CN102212513A - Proteolytic enzyme separated from cordyceps sinensis and purification method thereof - Google Patents
Proteolytic enzyme separated from cordyceps sinensis and purification method thereof Download PDFInfo
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Abstract
The invention relates to proteolytic enzyme separated from cordyceps sinensis and a purification method thereof. The proteolytic enzyme is separated from the cordyceps sinensis. In the proteolytic enzyme, the molecular weight is 43 kDa; the optimum temperature is 30 DEG C; and the optimum pH value is 9.5. The proteolytic enzyme obtained through separation and purification is brand-new proteolytic enzyme and belongs to alkaline proteolytic enzyme, wherein the optimum pH value is 9.5; the optimum temperature is 30 DEG C; and the enzyme cut basic sequence is -/-/-/K+-/-/PK/-. The proteolytic enzyme has specificity and tolerance on various protease inhibitors, metal ions, oxidants, surfactants and the like, and is applicable to labs and industry.
Description
Technical field
The present invention relates to a kind of new proteolytic ferment and purification process thereof, particularly relate to a kind of by separating proteolytic ferment and the purification process thereof that obtains in the Cordyceps fungus.
Background technology
Enzyme is the class protein that has special catalytic activity in bio-tissue or the cell.Wherein proteolytic ferment is the class of enzymes of protein hydrolysate peptide bond, bringing into play important physiological function in vivo, for example blood coagulation, fibrin dissolving, complement activation, polypeptide and proteohormone or organize the release of kassinin kinin, the fusion of cell and differentiation and macromolecular assembling or the like.Proteolytic ferment also is widely used in all respects of producing and living.Commerce or the industrial enzymes of the whole world more than 60% all is proteolytic ferment (Rao MB et al., Microbiol Mol Biol Rev1998; 62:597-634.).
At present, have about 40% in the enzyme of worldwide production, wherein come from bacterium, especially Bacillaceae greatly from microorganism.Because to proteolytic ferment, the demand that particularly has the proteolytic ferment of special property constantly increases, and makes people begin to seek new proteolysis enzyme source.Reasons such as fungi is easy to cultivate because it contains a large amount of proteolytic ferments, and the liquid nutrient medium filtration is convenient become new proteolysis enzyme source (Phadatare SU et al., the Enz Microb Technol 1993 that receives much concern; 15:72-76).
Cordyceps fungus (Cordyceps Sinensis) is the fungi of Clavicipitaceae Cordyceps, in parasitizing Chinese caterpillar fungus bat moth insect larvae bodies such as (Hepialusarm oricanus), occurring in nature forms complex body, be called as Cordyceps sinensis, have invigorate the lung and the kidney, the effect of hemostasis and phlegm, can cure mainly breathes hard breathes with cough spontaneous sweating, impotence and seminal emission, soreness of waist and knee joint, void does not wait disease again for a long time after being ill, is the traditional precious Chinese medicine of China.Cordyceps fungus is similar to the effect of Cordyceps sinensis, be rich in multiple effective bioactive ingredients such as cordycepin, cordycepic acid, Cordyceps polysaccharide (Noh EM et al., Rheumatology 2009; 48:45-48).The present Cordyceps fungus of discovering has effect (Wu JY et al., Phytomedicine2007 such as tumor suppression, immuno-stimulating, radioprotective; 14:43-49), be a kind of fungi that has very much pharmaceutical use.
Summary of the invention
The objective of the invention is to be purified into from Cordyceps fungus a kind of brand-new proteolytic ferment with special restriction enzyme site, it can be advantageously applied to laboratory and industrial production.
A kind of proteolytic ferment of the present invention obtains by separating in the Cordyceps fungus (CordycepsSinensis).Further, the molecular weight of described proteolytic ferment is 43kDa, and optimum temperuture is 30 ℃, and optimal pH is 9.5.
