CN102776160A - Method for separating and purifying microbial transglutaminase - Google Patents
Method for separating and purifying microbial transglutaminase Download PDFInfo
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- CN102776160A CN102776160A CN2012102767057A CN201210276705A CN102776160A CN 102776160 A CN102776160 A CN 102776160A CN 2012102767057 A CN2012102767057 A CN 2012102767057A CN 201210276705 A CN201210276705 A CN 201210276705A CN 102776160 A CN102776160 A CN 102776160A
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Abstract
The invention discloses a method for separating and purifying microbial transglutaminase. The method comprisies the following steps: adding 50-200mg/L dispase in streptomycete fermented liquid, subjecting to ethanol depositing, anion exchange chromatography and cation exchange chromatography, de-salting, concentrating, filtering, and freezing to be dry, so as to obtain the microbial transglutaminase, wherein the purity of the microbial transglutaminase is higher than 95% and the specific activity is 20000-50000U/g. The method disclosed by the invention is simple and is capable of obtaining high-purity transglutaminase from microbial fermentation product with the yield of higher than 70%.
Description
Technical field
The present invention relates to the separation purification method of biological technical field, be specifically related to a kind of separation purification method of glutamine of microbe transaminase.
Background technology
(Transglutaminase is called for short TG, EC2.3.2.13 to Transglutaminase EC2.3.2.13; Claim protein-L-glutamic acid-gamma-glutamyl amine transaminase again) be a kind of enzyme of catalyzing acyl shift reaction.TG can be used as the epsilon-amino effect of lysine residue in acyl acceptor and the protein; Form ε-(gamma-glutamyl) Lys key; Thereby catalytic proteins intramolecularly, intermolecular generation are crosslinked, the hydrolysis of glutamy amido in connection between protein and the amino acid and the protein molecule, and then change protein function character; Improve proteinic nutritive value, be widely used in fields such as food, bio-pharmaceuticals, weavings.Transglutaminase EC2.3.2.13 extensively is present in animal, plant and the mikrobe; Comprise Transglutaminase EC2.3.2.13 (the Guinea-pig Transglutaminase that is derived from animal; GTG) and be derived from mikrobe Transglutaminase EC2.3.2.13 (Microbial Transglutaminase, MTG).GTG mainly extracts from animal tissues, but because its separation and purification process is complicated, the source is less, cost an arm and a leg, greatly limits its application on industrial production.
Compare with GTG, MTG has Ca on using
2+Dependent/non-dependent, to the stability of heat, pH and be prone to advantage such as storage property.Research shows that MTG has and GTG similarly effect and function, can catalysis type i collagen albumen takes place crosslinkedly such as MTG, forms resistant to elevated temperatures glue (Nomura Y, et.al; Biosci Biotechnol Biochem. 2001 Apr; 65 (4): 982-5); Can cross-linked gelatin, form good perforated web and be used for hemostasis and wound bonding (Liu Y, et.al, J Biomed Mater Res B Appl Biomater.2009 Oct; 91 (1): 5-16; Chinese patent: 200780051215.4,200980131973.6; USP: 8133484; European patent: WO2012017415, EP2303344); The cross-linking agent that MTG and gelatin effect form is difficult for receiving heat of solution, has thermostability (Chen T, Embree HD, et.al preferably; Biomaterials. 2003 Aug; 24 (17): 2831-41, USP: 5,834,232) etc., so the glutamine of microbe transaminase linking agent that can be used as potential hemostatic material in medical use is used for surgical operations such as hemostasis, wound be bonding.But MTG in the market is mainly used in food processing field, and adopts traditional separation purifying technique preparation, and the yield of enzyme, purity and not higher than vigor still contain multiple foreign protein in the preparation process, are difficult to satisfy the requirement as medical material.
Summary of the invention
The present invention overcomes the above-mentioned defective of prior art; Innovation has proposed a kind of separation purification method of glutamine of microbe transaminase; Promptly utilize Dispase II at ambient temperature the pro-MTG in the streptomycete fermentation liquid (precursor forms of MTG) to be hydrolyzed and obtain having active MTG after handling, then, through ethanol sedimentation; In conjunction with the yin, yang ion exchange chromatography MTG is carried out purifying; When improving, obtained high purity (> 95%) and specific activity of enzyme (> 20000U/g) MTG, and improved active MTG yield.The mikrobe glutamine transaminase that the inventive method separation and purification obtains meets the requirement of medical material, can be used in hemostatic material in medical use and tackiness agent.
