CN105754892A - Separation and purification method of microbial transglutaminase - Google Patents
Separation and purification method of microbial transglutaminase Download PDFInfo
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Abstract
The invention discloses a separation and purification method of new microbial transglutaminase. The method comprises the following steps of carrying out alcohol precipitation on new streptomyces mobaraensis fermentation liquor with the strain number being CGMCC No.10804 obtained through natural screening, carrying out anion exchange chromatography, desalting, freezing and drying to obtain the microbial transglutaminase. The obtained transglutaminase has the purity being larger than or equal to 90 percent, the plasma endotoxin level being less than or equal to 0.02EU/mL, the specific enzyme activity being 25 to 30U/mg, and the recovery rate being larger than or equal to 70 percent. The separation and purification method of the new microbial transglutaminase is simple in process, low in cost, and high in enzyme recovery; the obtained transglutaminase is high in purity; the microbial transglutaminase obtained through fermenting the strain is stable in property, so that the industrial production is convenient; a feasible method is provided for satisfying medical grade MTGase scale production.
Description
Technical field
The invention belongs to the isolated and purified field of biotechnology, be specifically related to the separating and purifying technology of a kind of produced glutamine transaminage of new strain.
Background technology
Glutamine transaminage (TransglutaminaSe; it is called for short TGase; EC2.3.2.13) be a kind of catalyzing acyl transfer transferring enzyme; can catalytic proteins intramolecular and the formation of intermolecular ε-(γ-glutaminyl) lysine covalent bond; thus catalytic proteins intramolecular, intermolecular crosslink, and then change character and the function of protein.
Glutamine transaminage is widely present in plant, animal and microorganism.In Guinea Pig Liver, obtained TGase by scholar the earliest, its in Europe a very long time as the source of business TGase.The price that rare and animal origin the separation purifying technique complicated for TGase of resource causes enzyme is the highest, to such an extent as to is impossible to be applied in plant-scale food processing.So some scholars start to be devoted to obtain this enzyme from microorganism.Ando etc. found Microbial transglutaminase (microbial transglutaminase, MTGase) the earliest from streptomyces verticillatus culture medium later, it is achieved thereby that large-scale fermenting and producing.The most microbe-derived glutamine transaminage has many good qualities compared with the glutamine transaminage of plant and animal material, and if microbe-derived glutamine transaminage is calcium dependent/non-dependent enzyme, substrate specificity is low.These character make MTGase be widely used in every field.
After Ando in 1989 etc. have obtained glutamine transaminage from streptomyces verticillatus culture medium, experts and scholars find that other produce the streptomycete of TGase one by one, such as streptomyces hygroscopicus and cyclopentadienyl source streptomycete etc..The character of the MTGase that the MTGase in different streptomyces sources even belongs to strepto-secretion the most of the same race together is the most incomplete same.Their heat stability and pH stability have the biggest difference, thus affect the application in later stage.The streptomycete of the product MTGase screened at present is not all can be transformed in commercial production.So it is high to screen product enzyme efficiency, the task of the streptomycete that MTGase stability is high is extremely urgent.And the MTGase stability produced from the luxuriant source streptomycete that nature screens being previously mentioned in the present invention relatively before discovery will be high.
The most microbe-derived MTGase has applied in industrialized production.The MTGase that microorganism produces by fermentation just can apply in food processing after simple alcohol precipitation.But the MTGase purity that this method obtains is low, impurity content is many and complicated components.Studies have found that, MTGase has huge application prospect in field of biological pharmacy.Such as MTG can be catalyzed I-type collagen and crosslink, and forms resistant to elevated temperatures glue；Can also cross-linked gelatin, formed good perforated web for hemostasis and wound bond (Chinese patent: 200780051215.4,200980131973.6；United States Patent (USP): 8133484；European patent: WO2012017415, EP2303344)；Therefore Microbial transglutaminase can be used for stopping blooding as the cross-linking agent of potential hemostatic material in medical use, in the surgical operation such as wound bonding.But, apply to the purity of the MTGase of field of food and be unsatisfactory for the other requirement of pharmaceutical grade.The most existing tradition separation purifying technique is complicated, and cost is high, and in preparation process, the yield of enzyme, purity and Rate activity are the highest, still contain multiple foreign protein, it is difficult to meet the requirement as medical material.Instant invention overcomes the problem that presently, there are, obtain the purifying process of a simple low cost.