Another object of the present invention provides the purification process of above-mentioned proteolytic ferment:
A kind of purification process of proteolytic ferment, described purification process may further comprise the steps: (1) with the Cordyceps fungus fragmentation, and soaks with the Tris-HCl damping fluid; (2) with resulting soak solution in the described step (1) under 18000rpm centrifugal 30 minutes; (3) use HiTrap Q XL ion exchange column to carry out ion exchange chromatography resulting centrifugal supernatant in the described step (2); (4) use Superdex 200 prep grade molecular sieve chromatographies to carry out sieve chromatography resultant collection liquid in the step (3); (5) step (4) gained being collected liquid concentrates; (6) step (5) gained is collected liquid and carry out the nature gel electrophoresis, and electroelution reclaims albumen; (7) step (6) gained is collected liquid and carry out desalination, freeze-drying; (8) reverse chromatography is carried out in dissolving of step (7) gained dry powder and use SOURCE 15RPC reversed phase chromatography post.
Preferably, in described step (1), Cordyceps fungus is pulverized the back adopt mass/volume ratio to add 20mM Tris-HCl with 10 times, PH7.4,4 ℃ were stirred 24 hours.
Preferably, in described step (3), earlier soak solution is adjusted to 20mM Tris-HClPH7.4, and before ion exchange chromatography, regulates PH to 7.4 once more.
Preferably, in described step (3), with level pad 20mM Tris-HCl, PH 7.4 is eluted to baseline, adopts the method for gradient elution then earlier, and with the 20mM Tris-HCl that contains 0-1M NaCl, PH 7.4 carries out gradient elution, collects the target protein peak.
Preferably, in described step (4), the eluent that carries out sieve chromatography is the 20mM Tris-HCl that contains 500mMNaCl, and PH 7.4.
Preferably, used natural gel electrophoresis glue is 12% separation gel and 4% concentrated glue in described step (6), and electrophoretic buffer and electroelution damping fluid are Tris-Gly.
Preferably, in described step (8), earlier be eluted to baseline, adopt the method for gradient elution then, carry out gradient elution, collection target protein peak with the acetonitrile of 2%-70% with 2% acetonitrile.
Advantage of the present invention is:
The present invention at first slightly carries with ion exchange column HiTrap Q XL, removes most of foreign protein, makes the purifying of back more simple and effective.Reverse chromatography is adopted in final step, and purity of protein is increased greatly, and can not destroy protein-active.The last pure product of gained are single band through the SDS-PAGE electrophoresis detection.The protein that separation and purification of the present invention obtains is a brand-new proteolytic ferment, belong to the basic protein lytic enzyme, optimal pH 9.5,30 ℃ of optimum temperutures, enzyme cut motif-/-/-/K+-/-/PK/-, more single-minded, and multiple protein enzyme inhibitors, metal ion, oxygenant, tensio-active agent etc. are had tolerance, be fit to very much laboratory and industrial application.
Description of drawings
Fig. 1 is purified protein s DS polypropylene amine gel electrophoresis (SDS-PAGE) figure.Swimming lane 1 is that (72,55,43,34,26kDa), swimming lane 2 is the protein of purifying to protein molecular weight standard;
Fig. 2 is optimum temperuture figure;
Fig. 3 is optimal pH figure;
Fig. 4 A～4F is that directed peptide library mensuration enzyme is cut motif figure; Wherein, Fig. 4 D, 4E, 4F are that peptide library 1 enzyme is cut back N section sequencing result, and Fig. 4 A, 4B, 4C are that peptide library 2 enzymes are cut back N end sequencing result.
Embodiment
Embodiment 1: the purifying of proteolytic enzyme
1. raw material crushing and immersion
After the Cordyceps fungus pulverizing, than adding 20mMTris-HCl, transfer to PH and stir 24h for 7.4,4 ℃ with 10 times mass/volume.
2. centrifugal
4 ℃ of soak solutions, the centrifugal 30min of 18000rpm collects supernatant.Supernatant is through the 0.22mm membrane filtration.
3. ion exchange chromatography
Select HiTrap Q XL ion exchange column for use, with 10 column volume level pads (20mMTris-HCl, PH 7.4) balance.After treating the abundant balance of chromatography column,, wash chromatographic column with level pad stream behind the end of the sample, near A280 is stabilized to baseline with sample on the filtration supernatant of step 2 gained.Carry out gradient elution (0-100%) with elutriant (PH 7.4 for 1M NaCl, 20mM Tris-HCl), collect elution peak, and fully stream to be washed till baseline steady.After chromatographic column is finished using, wash 2 times of column volumes with 0.1M NaOH stream, deionized water fully stream is washed and is made column regeneration, and with 20% alcohol immersion chromatographic column prolonged preservation.