The present invention aims to provide a kind of separation purification method of glutamine of microbe transaminase, from the streptomycete mutant strain fermented liquid of luxuriant source, obtains highly purified MTG, as hemostatic material in medical use and binder materials.Among the present invention; The Transglutaminase EC2.3.2.13 (being MTG) that is derived from mikrobe belongs to extracellular enzyme, is mainly derived from the streptomycete in the mikrobe, like streptomyces hygroscopicus, luxuriant source streptomycete etc.; Its molecular weight size is about 38KD, and the active site of enzyme includes a free Cys sulfydryl.
The separation purification method of a kind of glutamine of microbe transaminase that the present invention proposes comprises, the Dispase II that in streptomycete fermentation liquid, adds 50-200mg/L is handled; Then, through ethanol sedimentation, anion-exchange chromatography, cation-exchange chromatography, again through desalination, concentrate, obtain said glutamine of microbe transaminase after the filtration, freeze-drying; Wherein, the purity of said glutamine of microbe transaminase is 20000-50000U/g greater than 95% than vigor.
Wherein, said streptomycete fermentation liquid is luxuriant source streptomycete STK4 mutant strain fermented liquid; The 10th amino acids of said mikrobe glutamine transaminase is Serine or Threonine; The 241st amino acids is L-Ala or glycocoll; The 284th amino acids is Xie Ansuan or tyrosine.
Among the present invention, " streptomycete fermentation liquid " is meant that luxuriant source streptomycete STK4 bacterial strain cultivates the fermented liquid after 48-72 hour in fermention medium.
Among the present invention, " luxuriant source streptomycete STK4 mutant strain " is meant the MTG mutant strain that luxuriant source streptomycete STK4 wild strain produces after uv irradiation mutagenesis.
Among the present invention, in the said anion-exchange chromatography step, sample-loading buffer is the Tris-HCl damping fluid of pH7.0-7.5; Elutriant is the NaCl solution of 0-2mol/L.
Among the present invention, in the said cation-exchange chromatography step, sample-loading buffer is the sodium hydrogen phosphate damping fluid of pH 5.5-6.5; Elutriant is pH 7.0, the NaCl solution of 0-1mol/L.
Among the present invention, the fiber filter film with 0.45 μ m-0.22 μ m filters before the ion exchange column on said sample.
Among the present invention, filter with nanofiltration membrane before the said sample freeze-drying.
Among the present invention, the yield of said glutamine of microbe transaminase is greater than 70%.
Among the present invention, the level of endotoxin < 4EU/>g of said glutamine of microbe transaminase.
Among the present invention, said glutamine of microbe transaminase can be used as wound hemostasis and medical jointing material.
Utilize the separation purification method of mikrobe paddy amine acyl transaminase of the present invention (MTG); Can from microbial fermentation product, obtain highly purified Transglutaminase EC2.3.2.13; The proteic purity of glutamine of microbe transaminase that obtains behind the purifying reaches 20000-50000U/g greater than 95% than vigor, and the yield of enzyme is more than 70%; Can either satisfy the requirement of hemostatic material in medical use and tackiness agent, realize High-efficient Production again.
Description of drawings
Cation-exchange chromatography color atlas in Fig. 1, the separation purification method of the present invention.Wherein, the foreign protein peak of the phosphate sodium dihydrogen buffer solution wash-out of peak 1 expression pH7.0; Peak 2 expression pH7.0 contain the Transglutaminase EC2.3.2.13 target protein peak of the phosphate sodium dihydrogen buffer solution wash-out of 200mM NaCl.
The electrophorogram of the Transglutaminase EC2.3.2.13 in Fig. 2, the separation and purification process of the present invention.Wherein, Marker: protein molecule marker;
Band 1: fermented liquid electrophorogram; Band 2: ethanol sedimentation product electrophorogram; Band 3: penetrate the liquid electrophorogram behind the anion-exchange chromatography; Band 4; MTG electrophorogram behind the cation-exchange chromatography.