Summary of the invention
It is an object of the invention to provide a kind of low cost, the response rate is high, purity is high, be applicable to industrialized production and meet the Microbial transglutaminase purification process of pharmaceutical grade requirement, solves the problem that current glutamine transaminage purifying process is complicated, cost is high, the response rate is low, purity is low.The isolated and purified microorganism casein hydrolyzated obtained of the inventive method meets the requirement of pharmaceutical grade.
In the inventive method, the strain numbered CGMCC No.10804 at China Committee for Culture Collection of Microorganisms's common micro-organisms center used, it it is the luxuriant source streptomycete obtained by Natural Selection, utilizing cyclopentadienyl source streptomycete producing microbial glutamine transaminage (MTGase) is a kind of the most general method, and the feature of this bacterial strain is that it produces enzyme efficiency height, compared with the MTGase produced MTGase produced with existing bacterial strain, stability is high, as in figure 2 it is shown, meet the requirement of pharmaceutical grade.
The present invention proposes a kind of cyclopentadienyl source streptomycete (Streptomyces sp.) bacterial strain, is the new streptomycete bacterial strain of strain that Natural Selection obtains from soil, and hypha form is as shown in Figure 1.Contrast through 16S rDNA sequence finds that it is different from other streptomycete bacterial strains of existing report, and the systematic evolution tree of the bacterial strain based on present invention proposition that built by neighbor-joining adjacent method and related strain 16S rDNA sequence is as shown in Figure 5.The bacterial strain that this patent proposes is in Streptomyces mobaraensis NBRC13819, unknown streptomycete between Streptomyces mobaraensis NRRL B-3729 and Streptomyces sp.J1S1, bacterial strain in black box i.e. represents the streptomycete that the present invention proposes, and can be speculated as new wild strain.
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation date is on May 13rd, 2015, and deposit number is CGMCC No.10804.The sequencing result of described cyclopentadienyl source streptomycete 16S rDNA is as shown in SEQ ID NO.1.
The invention allows for the cultural method of a kind of described cyclopentadienyl source streptomycete: described cyclopentadienyl source streptomycete is cultivated 24-48h in seed culture medium, cultivation temperature is 30 DEG C, shaking speed is 200r/min, then, it is transferred to again in fermentation medium cultivate 45-52h, obtains the fermentation liquid of described cyclopentadienyl source streptomycete；Wherein, described fermentation liquid contains the secondary metabolite MTGase of described cyclopentadienyl source streptomycete.
Specifically, the cultural method of described cyclopentadienyl source streptomycete is: bacterial strain screening obtained cultivates 24-48h in seed culture medium, consisting of (percentage by weight) of described seed culture medium: glycerol 1.5-2.5%, yeast extract 0.3-0.8%, fish meal protein peptone 2.1-2.7%, MgSO4·7H2O 0.15-0.4%, K2HPO40.15-2.3%, pH are 7.4, and cultivation temperature is 30 DEG C, and shaking speed is 200r/min.Then, then it is transferred in fermentation medium cultivate 45-52h, obtains the secondary metabolite MTGase of substantial amounts of cyclopentadienyl source streptomycete.Wherein, consisting of (percentage by weight) of described fermentation medium: glycerol 1.5-2.5%, yeast extract 0.3-0.8%, fish meal protein peptone 2.1-2.7%, MgSO4·7H2O 0.15-0.4%, K2HPO40.15-2.3%, accelerator 1-2%, pH are 7.4, and cultivation temperature is 30 DEG C, shaking speed is 200r/min.
The isolation and purification method of Microbial transglutaminase of the present invention comprises the following steps:
1, taking cyclopentadienyl source streptomycete fermentation liquid supernatant, with the dehydrated alcohol 1:1 process of pre-cooling, after standing 1h, 8000r/mim is centrifuged 15min, collects precipitation, obtains the crude extract of MTGase；
2, the crude extract of step 1 gained is dissolved in phosphate buffer, anion-exchange chromatography is carried out with AKTA avant 25 purification instrument under room temperature, the purification column model selected is Hitrap Q Sepharose Fast Flow (1mL), the flow velocity of level pad, sample and elution buffer is 1mL/min, collects first eluting peak occurred in elution process by the mode of linear elution；
3, step 2 being reclaimed the sample obtained and carry out desalination again, the flow velocity that desalting column model is Hiprep 26/10Desalting, sample and ultra-pure water of selection is 10mL/min；
4, step 3 is reclaimed the sample obtained and carry out lyophilization, finally obtain highly purified glutamine of microbe transaminase.
In purification step of the present invention, all of buffer and sample will be degerming through the membrane filtration of 0.22nm.
