CN105754892B - A kind of isolation and purification method of glutamine of microbe transaminase - Google Patents
A kind of isolation and purification method of glutamine of microbe transaminase Download PDFInfo
- Publication number
- CN105754892B CN105754892B CN201610070332.6A CN201610070332A CN105754892B CN 105754892 B CN105754892 B CN 105754892B CN 201610070332 A CN201610070332 A CN 201610070332A CN 105754892 B CN105754892 B CN 105754892B
- Authority
- CN
- China
- Prior art keywords
- mtgase
- elution
- streptomycete
- buffer
- glutamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/625—Streptoverticillium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
- C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/02—Aminoacyltransferases (2.3.2)
- C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention discloses a kind of isolation and purification method of new glutamine of microbe transaminase, the new strain number that Natural Selection is obtained is the luxuriant source streptomycete fermentation liquid of CGMCC No.10804 after alcohol precipitation, anion-exchange chromatography is carried out, using obtaining the glutamine of microbe transaminase after desalination, freeze-drying.Obtained glutamine transaminage Chun Du≤90%, endotoxin Shui Ping≤0.02EU/mL, specific enzyme activity is between 25-30U/mg, Hui Shou Shuai≤70%.Present invention process is simple; it is at low cost; the enzyme activity rate of recovery is high; obtained glutamine transaminage purity is high; and the glutamine of microbe transaminase property that this strain fermentation obtains is stablized; it is convenient for industrialized production, provides a practicable method to meet the large-scale production of MTGase of pharmaceutical grade.
Description
Technical field
Field is isolated and purified the invention belongs to biotechnology, and in particular to new the produced glutamine of strain of one kind turns ammonia
The separating and purifying technology of enzyme.
Background technique
Glutamine transaminage (TransglutaminaSe, abbreviation TGase, EC2.3.2.13) it is a kind of catalyzing acyl turn
The transferase of shifting, can catalytic proteins intramolecular and intermolecular ε-(γ-glutaminyl) lysine covalent bond formation, from
And catalytic proteins intramolecular, it is intermolecular crosslink, and then change protein property and function.
Glutamine transaminage is widely present in plant, animal and microorganism.It is obtained in Guinea Pig Liver by scholar earliest
TGase is obtained, in source of the European a very long time as business TGase.Resource is rare multiple with the TGase of animal origin
Miscellaneous separation purifying technique causes the price of enzyme very high, so that being impossible to be applied in plant-scale food processing.Institute
Start to be dedicated to some scholars to obtain this enzyme from microorganism.Later Ando etc. is earliest from streptomyces verticillatus culture medium
It was found that Microbial transglutaminase (microbial transglutaminase, MTGase), to realize extensive
Fermenting and producing.Glutamine transaminage microbe-derived simultaneously has much compared with the glutamine transaminage of plant and animal material
Advantage, such as microbe-derived glutamine transaminage is calcium dependent/non-dependent enzyme, and substrate specificity is low.These properties make
MTGase is widely used in every field.
From Ando in 1989 etc. after having obtained glutamine transaminage in streptomyces verticillatus culture medium, experts and scholars
Find that other produce the streptomycete of TGase, such as streptomyces hygroscopicus and luxuriant source streptomycete one by one.Different streptomyces sources
The MTGase MTGase that even belongs to strepto- secretion not of the same race property it is also not exactly the same.Their thermal stability and
PH stability has very big difference, to influence the application in later period.The streptomycete of the production MTGase screened at present is not
It is all to be transformed into industrial production.So screening, producing enzyme is high-efficient, the task of the high streptomycete of MTGase stability
It is extremely urgent.And the luxuriant source streptomycete produced MTGase stability mentioned in the present invention screened from nature compared with
Preceding discovery will be high.
MTGase microbe-derived at present has applied in industrialized production.Microorganism generates through everfermentation
MTGase can apply in food processing after simple alcohol precipitation.But the MTGase purity that this method obtains
Low, impurity content is more 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 form glue resistant to high temperature；It can also be formed good porous with cross-linked gelatin
Mesh bonds (Chinese patent: 200780051215.4,200980131973.6 with wound for stopping blooding；United States Patent (USP):
8133484；European patent: WO2012017415, EP2303344)；Therefore Microbial transglutaminase can be used as potential
In the surgical operations such as the crosslinking agent of hemostatic material in medical use is for stopping blooding, wound bonds.But apply to field of food
The purity of MTGase is simultaneously unsatisfactory for the other requirement of pharmaceutical grade.Traditional separation purifying technique existing simultaneously is complicated, at high cost, system
Yield, purity and the Rate activity of enzyme be not high during standby, still containing there are many foreign proteins, it is difficult to meet and be used as medical material
Requirement.The present invention overcomes presently, there are the problem of, obtained a simple purifying process at low cost.