Sudden change MTG that separation and purification of the present invention obtains under Fig. 3, the differing temps and the enzyme of wild MTG are lived relatively.
Embodiment
In conjunction with following specific embodiment and accompanying drawing, the present invention is done further detailed description, protection content of the present invention is not limited to following examples.Under spirit that does not deviate from inventive concept and scope, variation and advantage that those skilled in the art can expect all are included in the utility model, and are protection domain with the appending claims.The process of embodiment of the present invention, condition, reagent, experimental technique etc. except that the following content of mentioning specially, are the universal knowledege and the common practise of this area, and the present invention does not have special limiting content.As according to people such as Sambrook, molecular cloning, laboratory manual (New York:Cold Spring Harbor Laboratory Press, the 1989) content of putting down in writing, or according to the suggestion condition of manufacturer.
Separation purification method of the present invention is handled the MTG in the fermented liquid through the process of the so special setting of Dispase II processing → ethanol sedimentation → anion-exchange chromatography → cation-exchange chromatography and is carried out separation and purification; Obtain the object of the invention product glutamine of microbe transaminase, i.e. MTG.
Among the present invention, at first add an amount of Dispase II fermented liquid is handled, Dispase II catalysis pro-MTG hydrolysis formation under given conditions has active MTG.Handle to make the pro-MTG in the fermented liquid be converted into MTG through Dispase II, thereby what farthest guarantee to obtain all is to have active MTG (shown in the table 1), has increased the yield of active MTG.Then, with ethanol sedimentation the MTG in the fermented liquid is carried out preliminary purification.Through regulating alcohol concn and the pH in the fermented liquid, make it reach the threshold value that MTG albumen is separated out, through centrifugal it is carried out initial gross separation from fermented liquid; Make the MTG purity of protein by bringing up to more than 30%; As shown in Figure 2, improved 7 times than vigor, as shown in table 2.Then, with anion-exchange chromatography and cation-exchange chromatography the purpose product is carried out polishing purification.That is, earlier remove iso-electric point less than 7.0 Partial Protein with anion-exchange chromatography, the purity of Transglutaminase EC2.3.2.13 is brought up to more than 70%, MTG improves more than 4 times than vigor; Then, with cation-exchange chromatography MTG is further purified again, the purity of Transglutaminase EC2.3.2.13 is brought up to more than 95%, MTG improves more than 1.4 times, finally than vigor than vigor>25000U/g, shown in Fig. 2, table 2, table 3.
Protein is polyvalent Amphiphatic high polymer ionogen, and therefore different proteins, can use ion exchange chromatography purifying MTG owing to its amino acid composition difference has different iso-electric point PI.As environment pH during greater than PI, the protein band negative electricity can be adsorbed by anionite; As pH during less than PI, protein band positive electricity can be adsorbed by cationite.Ionic group or ionogen are contained in the ionite surface, adsorb the ion that has opposite charges through electrostatic attraction, and adsorbent can be through changing the method wash-out of PH or raising ionic strength.Among the present invention, the iso-electric point of MTG is about 8.9, and is positively charged under the condition of pH5.5-7.5, can be adsorbed by cationite; Can this albumen and other albumen be separated according to this, reach the purpose of separation and purification.
Separation purification method of the present invention comprises following concrete steps:
The processing of bacterial strain fermentation liquor:
Luxuriant source streptomycete mutant strain through fermentation after (4 ℃ of thalline → filtered solution low-temperature centrifugation 15min are removed in press filtration; 12000rmp) add Dispase II by 50-200mg Dispase II/L and handle, obtain the fermented liquid supernatant after Dispase II is handled to remove supernatant behind residual bacterial chip and the part high molecular weight protein → centrifugal.Dispase II is handled the yield that helps to improve active MTG.Preferably, the proportional range of bacterial strain fermentation liquor and Dispase II is 50-100mg enzyme/1L fermented liquid.Handling with Dispase II is at room temperature to stir 30-60min.