Described step 2 is purified by anion-exchange chromatography method, compared with existing purification process, method is novel, step is simple, have only to the main anion-exchange chromatography of a step and can be obtained by meeting the MTGase of pharmaceutical grade purity requirement, and then can obtain salt-free pure fine work MTGase by desalination and cryodesiccated method, and the powder after lyophilizing is more suitable for later stage application, storage and transport.The used sodium phosphate buffer that level pad is 15-25mM pH6.5-8.0, elution buffer is the sodium phosphate buffer+1M sodium chloride of 15-25mM pH6.5-8.0, elution process is linear elution, 0-20% elution buffer, elution volume is 25 column volumes, and the concentration of sample is 0.4-0.5mg/mL, and unit enzyme is lived as 5-6U/mL, the total protein of loading is 10-15mg, and total enzyme is lived as 100-300U.
In described step 3, the solution used by desalination is ultra-pure water, and sample loading volume is less than or equal to the 30% of column volume.
In freezing dry process in described step 4, sublimation temperature is-10 DEG C for the first time, and sublimation temperature is 4 DEG C for the second time.
In the present invention, use the response rate 70% of MTGase that the inventive method finally gives, specific enzyme activity between 25-30U/mg, endotoxin content 0.02EU/mL, purity 90%.Present invention process is simple; low cost; the enzyme response rate alive is high; the glutamine transaminage purity obtained is high; and glutamine of microbe transaminase's stable in properties that this strain fermentation obtains; it is easy to industrialized production, provides a practicable method for meeting the large-scale production of the MTGase of pharmaceutical grade.
The present invention proposes the application that cyclopentadienyl source streptomycete is used for producing Microbial transglutaminase.
The present invention proposes the Microbial transglutaminase obtained by above-mentioned method for purifying and separating.The invention allows for the medical usage of the Microbial transglutaminase obtained by said method.The MTGase finally given after purification by the present invention can work for wound hemostasis and adhesive of medical with gelatin one, it is also possible to for the bio-modification etc. of pharmaceutical protein.
In sum, the beneficial effects of the present invention is: bacterial strain the produced MTGase stable in properties that (1) Natural Selection obtains, cause the enzyme impact lost alive less due to self stability in fermentation liquid last handling process.(2) MTGase isoelectric point, IP is about 8.0, at pH < MTGase positively charged in theory when 8.0, ion exchange layer analysis method used in existing scheme is all cation-exchange chromatography, and the purification process mentioned in the present invention is for use anion-exchange chromatography under the conditions of pH6.5-8.0.This is because in the present invention propose bacterial strain under the conditions of pH6.5-8.0 can on the band of surface substantial amounts of negative charge, thus be combined with anion column, thus broken purification process and the Purification: Principles of our routine, provide new approaches for MTGase purification and other protein purifications.(3) in existing scheme, MTMase purification process is and is purified by cation-exchange chromatography or Gel filtration analysis method under the conditions of pH 7.0, and one step purifying method cannot obtain purity higher than the MTGase of 20U/mg, the MTGase loss simultaneously obtained is relatively big, complex steps, cannot apply to commercial production.The purification schemes proposed in the present invention, can obtain specific enzyme activity more than the MTGase of 25U/mg by method one step of anion-exchange chromatography under the conditions of pH6.5-8.0, ult rec can reach more than 70%, i.e., the purity using the enzyme of the purification process acquisition of the present invention is high, and the response rate is high.Method is simple, low cost, can industry amplify, it is adaptable to industrialized production.
Accompanying drawing explanation
Fig. 1 is that the bacterial strain that proposes of present invention growthform in ISP culture medium is observed.
The produced MTGase of the luxuriant source streptomycete (experimental group) that Fig. 2 proposes by the present invention and the comparison of existing report Zhong Mao source the produced MTGase of streptomycete (matched group 1:40587, matched group 2:40847) enzyme stability at different temperatures.A is the result of different MTGase stability under the conditions of being 70 DEG C, and B is the result of different MTGase stability under the conditions of being 60 DEG C, and C is the result of different MTGase stability under the conditions of being 50 DEG C, and D is the result of different MTGase stability under the conditions of being 40 DEG C.
Fig. 3 is anion-exchange chromatography chromatogram.Penetrate peak and represent the foreign protein peak not being combined with anion chromatography post；Eluting peak 1 represents destination protein peak；Eluting peak 2 represents the foreign protein peak with anion chromatography post strong bonded.