4. sieve chromatography
Select Superdex 200prep grade molecular sieve chromatography for use, with 5 column volume level pads (PH 7.4 for 500mM NaCl, 20mM Tris-HCl) balance.After treating the abundant balance of chromatography column, with 4 ℃ of the collection liquid of step 3 gained, the centrifugal 30min of 12000rpm, sample on the centrifugal supernatant.Continue to carry out stream behind the end of the sample and wash, collect chromatographic peak respectively with buffer system.After chromatographic column is finished using, with deionized water fully stream wash, and with 20% alcohol immersion chromatographic column prolonged preservation.
5. concentrate
The collection liquid of step 4 gained is through 4 ℃ of 5kDa ultrafiltration and concentration centrifuge tubes, and 12000rpm is centrifugal to be concentrated.
6. natural gel electrophoresis also reclaims
Step 5 gained concentrated solution carries out the nature gel electrophoresis at 4 ℃, being formulated as follows of running gel and damping fluid:
The running gel preparation:
The preparation of 1 * electrophoretic buffer:
Behind the last sample, fixed voltage 80V, 30min, 120V, 90min carries out electrophoresis.After the nature gel electrophoresis is finished, use mini whole glue electroelution instrument (bio-rad mini whole gel eluter) to carry out electroelution, fixed current 75mA, 20min reclaims and concentrates.
7. desalination concentrates
Select for use the desalting desalting column of GE to carry out desalination.With 5 column volumes of deionized water balance, step 6 gained is collected sample on the liquid, the deionization current are washed, and collect chromatographic peak, freeze-drying.After desalting column is finished using, with deionized water fully stream wash, and with 20% alcohol immersion chromatographic column prolonged preservation.
8. reverse chromatography
Select SOURCE 15RPC reversed phase chromatography post for use, with 10 column volumes, 2% acetonitrile balance, treat the abundant balance of chromatography column after, with step 7 gained dry powder with 2% acetonitrile dissolving and go up sample.Wash chromatographic column with 2% acetonitrile stream behind the end of the sample, near A280 is stabilized to baseline.Carry out gradient elution (0-100%) with elutriant (70% acetonitrile), collect elution peak, and fully stream to be washed till baseline steady.After chromatographic column is finished using, wash 15ml with 0.5M NaOH stream, 0.5MNaOH is to 50% Virahol gradient elution 5ml, and 50% Virahol stream is washed 15ml, and deionized water wash-out 5ml is kept at chromatography column in 20% ethanol then.
The purge process income is as follows:
The proteolytic enzyme amount of a unit is equivalent to the enzyme amount of energy hydrolysis 1 μ M/ml/mim polypeptide (ARKQYP) under the standard test condition.
The purifying end product is through SDS polypropylene amine gel electrophoresis analysis, as Fig. 1, and swimming lane 1: protein molecular weight standard, 130,95,72,55,43,34 and 26kDa; Swimming lane 2, the proteolytic enzyme of purifying.The molecular weight of proteolytic enzyme is about 43kDa.
The protein of purifying is by the order-checking of N end, tandem mass spectrum (MS-MS), and lining matter auxiliary laser is resolved with ionization-flight time mass spectrum (MALDI-TOF MS) and de novo sequencing (De novoSequencing) and analyzed, and the result is as follows:
Embodiment 3 proteolytic enzyme property testings
1. optimum temperuture
Enzyme and protein (or polypeptide A RKQYP) are in the phosphate buffered saline buffer of pH9.5, hatch 30min 0,10,20,30,40,50,60 ℃ of mixing respectively, add 0.02M NaOH termination reaction, reactant is analyzed through reversed-phase column SOURCE 15RPC (or SOURCE 5RPC), draw enzyme and cut the product amount, obtaining optimal reactive temperature at last is 30 ℃.As shown in Figure 2.