The preliminary purification of Transglutaminase EC2.3.2.13:
The absolute ethyl alcohol that adds-20 ℃ of precoolings in the fermented liquid of luxuriant source streptomycete mutant strain after above-mentioned Dispase II is handled; Make that the ethanol final concentration is 20% in the fermented liquid, then, the regulator solution pH value is between the 8.5-9.0; Mild stirring 60-120min under 4 ℃ of conditions; Warp is 4 ℃ again, centrifugal 30min under the 8000rmp condition, and the deposition that obtains is the glutamine of microbe transaminase of preliminary purification.Utilize isoelectric precipitation, organic solvent deposit can remove the part foreign protein in the fermented liquid, improve MTG purity to a certain extent, and Ethanol Treatment have certain deactivation to pathogenic micro-organism and endogenous toxic material.
The pre-treatment of ion exchange column:
For preventing the pollution of pyrogen material, the yin, yang ion exchange column is handled with the NaOH of 0.5N before use.3-5 times of column volume of NaOH solution flushing with 0.5N left standstill 3 hours; Use the flushing of water for injection (WFI) or sample-loading buffer to be neutrality to penetrating liquid more than the column volume for 10 times then, subsequent use.
The preparation of all article solution on the anion-exchange chromatography:
The Tris-HCl damping fluid that will the thick enzyme prepn of MTG behind ethanol sedimentation be dissolved in pH7.0-7.5, preferably, with the Tris-HCl damping fluid of pH 7.0.Slowly stir 30-60min down at 4 ℃, the thick enzyme prepn of MTG is fully dissolved.To dissolve completely the thick enzyme prepn of MTG at 4 ℃, centrifugal 5min under the 8000rmp condition, thus remove oarse-grained insolubles or impurity in the solution.
Further, before carrying out anion-exchange chromatography, filter with 0.45-0.22 μ m tunica fibrosa.Preferably, with 0.22 μ m tunica fibrosa.Thereby further remove PM for particulate matter more tiny in the sample or insolubles,, also reduced mikrobe that maybe be residual in the sample solution simultaneously to greatest extent in order to avoid cause the obstruction and the pollution of ion exchange column.
Anion-exchange chromatography:
With anion-exchange column HiPrep DEAE FF on the MTG sample after the above-mentioned processing.Exchange column is with 4 ℃, and the Tris-HCl damping fluid of pH 7.0 carries out balance, the about 8-10 of balance times column volume.Begin to go up appearance behind the good pillar of balance, last appearance flow rate control is at 4-10ml/min, and pressure is less than 0.3MPa.Preferably, last appearance flow velocity is 4ml/min.Low flow velocity can make albumen have adequate time and exchanger to combine.Behind the end of the sample,, use the NaCl solution (urea that contains 60mM) of 0-2 mol/L that pillar is carried out wash-out then with the level pad flushing cylinder of 3 times of column volumes, preferably, with the NaCl eluant solution of 2mol/L.Can also use the NaCl eluant solution of 1mol/L.Collect respectively and penetrate liquid and elutriant, SDS-PAGE identifies the distribution situation of Transglutaminase EC2.3.2.13.In the buffer system of PH7.0, MTG is positively charged, can not be adsorbed by the DEAE cylinder; Iso-electric point then is adsorbed on the DEAE colloid surface less than 7.0 albumen, thereby this part albumen and MTG are separated.
Cation-exchange chromatography:
With the above-mentioned MTG of containing penetrate liquid with 4 ℃, the phosphate sodium dihydrogen buffer solution of pH6.0-7.0 is replaced, preferably, with the phosphate sodium dihydrogen buffer solution of pH6.0.Then, filter with 0.22 μ m filter.Then, last HiPrep SP XL post, last appearance flow rate control is preferably appearance flow velocity 2ml/min at 2-5ml/min.Adopt low flow velocity can guarantee that the yellow bright basic ionic group on MTG and the HiPrep SP XL post fully combines.After treating end of the sample, with the level pad flushing cylinder of 3 times of column volumes, use pH7.0 then, the elutriant that contains 0-1mol/L NaCl carries out wash-out, collects the elutriant with activeconstituents.Through the pH and the ionic strength of regulating elutriant target protein MTG and foreign protein are separated then.