Fig. 4 is glutamine of microbe transaminase electrophoretogram in purge process.M represents protein standard maker；Swimming lane 1 represents fermentation liquid electrophoretogram；Swimming lane 2 represents alcohol precipitation product electrophoretogram；Swimming lane 3 represents purpose peak electrophoretogram after anion-exchange chromatography；Swimming lane 4 represents MTGase electrophoretogram after desalination；Swimming lane 5 represents MTGase electrophoretogram after lyophilization.
Fig. 5 is the bacterial strain proposed based on the present invention and the systematic evolution tree of related strain 16S rDNA sequence built by neighbor-joining adjacent method.
Detailed description of the invention
In conjunction with specific examples below and accompanying drawing, the present invention is described in further detail, and the protection content of the present invention is not limited to following example.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change and advantage be all included in the present invention, and with appending claims as protection domain.Implementing the process of the present invention, condition, reagent, experimental technique etc., outside the lower content mentioned specially, be universal knowledege and the common knowledge of this area, the present invention is not particularly limited content.
Embodiment 1 alcohol precipitation obtains MTGase crude extract
(1) fermentation liquid 8000g fermentation obtained, 4 DEG C of centrifugal 10min, remove precipitation；Slowly adjust PH to 5.0,8000g, 4 DEG C of centrifugal 10min with phosphoric acid, remove precipitation；The dehydrated alcohol of pre-cooling in advance is slowly added to 1:1, is placed in ice chest and places 1h, 8000g, 4 DEG C of centrifugal 10min, remove supernatant.
(2) SDS-PAGE testing result is as shown in swimming lane 2 in Fig. 4, and MTGase molecular weight is about 38KDa, can be seen that the crude extract that alcohol precipitation obtains containing substantial amounts of foreign protein from swimming lane 2.It is 2760.6U that the total enzyme of alcohol precipitation primary fermentation liquid is lived, and after alcohol precipitation, total enzyme is lived is 2484.5U, and concrete outcome is as shown in table 1.Enzyme activity determination result of living shows that the total enzyme of this step response rate alive is 90%.
The detection of embodiment 2MTGase crude extract temperature stabilization
(1) it is dissolved in the phosphate buffer of pH6.0 with 3U/mL after MTGase (experimental group) lyophilizing that embodiment 1 alcohol precipitation is obtained, choose two currently reported any two kinds of MTGase (matched group 1:40587, matched group 2:40847 are all from Taixing Dongsheng Food Science & Technology Co., Ltd.) conduct comparisons simultaneously, be dissolved in the phosphate buffer of pH6.0 with 3U/mL equally.
(2) being respectively placed in by three kinds of MTGase in the water-bath of 40 DEG C, 50 DEG C, 60 DEG C and 70 DEG C, the time interval under each temperature conditions as shown in Figure 2 preserves MTGase, and after certain time, the enzyme of the unified MTGase of survey is lived.A is the result of different MTGase stability under the conditions of being 70 DEG C, and B is the result of different MTGase stability under the conditions of being 60 DEG C, and C is the result of different MTGase stability under the conditions of being 50 DEG C, and D is the result of different MTGase stability under the conditions of being 40 DEG C.
As in figure 2 it is shown, under the conditions of 70 DEG C, the stability of the MTGase heretofore described when placing 75s is higher than the 40587 of matched group 1 by 23%, higher than the 40847 of matched group 2 by 19%；Under the conditions of 60 DEG C, the stability of the MTGase heretofore described when placing 5min is higher by 22% than 40587, higher than the 40847 of matched group 2 by 25%；Under the conditions of 50 DEG C, the stability of the MTGase heretofore described when placing 120min is higher than the 40587 of matched group 1 by 18%, higher than the 40847 of matched group 2 by 21%；Under the conditions of 40 DEG C, the stability of the MTGase heretofore described when placing 4d is higher than the 40587 of matched group 1 by 14%, higher than the 40847 of matched group 2 by 15%.In sum, under the conditions of uniform temperature, the stability of heretofore described MTGase is above other two kinds of enzyme powders.
Embodiment 3 anion-exchange chromatography method obtains high-purity MTGase after purification
(1) the purification instrument selected is AM General companyAvant 25, the chromatographic column used by purification is prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) purchased from AM General company.