2. optimal pH
Enzyme and protein (or polypeptide A RKQYP) are at 30 ℃, mix at pH5,6,7,8,9,9.5,10,11,12 respectively and hatch 30min, add 0.02M NaOH termination reaction, reactant is analyzed through reversed-phase column SOURCE 15RPC (or SOURCE 5RPC), draw enzyme and cut the product amount, obtaining optimal pH at last is 9.5.As shown in Figure 3.
3. proteinase inhibitor is to activity influence
Enzyme respectively with proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), aprotinin (Aprotinin), Trypsin inhibitor SBTI (SBTI), pepstatin A (Pepstatin A), leupeptin (Leupeptin), Na-p-tosyl-L-arginine methyl ester hydrochloride (TAME), ethylenediamine tetraacetic acid (EDTA) (EDTA) and ethyleneglycolbis(2-aminoethylether)tetraacetic acid (EGTA) are at 30 ℃, 20mM Tris-HCl, hatch 15min under the condition of pH9.0, mix with protein (or polypeptide A RKQYP) then and hatch 30min, add 0.02M NaOH termination reaction, reactant is analyzed through reversed-phase column SOURCE 15RPC (or SOURCE 5RPC), draw the influence of proteinase inhibitor to this protease activity, as follows:
4. metal ion is to activity influence
Enzyme respectively with metallic ions Ca 2+ (CaCl2), Mn2+ (MnCl2), Mg2+ (MgCl2), Zn2+ (ZnSO4) at 30 ℃, 20mM Tris-HCl, hatch 15min under the condition of pH9.0, mix with protein (or polypeptide A RKQYP) then and hatch 30min, add 0.02M NaOH termination reaction, reactant is analyzed through reversed-phase column SOURCE 15RPC (or SOURCE 5RPC), draws the influence of metal ion to this protease activity, and is as follows:
5. inhibitor such as oxygenant and tensio-active agent is to activity influence
Enzyme gathers ethoxy ethanol (Triton X-100), sodium lauryl sulphate (SDS) and hydrogen peroxide (H2O2) at 30 ℃ with beta-mercaptoethanol, tween 80 (Tween 80), Octylphenoxy respectively, 20mM Tris-HCl, hatch 15min under the condition of pH9.0, mix with protein (or polypeptide A RKQYP) then and hatch 30min, add 0.02M NaOH termination reaction, reactant is analyzed through reversed-phase column SOURCE 15RPC (or SOURCE 5RPC), draw the influence of inhibitor to this protease activity, as follows:
1. synthetic peptide library 1
Synthetic dodecapeptide storehouse Ac-XXXXXXXXXXXX at random, N holds sealing.
2. enzyme is cut
Enzyme is carried out in step 1 a synthetic rondom polypeptide storehouse cut the enzyme tangent condition: 30 ℃, pH9.5, enzyme and peptide library are hatched 30min.
3.N end order-checking
The enzyme of step 2 gained is cut product carry out the order-checking of N end, using method is the Edman edman degradation Edman.
4. synthetic peptide library 2
According to step 3 sequencing result, design peptide library 2:MXXXQYPKHK (vitamin H), the N end does not seal, and the C end connects vitamin H.
5. enzyme is cut
Enzyme is carried out in step 4 a synthetic rondom polypeptide storehouse cut the enzyme tangent condition: 30 ℃, pH9.5, enzyme and peptide library are hatched 30min.
6.N end order-checking
The enzyme of step 5 gained is cut product carry out the order-checking of N end, using method is the Edman edman degradation Edman.
7. enzyme is cut motif and is determined
According to the measurement result of step 3 and 6, determine enzyme cut motif for-/-/-/K+-/-/PK/-.When the P1 site is a Methionin, when P3 ' site was proline(Pro) or Methionin, albumen can digestedly be opened.In addition, also there are certain selectivity in P2 and P1 ' site, and when the P2 site is arginine or Methionin, when P3 ' site was L-Ala, enzyme was cut and may be more prone to.Figure 4 shows that directed peptide library measures enzyme and cut the motif result.Wherein Fig. 4 D, 4E, 4F are that peptide library 1 enzyme is cut back N section sequencing result, and Fig. 4 A, 4B, 4C are that peptide library 2 enzymes are cut back N end sequencing result.