Desalination, concentrated:
Use the aperture to concentrate the active elutriant of glutamine transaminage that contains of above-mentioned acquisition, be concentrated into 1/10 of initial volume, use then greater than the 100mM sodium citrate buffer solution of 10 times of volume pH7.0 and replace as the ultra-filtration membrane of 10KD.Ratio according to 20 μ g maltodextrin/1U enzymes adds maltodextrin, and fully usefulness is filtered with the filter of 0.45 and 0.22 μ m respectively behind the mixing.
Freeze-drying:
The sample that above-mentioned filtration is good filters with nanofiltration membrane, installs in the penicillium mould bottle of 10ml according to every bottle of branch of 2ml then, carries out vacuum-freeze-dry and handles.MTG after the lyophilize redissolves, detects enzyme activity.The pure article of purpose product glutamine transaminage that obtain, its purity is 20000-50000U/g greater than 95% than vigor.
The fermentation of embodiment 1 Transglutaminase EC2.3.2.13 enzyme
The production bacterial strain of MTG is luxuriant source streptomycete STK4 (Streptomyces mobaraensis) mutant strain in the present embodiment, and this mutant strain is produced through uv irradiation mutagenesis by the STK4 wild strain, and this mutant strain excretory MTG undergos mutation.Amino acid on the 10th, 241 and 284 of two mutants MTG is undergone mutation, and the 10th amino acids wherein is Serine or Threonine; The 241st amino acids is L-Ala or glycocoll; The 284th amino acids is Xie Ansuan or tyrosine.And the amino acid on the 10th, 241,284 of wild-type MTG is respectively L-Ala, proline(Pro), Serine.MTG after the present invention's sudden change has the wild MTG height of specific activity at 25 ℃ and 37 ℃.
At first, bacterial classification inoculation was cultivated 5-7 days in 30 ℃ to the slant medium, collected all microbial inoculants in fermentation culture, 150rpm cultivated 24 hours, and 250-300rpm cultivated 24-48 hour then, and it is subsequent use to collect fermented liquid.
The slant culture based formulas: agar 20g, starch 20g, KNO3 1g, MgSO47H2O 0.5g, K2HPO43H2O 0.5g, NaCl 0.5g, FeSO47H2O 0.01g is dissolved in the 1L water.Fermentative medium formula: peptone 25g, yeast extract paste 6g, glycerine 20ml, MgSO47H2O 2g, K2HPO43H2O 10g is dissolved in the 1L water.
Pressing Grossowicz colorimetric method for determining enzyme and live, serves as the effect substrate with NA-CBZ-GLN-GLY and azanol, makes typical curve with L-L-glutamic acid-γ-single hydroximic acid.To act on substrate (NA-CBZ-GLN-GLY and azanol) and put into water preheating 10-15min with the 40ul testing sample, and in sample, add the 100ul substrate, effect 10min adds 40ul stop buffer (trichoroacetic acid(TCA) and iron trichloride) termination reaction then.Take out sample, the centrifugal 5-10min of 8000 * g lives with protein nucleic acid quantitative determination instrument colorimetric method for determining enzyme under 562nm.The enzyme activity unit definition: this enzyme is under 37 ℃ of conditions, and the 1min catalytic substrate generates 1molL-L-glutamic acid-γ-needed enzyme amount of single hydroximic acid.
Thalline is removed in the fermented liquid press filtration, 4 ℃ of filtered liqs, and the centrifugal 15min of 12000rmp is to remove residual bacterial chip and part high molecular weight protein; Supernatant is pressed 50-200mg/L and is added the Dispase II processing, and room temperature slowly stirs 30-60min.Fermented liquid supernatant after the processing, enzyme activity is brought up to 2.4U/ml by original 1.9U/ml.
Table 1, Dispase II are to the influence of active MTG in the fermented liquid
| ? | Volume | Enzyme activity |
| Original fermented liquid | 450ml | 1.9U/ml |
| Fermented liquid after Dispase II is handled | 400ml | 2.4U/ml |
Table 1 explanation, Dispase II is handled can be converted into the pro-MTG in the fermented liquid active MTG, thereby improves the yield of active MTG.