(2) prepare the sodium dihydrogen phosphate of appropriate 20mM and the disodium hydrogen phosphate of 20mM respectively, two kinds of buffer are mixed in proportion the phosphate buffer being finally formulated as pH6.5, conductance about 2.5mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(3) on the basis of level pad, add 1M sodium chloride, with phosphoric acid, its pH is adjusted to 6.5 again after fully dissolving, conductance about 75.0mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(4) the MTGase crude extract obtained by alcohol precipitation is dissolved in the 20mM phosphate buffer of pH6.5,4 DEG C of centrifugal 10min of 8000r/min after fully dissolving, and then filters enzyme liquid with the filter membrane of 0.22um, and then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.The sample protein concentration that preparation obtains is 0.36mg/mL, and unit enzyme is lived as 4.81U/mL.
(5) first with the level pad of 20CV, anion chromatographic column is balanced, take the enzyme liquid 30mL loading prepared in advance, then will not be combined with chromatographic column with the level pad of 10CV or combine unstable albumen and elute, as shown in Figure 3 penetrate peak, eluting is carried out with the method for linear elution again with the elution buffer of 25CV, now sodium chloride ionic strength increases to 0.2M from 0, the eluting peak obtained eluting peak 1 as shown in Figure 3, finally with the foreign protein of the sodium chloride eluting of 1M with chromatographic column strong bonded, the eluting peak obtained eluting peak 2 as shown in Figure 3.Flow velocity in all processs of the test is all 1mL/min.Collect eluting peak 1 and be MTGase after purification.
(6) SDS-PAGE testing result is as shown in the swimming lane 3 in Fig. 4, and the sample that purification obtains is 92% through gray scale scanning purity, and all samples obtained after alcohol precipitation total enzyme after this step response rate alive is 89.6%, and specific enzyme activity is 26.34U/mg.
Embodiment 4 anion-exchange chromatography method obtains high-purity MTGase after purification
(1) the purification instrument selected is AM General companyAvant 25, the chromatographic column used by purification is prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) purchased from AM General company.
(2) prepare the sodium dihydrogen phosphate of appropriate 20mM and the disodium hydrogen phosphate of 20mM respectively, two kinds of buffer are mixed in proportion the phosphate buffer being finally formulated as pH7.0, conductance about 2.5mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(3) on the basis of level pad, add 1M sodium chloride, with phosphoric acid, its pH is adjusted to 7.0 again after fully dissolving, conductance about 75.0mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(4) the MTGase crude extract obtained by alcohol precipitation is dissolved in the 20mM phosphate buffer of pH7.0,4 DEG C of centrifugal 10min of 8000r/min after fully dissolving, and then filters enzyme liquid with the filter membrane of 0.22um, and then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.The sample protein concentration that preparation obtains is 0.42mg/mL, and unit enzyme is lived as 5.09U/mL.
(5) first with the level pad of 20CV, anion chromatographic column is balanced, take the enzyme liquid 30mL loading prepared in advance, then will not be combined with chromatographic column with the level pad of 10CV or combine unstable albumen and elute, as shown in Figure 3 penetrate peak, eluting is carried out with the method for linear elution again with the elution buffer of 25CV, now sodium chloride ionic strength increases to 0.2M from 0, the eluting peak obtained eluting peak 1 as shown in Figure 3, finally with the foreign protein of the sodium chloride eluting of 1M with chromatographic column strong bonded, the eluting peak obtained eluting peak 2 as shown in Figure 3.Flow velocity in all processs of the test is all 1mL/min.Collect eluting peak 1 and be MTGase after purification.
(6) SDS-PAGE testing result is as shown in the swimming lane 3 in Fig. 4, the sample that purification obtains is 90% through gray scale scanning purity, the all samples obtained after alcohol precipitation total enzyme after this step response rate alive is 92.0%, and specific enzyme activity is 28.61U/mg, and concrete outcome is as shown in table 1.
Embodiment 5 anion-exchange chromatography method obtains high-purity MTGase after purification
(1) the purification instrument selected is AM General companyAvant 25, the chromatographic column used by purification is prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) purchased from AM General company.
(2) prepare the sodium dihydrogen phosphate of appropriate 20mM and the disodium hydrogen phosphate of 20mM respectively, two kinds of buffer are mixed in proportion the phosphate buffer being finally formulated as pH7.5, conductance about 2.5mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(3) on the basis of level pad, add 1M sodium chloride, with phosphoric acid, its pH is adjusted to 7.5 again after fully dissolving, conductance about 75.0mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(4) the MTGase crude extract obtained by alcohol precipitation is dissolved in the 20mM phosphate buffer of pH7.5,4 DEG C of centrifugal 10min of 8000r/min after fully dissolving, and then filters enzyme liquid with the filter membrane of 0.22um, and then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.The sample protein concentration that preparation obtains is 0.4mg/mL, and unit enzyme is lived as 5.19U/mL.