Summary of the invention
The purpose of the present invention is to provide it is a kind of it is at low cost, the rate of recovery is high, it is with high purity, be suitable for industrialized production and satisfaction
The Microbial transglutaminase purification process that pharmaceutical grade requires, solve current glutamine transaminage purifying process it is complicated,
Problem at high cost, the rate of recovery is low, purity is low.The microorganism casein hydrolyzated symbol that the method for the present invention isolates and purifies
Close the requirement of pharmaceutical grade.
In the method for the present invention, volume of the strain used in China Committee for Culture Collection of Microorganisms's common micro-organisms center
Number be CGMCC No.10804, be the luxuriant source streptomycete obtained by Natural Selection, utilize luxuriant source streptomycete producing microbial paddy ammonia
Amide transaminase (MTGase) is a kind of method general at present, and the characteristics of this bacterial strain is that its producing enzyme is high-efficient, produced
MTGase is compared with the produced MTGase of existing bacterial strain, and stability is high, as shown in Fig. 2, meeting the requirement of pharmaceutical grade.
The invention proposes a kind of luxuriant source streptomycete (Streptomyces sp.) bacterial strains, are that Natural Selection obtains from soil
The one plant of new streptomycete bacterial strain arrived, hypha form are as shown in Figure 1.It is existing that comparison by 16S rDNA sequence finds that it is different from
Other streptomycete bacterial strains having been reported that, and by neighbor-joining adjacent method construct based on bacterium proposed by the present invention
The systematic evolution tree of strain and related strain 16S rDNA sequence is as shown in Figure 5.The bacterial strain that this patent proposes is in
Streptomyces mobaraensis NBRC13819, Streptomyces mobaraensis NRRL B-3729 and
Unknown streptomycete between Streptomyces sp.J1S1, the bacterial strain in black box indicate strepto- proposed by the present invention
Bacterium can be speculated as new wild strain.
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and the deposit date is 2015 5
The moon 13, deposit number are CGMCC No.10804.The sequencing result such as SEQ ID of the cyclopentadienyl source streptomycete 16S rDNA
Shown in NO.1.
The invention also provides a kind of cultural methods of luxuriant source streptomycete: by the luxuriant source streptomycete in seed culture
24-48h is cultivated in base, cultivation temperature is 30 DEG C, then shaking speed 200r/min, then is transferred in fermentation medium and trains
45-52h is supported, the fermentation liquid of the luxuriant source streptomycete is obtained；Wherein, time containing the luxuriant source streptomycete in the fermentation liquid
Grade metabolite MTGase.
Specifically, the cultural method of the luxuriant source streptomycete are as follows: cultivate the bacterial strain that screening obtains in seed culture medium
The group of 24-48h, the seed culture medium become (weight percent): glycerol 1.5-2.5%, yeast extract 0.3-0.8%, fish meal
Peptone 2.1-2.7%, MgSO4·7H2O 0.15-0.4%, K2HPO40.15-2.3%, pH 7.4, cultivation temperature 30
DEG C, shaking speed 200r/min.Then, it then is transferred in fermentation medium and cultivates 45-52h, obtain a large amount of luxuriant source strepto-
The secondary metabolite MTGase of bacterium.Wherein, the group of the fermentation medium becomes (weight percent): 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%, promotor 1-2%, pH 7.4, cultivation temperature is 30 DEG C, shaking speed 200r/min.
The isolation and purification method of Microbial transglutaminase of the present invention the following steps are included:
1, take luxuriant source streptomycete fermentation liquid supernatant, handled with the dehydrated alcohol 1:1 of pre-cooling, after standing 1h, 8000r/mim from
Heart 15min collects precipitating, obtains the crude extract of MTGase；
2, the resulting crude extract of step 1 is dissolved in phosphate buffer, purifies instrument with AKTA avant 25 at room temperature and carries out
Anion-exchange chromatography, the purification column model Hitrap Q Sepharose Fast Flow (1mL) of selection, equalizing and buffering
The flow velocity of liquid, sample and elution buffer is 1mL/min, is collected with the mode of linear elution and to be occurred in elution process
First eluting peak；
3, the sample that step 2 recycling obtains is subjected to desalination, the desalting column model Hiprep 26/ of selection again
The flow velocity of 10Desalting, sample and ultrapure water is 10mL/min；
4, the sample that step 3 recycling obtains is freeze-dried, the glutamine of microbe for finally obtaining high-purity turns
Adnosine deaminase.