In the fermented liquid supernatant after the Dispase II that embodiment 3 obtains is handled, adding absolute ethyl alcohol to ethanol final concentration is 20%; The pH value of regulator solution is between the 8.5-9.0, and 4 ℃ are stirred 60-120min, the centrifugal 30min of 8000rmp, and the gained deposition is the preliminary purification product of MTG, and it is subsequent use that it is dissolved in 200ml Tris-HCl damping fluid.As shown in Figure 2, swimming lane 2 is the preliminary purification product of MTG, shows behind ethanol sedimentation, and high molecular weight protein and a part of small molecules foreign protein are removed, and the purity of MTG increases.Shown in following table 2, its purity rises to more than 30% by 7.5%, than vigor by original 0.67U/mg
Bring up to 4.8U/mg, show that the purity of MTG and the vigor that compares all are significantly improved.
Table 2, ethanol sedimentation, ion exchange chromatography are to the MTG purity of protein and than the influence of vigor
| ? | Volume | Protein electrophoresis purity | Compare vigor |
| Fermented liquid after Dispase II is handled | 400ml | 7.5% | 0.67U/mg |
| MTG solution behind the ethanol sedimentation | 200ml | >;30% | 4.8U/mg |
| MTG behind the anion-exchange chromatography | 180ml | >;70% | 19.3U/mg |
| MTG behind the cation-exchange chromatography | 40ml | >;95% | 27.1U/mg |
Embodiment 5 anion-exchange chromatographies
With going up anion-exchange column HiPrep DEAE FF through the deposition that ethanol sedimentation obtains with the dissolving of Tris-HCl damping fluid and after filtering among the embodiment 4, last appearance flow rate control is at 4ml/min, and pressure is less than 0.3MPa, and it is subsequent use that collection penetrates liquid.NaCl solution (urea that contains 60mM) with 2mol/L carries out wash-out and regeneration to pillar.Through the SDS-PAGE electrophoretic analysis; As shown in Figure 2; What swimming lane 3 was represented is the product behind anion-exchange chromatography, compares the albumen (less than 15KD) of small molecular weight with ethanol sedimentation product (swimming lane 2) and is effectively removed, and the MTG purity of protein behind the anion-exchange chromatography purifying reaches more than 70%; Reach 19.3U/ml than vigor, shown in above table 2.
Embodiment 6 cation-exchange chromatographies
The liquid that penetrates that obtains among the embodiment 4 is replaced with the phosphate sodium dihydrogen buffer solution of pH6.0, go up HiTrap SP XL post then.Last appearance flow rate control is at 2ml/min, and last appearance pressure-controlling is in 0.3MPa.Phosphate buffered saline buffer (pH7.0) with containing 0-0.2mol/L NaCl carries out gradient elution, and elution flow rate is controlled in the 2ml/min, collects active elution peak.As shown in Figure 2, the 4th swimming lane is represented the MTG behind the cation-exchange chromatography purifying, obtains purity greater than 95% through cation-exchange chromatography, and it reaches the MTG of 27.1U/mg than vigor, shown in above table 2.
Table 2 shows after ethanol sedimentation, anion-exchange chromatography, cation-exchange chromatography are handled; The proteic purity of MTG has been brought up to more than 95% by primary 7.5%; And the ratio vigor of enzyme is also brought up to 27.1U/mg from 0.67U/mg, and the ratio vigor of purity of protein and enzyme has all obtained greatly improving.
Fig. 1 is the color atlas of the cation-exchange chromatography in the separation purification method of the present invention.Fig. 1 shows, the concentration of the pH value through regulating elutriant and the NaCl of elutriant can be separated MTG (peak 2) and other foreign proteins (peak 1), reaches the purpose of separation and purification.