(5) first with the level pad of 20CV, anion chromatographic column is balanced, take the enzyme liquid 30mL loading prepared in advance, then will not be combined with chromatographic column with the level pad of 10CV or combine unstable albumen and elute, as shown in Figure 3 penetrate peak, eluting is carried out with the method for linear elution again with the elution buffer of 25CV, now sodium chloride ionic strength increases to 0.2M from 0, the eluting peak obtained eluting peak 1 as shown in Figure 3, finally with the foreign protein of the sodium chloride eluting of 1M with chromatographic column strong bonded, the eluting peak obtained eluting peak 2 as shown in Figure 3.Flow velocity in all processs of the test is all 1mL/min.Collect eluting peak 1 and be MTGase after purification.
(6) SDS-PAGE testing result is as shown in the swimming lane 3 in Fig. 4, and the sample that purification obtains is 93% through gray scale scanning purity, and all samples obtained after alcohol precipitation total enzyme after this step response rate alive is 91.4%, and specific enzyme activity is 28.21U/mg.
Embodiment 6 anion-exchange chromatography method obtains high-purity MTGase after purification
(1) the purification instrument selected is AM General companyAvant 25, the chromatographic column used by purification is prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) purchased from AM General company.
(2) prepare the sodium dihydrogen phosphate of appropriate 20mM and the disodium hydrogen phosphate of 20mM respectively, two kinds of buffer are mixed in proportion the phosphate buffer being finally formulated as pH8.0, conductance about 2.5mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(3) on the basis of level pad, add 1M sodium chloride, with phosphoric acid, its pH is adjusted to 8.0 again after fully dissolving, conductance about 75.0mS/cm.With the membrane filtration of 0.22um after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.
(4) the MTGase crude extract obtained by alcohol precipitation is dissolved in the 20mM phosphate buffer of pH8.0,4 DEG C of centrifugal 10min of 8000r/min after fully dissolving, then filtering enzyme liquid with the filter membrane of 0.22um, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of Refrigerator stores are standby.The sample protein concentration that preparation obtains is 0.42mg/mL, and unit enzyme is lived as 5.09U/mL.
(5) first with the level pad of 20CV, anion chromatographic column is balanced, take the enzyme liquid 30mL loading prepared in advance, then will not be combined with chromatographic column with the level pad of 10CV or combine unstable albumen and elute, as shown in Figure 3 penetrate peak, eluting is carried out with the method for linear elution again with the elution buffer of 25CV, now sodium chloride ionic strength increases to 0.2M from 0, the eluting peak obtained eluting peak 1 as shown in Figure 3, finally with the foreign protein of the sodium chloride eluting of 1M with chromatographic column strong bonded, the eluting peak obtained eluting peak 2 as shown in Figure 3.Flow velocity in all processs of the test is all 1mL/min.Collect eluting peak 1 and be MTGase after purification.
(6) SDS-PAGE testing result is as shown in the swimming lane 3 in Fig. 4, and the sample that purification obtains is 87% through gray scale scanning purity, and all samples obtained after alcohol precipitation total enzyme after this step response rate alive is 94.7%, and specific enzyme activity is 27.81U/mg.
Obtain, after embodiment 7 desalination, the MTGase that high-purity is salt-free
(1) the purification instrument selected is AM General companyAvant 25, desalting column is the prepacked column Hiprep 26/10Desalting purchased from AM General company.
(2) being first balanced desalting column with the flow velocity of 10mL/min with ultra-pure water, the direct loading of sample then obtained by anion-exchange chromatography in embodiment 5, loading cumulative volume is 12mL, and it is 100U that the total enzyme of loading is lived, and loading total protein is 5mg.
(3) sample that protein peak is corresponding is collected, SDS-PAGE testing result is as shown in swimming lane 4 in Fig. 4, sample after desalination is 92% through gray scale scanning purity, and all samples obtained after anion-exchange chromatography total enzyme after this step response rate alive is 95%, and concrete outcome is as shown in table 1.
The glutamine transaminage response rate during the present invention is isolated and purified, as shown in table 1:
Table 1 MTGase purification result
Note: alcohol precipitation is the result in embodiment 1；Anion-exchange chromatography is the result in embodiment 4；Desalination is the result in embodiment 7.
