process step and 2. middle shake-flask culture time of step are 8～9 days, and the 4. middle fermentation culture conditions of step is to cultivate 13～20 days under ℃ temperature of relative humidity≤70%, 24～28.

8. the preparation method of ganoderma sinensis streptokinase according to claim 1 is characterized in that:

The soya bean of adopting in the described purple sesame solid ferment process obtains through the process that bubble rises, removes the peel, drains.

9. the preparation method of ganoderma sinensis streptokinase according to claim 8 is characterized in that:

The bubble condition of rising of described soya bean is: soaked 7～9 hours under 20～25 ℃ of conditions; Or under 30～35 ℃ condition, soaked 5～7 hours; Or under condition more than 35 ℃, soaked 4～6 hours.

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