Embodiment 7 SDS-PAGE detect purity of protein
With the stream of MTG sample solution, ion exchange chromatography wear liquid, elutriant is collected respectively, carries out the SDS-PAGE electrophoretic analysis respectively.Electrophoresis is used 10% separation gel, and 120V electrophoresis 100 minutes dyes with the Xylene Brilliant Cyanine G dye liquor then.The SDS-PAGE electrophorogram in each stage is as shown in Figure 2 in the Transglutaminase EC2.3.2.13 separation and purification process, through the ethanol sedimentation gained, like the band among Fig. 22, that is, removed the part foreign protein in the fermented liquid, the gray scale scanning analysis shows that MTG accounts for 30% of total protein.Through the anion-exchange chromatography gained, like the band among Fig. 23, promptly MTG accounts for more than 70% of total protein content.Through the cation-exchange chromatography gained, like the band among Fig. 24, MTG accounts for total protein content more than 95%, its purity and reach the requirement of hemostatic material in medical use and tackiness agent than vigor.
Mutant MTG that embodiment 8 separation and purification of the present invention obtain and the specific activity of wild-type MTG are
Under condition of different temperatures, that is, under 25 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃, 75 ℃, measure mutant MTG that separation and purification of the present invention obtains and the enzyme of wild MTG respectively and live, and carry out enzyme activity relatively.Measuring method is with embodiment 2.Mensuration result is as shown in Figure 3, and the MTG that separation and purification of the present invention obtains has more than 25 ℃ than the height ratio enzyme activity, preferably, between 25-65 ℃, has than the height ratio enzyme activity.25 ℃ (envrionment temperatures during anthemorrhagic operation), 37 ℃ (human body temperature during anthemorrhagic operation), 65 ℃, 75 ℃ (under the higher temperature conditions) all have higher ratio enzyme activity, show better stability.And wild MTG more than 55 ℃ the time than the rapid decline of enzyme activity, loss of activity almost in the time of more than 65 ℃.
The MTG of embodiment 9 separation and purification of the present invention and the crosslinked experiment of gelatin
To contain 20% fishskin gelatin solution in the sodium citrate soln of 200mM, 37 ℃ subsequent use.The glutamine of microbe transaminase MTG of above-mentioned purifying is dissolved in the sodium citrate soln of 200mM subsequent use according to the concentration of 10mg/mL.The gelatin solution of getting the above-mentioned preparation of 30ml respectively with contain 0mg, 1mg, 2mg, 5mg, MTG solution (15ml) reaction of 10mg is observed the gelling situation and is also measured gel viscosity.
Viscometer test: MT reconnaissance G and gelatin solution thorough mixing are placed in the beaker viscosity when following the trail of the gelling of blended MTG-gelatin solution experience.Write down each test group and reach maximum 30% and 90% required time of coagulating degree, said peak viscosity can detect through the specific speed of certain rotor in the viscometer.DV II+PRO the Digital Viscometer (Brolkfield company) with T-E " t-bar " rotor is adopted in this experiment, and it is 10 * 106cP that the rotor maximum can write down viscosity, and therefore 30% and 90% viscosity points is respectively 3 * 106cP and 9 * 106cP.The entire reaction course test soln need be immersed in 37 ℃ of water-baths and carry out.The result is as shown in table 4, the gelation time under the different enzyme concns.
Table 3 different concns MTG is to the influence of gel time
| Group (MTG) | 30% time | 90% time |
| The 0mg control group | No gelling situation takes place | No gelling situation takes place |
| The 1mg experimental group | 194 seconds | 486 seconds |
| The 2mg experimental group | 136 seconds | 247 seconds |
| The 5mg experimental group | 69 seconds | 107 |
| The 10mg experimental group | < 15 seconds | 47 seconds |
Table 3 shows that the MTG after the separation and purification can crosslinked, generation gelling phenomenon take place with gelatin, and in 0-10mg scope interval, along with the increase of MTG amount, MTG and gelatin generation agglomerative time are short more.
The yield of Transglutaminase EC2.3.2.13 in the embodiment 10 separation and purification processes of the present invention
As shown in table 4, total enzyme activity of whole separation and purification process of the present invention, the yield of compare vigor and MTG have been carried out computational analysis, the ratio vigor of MTG rises to 26.9U/mg by original 0.67U/mg, has increased more than 30 times, and yield reaches 73.7%.