In sum, the isolation and purification method of a kind of new glutamine of microbe transaminase that the present invention proposes has obvious beneficial effect.First, from embodiment 2 it will be seen that the MTGase stability proposed the present invention is apparently higher than other kinds of MTGase, different temperatures stability inferior exceeds 10-20%, stable in properties, all has higher stability in fermentation liquid post processing or later stage medical applications, meets pharmaceutical grade requirement.Secondly, by embodiment 2-6 it appeared that, the ratio MTGase lived more than 25U/mg can be obtained by method one step of anion-exchange chromatography under the conditions of pH6.5-8.0, the ult rec finally giving the solid enzyme powder meeting pharmaceutical grade requirement through a series of process can reach more than 70%, compared with existing document report, there is obvious advantage.Existing document report obtains the response rate of the higher MTGase of purity all below 70% by different purification schemes, and this result is not have the result before desalination and lyophilization, and the response rate that the method mentioned in the present invention obtains merely highly purified MTGase can reach more than 80%, obtain, even across desalination and lyophilization, the MTGase enzyme powder ult rec being dried and also reach more than 70%, the response rate is high, method is simple, low cost, can industry amplify, it is adaptable to industrialized production.Finally, MTGase isoelectric point, IP is 8.0, ion exchange layer analysis method used in existing scheme is all cation-exchange chromatography, and the purification process mentioned in the present invention is for using anion-exchange chromatography under the conditions of pH6.5-8.0, break purification process and the Purification: Principles of our routine, provide new approaches for MTGase purification and other protein purifications.
Claims (10)
1. luxuriant source streptomycete (Streptomyces sp.) bacterial strain, it is characterized in that, it it is the new streptomycete bacterial strain of strain that Natural Selection obtains from soil, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on May 13rd, 2015, and deposit number is CGMCC No.10804.
2. the isolation and purification method of a glutamine of microbe transaminase, it is characterised in that said method comprising the steps of:
(1) taking the luxuriant source streptomycete fermentation liquid supernatant described in claim 1, with the dehydrated alcohol 1:1 process of pre-cooling, after standing 1h, 8000r/mim is centrifuged 15min, collects precipitation, obtains the crude extract of MTGase；
(2) crude extract of step (1) gained is dissolved in phosphate buffer, anion-exchange chromatography is carried out with AKTA avant 25 purification system under room temperature, the purification column model selected is Hitrap Q Sepharose Fast Flow (1mL), the flow velocity of level pad, sample and elution buffer is 1mL/min, collects first eluting peak occurred in elution process by the mode of linear elution；
(3) step (2) being reclaimed the sample obtained and carry out desalination again, the flow velocity that desalting column model is Hiprep 26/10Desalting, sample and ultra-pure water of selection is 10mL/min；
(4) step (3) is reclaimed the sample obtained and carry out lyophilization, finally obtain highly purified glutamine of microbe transaminase.
The isolation and purification method of glutamine of microbe transaminase the most according to claim 2, it is characterised in that in described method, all of sample and used buffer will be degerming through the membrane filtration of 0.22um.
The isolation and purification method of glutamine of microbe transaminase the most according to claim 2, it is characterised in that the luxuriant source streptomycete numbered CGMCC No.10804 in described step (1).
The isolation and purification method of glutamine of microbe transaminase the most according to claim 2, it is characterized in that, the sodium phosphate buffer of the 15-25mM that level pad is pH6.5-8.0 in described step (2), elution buffer is the sodium phosphate buffer+1M sodium chloride of the 15-25mM of pH6.5-8.0, elution process is linear elution, 0-20% elution buffer, 25 column volumes of eluting, the concentration of sample is 0.4-0.5mg/mL, unit enzyme is lived as 5-6U/mL, the total protein of loading is 10-15mg, and total enzyme is lived as 100-300U.
The isolation and purification method of glutamine of microbe transaminase the most according to claim 2, it is characterised in that in described step (3), the solution used by desalination is ultra-pure water, sample loading volume is less than or equal to the 30% of column volume.
The isolation and purification method of glutamine of microbe transaminase the most according to claim 2, it is characterised in that in the freezing dry process in described step (4), sublimation temperature is-10 DEG C for the first time, sublimation temperature is 4 DEG C for the second time.
8. the cultural method of cyclopentadienyl source as claimed in claim 1 streptomycete, it is characterised in that described cyclopentadienyl source streptomycete is cultivated in seed culture medium 24-48h, and cultivation temperature is 30 DEG C, and shaking speed is 200r/min；Then, then it is transferred in fermentation medium cultivate 45-52h, obtains the fermentation liquid of described cyclopentadienyl source streptomycete；Wherein, described fermentation liquid contains the secondary metabolite MTGase of described cyclopentadienyl source streptomycete.