Claims (9)
1. proteolytic ferment is characterized in that: this proteolytic ferment obtains by separating in the Cordyceps fungus (Cordyceps Sinensis).
2. proteolytic ferment according to claim 1 is characterized in that: the molecular weight of described proteolytic ferment is 43kDa, and optimum temperuture is 30 ℃, and optimal pH is 9.5.
3. the purification process of a proteolytic ferment, it is characterized in that: described purification process may further comprise the steps:
(1), and soaks with the Tris-HCl damping fluid with the Cordyceps fungus fragmentation;
(2) with resulting soak solution in the described step (1) under 18000rpm centrifugal 30 minutes;
(3) use HiTrap Q XL ion exchange column to carry out ion exchange chromatography resulting centrifugal supernatant in the described step (2);
(4) use Superdex 200 prep grade molecular sieve chromatographies to carry out sieve chromatography resultant collection liquid in the step (3);
(5) step (4) gained being collected liquid concentrates;
(6) step (5) gained is collected liquid and carry out the nature gel electrophoresis, and electroelution reclaims albumen;
(7) step (6) gained is collected liquid and carry out desalination, freeze-drying;
(8) reverse chromatography is carried out in dissolving of step (7) gained dry powder and use SOURCE 15RPC reversed phase chromatography post.
4. the purification process of proteolytic ferment according to claim 3 is characterized in that: in described step (1), will Cordyceps fungus pulverizes the back and adopts with 10 times mass/volume than adding 20mM Tris-HCl, and PH7.4,4 ℃ were stirred 24 hours.
5. according to the purification process of claim 3 or 4 described proteolytic ferments, it is characterized in that: in described step (3), earlier soak solution is adjusted to 20mM Tris-HCl PH7.4, and before ion exchange chromatography, regulates PH to 7.4 once more.
6. the purification process of proteolytic ferment according to claim 5, it is characterized in that: in described step (3), earlier with level pad 20mM Tris-HCl, PH 7.4 is eluted to baseline, adopt the method for gradient elution then, with the 20mM Tris-HCl that contains 0-1M NaCl, PH 7.4 carries out gradient elution, collects the target protein peak.
7. the purification process of proteolytic ferment according to claim 3, it is characterized in that: in described step (4), the eluent that carries out sieve chromatography is the 20mMTris-HCl that contains 500mM NaCl, PH7.4.
8. the purification process of proteolytic ferment according to claim 3 is characterized in that: used natural gel electrophoresis glue is 12% separation gel and 4% concentrated glue in described step (6), and electrophoretic buffer and electroelution damping fluid are Tris-Gly.
9. the purification process of proteolytic ferment according to claim 3, it is characterized in that: in described step (8), be eluted to baseline with 2% acetonitrile earlier, adopt the method for gradient elution then, acetonitrile with 2%-70% carries out gradient elution, collects the target protein peak.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113999832A (en) * | 2021-11-30 | 2022-02-01 | 上海市农业科学院 | Neutral protease of straw mushroom fruiting body, extraction and purification method and application thereof |
| CN114292833A (en) * | 2021-11-30 | 2022-04-08 | 上海市农业科学院 | Method for extracting and purifying protease from straw mushroom fruiting body |
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| CN1500873A (en) * | 2002-11-15 | 2004-06-02 | 敏 洪 | Nereides protease, separating and purifying method and application thereof |
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| CN1500873A (en) * | 2002-11-15 | 2004-06-02 | 敏 洪 | Nereides protease, separating and purifying method and application thereof |
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| BO BI等: "Purification and characterisation of a novel protease from Cordyceps sinensis and determination of the cleavage site motifs using oriented peptide library mixtures", 《FOOD CHEMISTRY》, vol. 126, 31 December 2011 (2011-12-31), pages 46 - 53, XP027557467 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999832A (en) * | 2021-11-30 | 2022-02-01 | 上海市农业科学院 | Neutral protease of straw mushroom fruiting body, extraction and purification method and application thereof |
| CN114292833A (en) * | 2021-11-30 | 2022-04-08 | 上海市农业科学院 | Method for extracting and purifying protease from straw mushroom fruiting body |
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