Table 4 MTG purifying yield table
| Step | Total enzyme is lived | Compare vigor | Yield |
| Fermented liquid | 1209U | 0.67U/mg | ? |
| First pure products | 1054U | 4.8U/ mg | 87.17% |
| Anion-exchange chromatography | 946U | 19.3U/ mg | 78.24% |
| Cation-exchange chromatography | 897U | 27.1U/ mg | 74.2% |
| Lyophilize | 891U | 26.9U/ mg | 73.7% |
Claims (9)
1. the separation purification method of a glutamine of microbe transaminase; It is characterized in that; The Dispase II that in streptomycete fermentation liquid, adds 50-200mg/L is handled; Through ethanol sedimentation, anion-exchange chromatography, cation-exchange chromatography, again through desalination, concentrate, obtain said glutamine of microbe transaminase after the filtration, freeze-drying; Wherein, the purity of said glutamine of microbe transaminase is 20000-50000U/g greater than 95% than vigor.
2. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, said streptomycete fermentation liquid is luxuriant source streptomycete STK4 mutant strain fermented liquid; The 10th amino acids of said mikrobe glutamine transaminase is Serine or Threonine; The 241st amino acids is L-Ala or glycocoll; The 284th amino acids is Xie Ansuan or tyrosine.
3. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, in the said anion-exchange chromatography step, sample-loading buffer is the Tris-HCl damping fluid of pH 7.0-7.5; Elutriant is the NaCl solution of 0-2mol/L.
4. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, in the said cation-exchange chromatography step, sample-loading buffer is the PB damping fluid of Ph5.5-6.5; Elutriant is pH 7.0, the NaCl solution of 0-1mol/L.
5. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, the fiber filter film with 0.45 μ m-0.22 μ m filters before the ion exchange column on said sample.
6. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, filters with nanofiltration membrane before the said sample freeze-drying.
7. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, the yield of said glutamine of microbe transaminase is greater than 70%.
8. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, the level of endotoxin < 4EU/>g of said glutamine of microbe transaminase.
9. the separation purification method of glutamine of microbe transaminase as claimed in claim 1 is characterized in that, said glutamine of microbe transaminase can be used as wound hemostasis and medical jointing material.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480094A (en) * | 2014-11-27 | 2015-04-01 | 苏州嘉禧萝生物科技有限公司 | Method for separating and purifying glutamate decarboxylase |
| CN105754892A (en) * | 2016-02-01 | 2016-07-13 | 华东师范大学 | Separation and purification method of microbial transglutaminase |
| CN106011099A (en) * | 2016-06-23 | 2016-10-12 | 华东理工大学 | Separation and purification method of insect transglutaminase |
| CN108570491A (en) * | 2017-03-14 | 2018-09-25 | 华东师范大学 | A kind of detection method of Microbial transglutaminase stability |
| CN113528481A (en) * | 2021-07-21 | 2021-10-22 | 华东理工大学 | With temperature-responsive random copolyether EO20PO80Method for separating glutamine transaminase by precipitation method |
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2012
- 2012-08-06 CN CN2012102767057A patent/CN102776160A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480094A (en) * | 2014-11-27 | 2015-04-01 | 苏州嘉禧萝生物科技有限公司 | Method for separating and purifying glutamate decarboxylase |
| CN105754892A (en) * | 2016-02-01 | 2016-07-13 | 华东师范大学 | Separation and purification method of microbial transglutaminase |
| CN105754892B (en) * | 2016-02-01 | 2019-09-06 | 华东师范大学 | A kind of isolation and purification method of glutamine of microbe transaminase |
| CN106011099A (en) * | 2016-06-23 | 2016-10-12 | 华东理工大学 | Separation and purification method of insect transglutaminase |
| CN108570491A (en) * | 2017-03-14 | 2018-09-25 | 华东师范大学 | A kind of detection method of Microbial transglutaminase stability |
| CN108570491B (en) * | 2017-03-14 | 2022-01-11 | 华东师范大学 | Method for detecting stability of microbial transglutaminase |
| CN113528481A (en) * | 2021-07-21 | 2021-10-22 | 华东理工大学 | With temperature-responsive random copolyether EO20PO80Method for separating glutamine transaminase by precipitation method |
| CN113528481B (en) * | 2021-07-21 | 2023-01-03 | 华东理工大学 | With temperature-responsive random copolyether EO 20 PO 80 Method for separating glutamine transaminase by precipitation method |
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