9. cultural method as claimed in claim 8, it is characterised in that consisting of (percentage by weight) of described seed culture medium: glycerol 1.5-2.5%, yeast extract 0.3-0.8%, fish meal protein peptone 2.1-2.7%, MgSO4·7H2O 0.15-0.4%, K2HPO40.15-2.3%, pH are 7.4.
10. cultural method as claimed in claim 8, it is characterised in that consisting of (percentage by weight) of described fermentation medium: glycerol 1.5-2.5%, yeast extract 0.3-0.8%, fish meal protein peptone 2.1-2.7%, MgSO4·7H2O 0.15-0.4%, K2HPO40.15-2.3%, accelerator 1-2%, pH are 7.4.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106244567A (en) * | 2016-08-31 | 2016-12-21 | 安徽珍味奇调味食品有限公司 | A kind of glutamine transaminage process for separation and purification |
| CN108570491A (en) * | 2017-03-14 | 2018-09-25 | 华东师范大学 | A kind of detection method of Microbial transglutaminase stability |
| CN110452890A (en) * | 2019-07-30 | 2019-11-15 | 泰兴市东圣生物科技有限公司 | It is a kind of through acidolysis or the pretreated Fermentation Medium for Transglutaminase of enzymatic hydrolysis and its preparation method and application |
| CN110616208A (en) * | 2019-11-04 | 2019-12-27 | 泰兴市东圣生物科技有限公司 | Optimized fermentation method of glutamine transaminase based on hydrodynamics |
| CN112553176A (en) * | 2020-12-29 | 2021-03-26 | 江南大学 | Glutamine transaminase with improved thermal 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 |
| CN114686389A (en) * | 2020-12-25 | 2022-07-01 | 江苏东汇生物科技有限公司 | Glutamine transaminase high-producing strain for enhancing vgbS gene transcription level and preparation and fermentation methods thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0726317A2 (en) * | 1995-02-09 | 1996-08-14 | Ajinomoto Co., Inc. | Bacillus-derived transglutaminase |
| CN102776160A (en) * | 2012-08-06 | 2012-11-14 | 华东师范大学 | Method for separating and purifying microbial transglutaminase |
-
2016
- 2016-02-01 CN CN201610070332.6A patent/CN105754892B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0726317A2 (en) * | 1995-02-09 | 1996-08-14 | Ajinomoto Co., Inc. | Bacillus-derived transglutaminase |
| CN102776160A (en) * | 2012-08-06 | 2012-11-14 | 华东师范大学 | Method for separating and purifying microbial transglutaminase |
Non-Patent Citations (2)
| Title |
|---|
| Y. ZHU ET AL.,: ""Medium Design Based on Stoichiometric Analysis of Microbial Transglutaminase Production by Streptoverticillium mobaraense"", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| 汪燕等: ""响应面法优化茂源链霉菌产"", 《生物工程》 * |
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| CN106244567A (en) * | 2016-08-31 | 2016-12-21 | 安徽珍味奇调味食品有限公司 | A kind of glutamine transaminage process for separation and purification |
| 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 |
| CN110452890A (en) * | 2019-07-30 | 2019-11-15 | 泰兴市东圣生物科技有限公司 | It is a kind of through acidolysis or the pretreated Fermentation Medium for Transglutaminase of enzymatic hydrolysis and its preparation method and application |
| CN110616208A (en) * | 2019-11-04 | 2019-12-27 | 泰兴市东圣生物科技有限公司 | Optimized fermentation method of glutamine transaminase based on hydrodynamics |
| CN114686389A (en) * | 2020-12-25 | 2022-07-01 | 江苏东汇生物科技有限公司 | Glutamine transaminase high-producing strain for enhancing vgbS gene transcription level and preparation and fermentation methods thereof |
| CN114686389B (en) * | 2020-12-25 | 2024-05-07 | 泰兴市东圣生物科技有限公司 | Glutamine transaminase high-yield strain for enhancing transcription level of vgbS gene and preparation and fermentation methods thereof |
| CN112553176A (en) * | 2020-12-29 | 2021-03-26 | 江南大学 | Glutamine transaminase with improved thermal stability |
| CN112553176B (en) * | 2020-12-29 | 2022-04-29 | 江南大学 | Glutamine transaminase with improved thermal 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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