All buffers and sample will pass through the membrane filtration degerming of 0.22nm in purification step of the present invention.
Purified in the step 2 with anion-exchange chromatography method, compared with existing purification process, method is new
Grain husk, step are simple, it is only necessary to which the main anion-exchange chromatography of a step, which can be obtained by, meets pharmaceutical grade purity requirement
MTGase, and then the fine work MTGase of the available salt-free free from admixture of method by desalination and freeze-drying, and after freeze-drying
It is powdered to be more suitable for later period application, storage and transport.Used equilibration buffer is the phosphoric acid of 15-25mM pH6.5-8.0
Sodium buffer, elution buffer are the sodium phosphate buffer+1M sodium chloride of 15-25mM pH6.5-8.0, and elution process is linear
Elution, 0-20% elution buffer, elution volume are 25 column volumes, and the concentration of sample is 0.4-0.5mg/mL, unit enzyme activity
For 5-6U/mL, the total protein of loading is 10-15mg, total enzyme activity 100-300U.
Solution used in desalination is ultrapure water in the step 3, and sample loading volume is less than or equal to the 30% of column volume.
In freezing dry process in the step 4, first time sublimation temperature is -10 DEG C, and second of sublimation temperature is 4
℃。
In the present invention, using Hui Shou Shuai≤70% of the finally obtained MTGase of the method for the present invention, specific enzyme activity is in 25-30U/
Between mg, endogenous toxic material cellulose content≤0.02EU/mL, pure degree≤90%.Present invention process is simple, at low cost, and the enzyme activity rate of recovery is high,
Obtained glutamine transaminage purity is high, and the glutamine of microbe transaminase property that this strain fermentation obtains is stablized, just
In industrialized production, a practicable method is provided to meet the large-scale production of MTGase of pharmaceutical grade.
The invention proposes the applications that luxuriant source streptomycete is used to produce Microbial transglutaminase.
The invention proposes the Microbial transglutaminases obtained by above-mentioned method for purifying and separating.The present invention also proposes
Pass through the medical usage of Microbial transglutaminase obtained by the above method.It finally obtains after purification through the invention
MTGase can work with gelatin one for wound hemostasis and adhesive of medical, can be used for the biology of pharmaceutical protein
Modification etc..
In conclusion the beneficial effects of the present invention are: (1) the produced MTGase property of the bacterial strain that Natural Selection obtains is steady
It is fixed, since self stability causes the influence of enzyme activity loss smaller in fermentation liquid last handling process.(2) MTGase isoelectric point
About 8.0, in pH < 8.0, MTGase is theoretically positively charged, and used ion-exchange chromatography method is all sun in existing scheme
Ion-exchange chromatography, and purification process mentioned in the present invention is anion-exchange chromatography under the conditions of pH6.5-8.0.This
It is because the bacterial strain proposed in the present invention can take a large amount of negative electrical charge on surface under the conditions of pH6.5-8.0, thus with yin
Ion column combines, thus broken our conventional purification process and Purification: Principles, it is pure for MTGase purifying and other protein
Change provides a new approaches.(3) MTMase purification process is to pass through cation under the conditions of pH≤7.0 in existing scheme
Displacement chromatography or gel permeation chromatography method are purified, and a step purifying method is unable to get purity higher than 20U/mg's
MTGase, while obtained MTGase loss is larger, complex steps can not apply to industrial production.What is proposed in the present invention is pure
Change scheme, the available specific enzyme activity of one step of method of anion-exchange chromatography is greater than 25U/mg's under the conditions of pH6.5-8.0
MTGase, ult rec can achieve 70% or more, that is, using the purity is high for the enzyme that purification process of the invention obtains, return
High income.Method is simple, at low cost, industrial can amplify, be suitable for industrialized production.
Detailed description of the invention
Fig. 1 is growthform observation of the bacterial strain proposed by the present invention on ISP culture medium.
Fig. 2 produces MTGase (experimental group) and the existing source report Zhong Mao streptomycete institute by luxuriant source streptomycete proposed by the present invention
Produce the comparison of MTGase (control group 1:40587, control group 2:40847) enzyme stability at different temperatures.A is 70 DEG C of conditions
Lower difference MTGase stability as a result, B be 60 DEG C under the conditions of difference MTGase stability as a result, C be 50 DEG C under the conditions of not
With MTGase stability as a result, D is the result of difference MTGase stability under the conditions of 40 DEG C.
Fig. 3 is anion-exchange chromatography chromatogram.Penetrate foreign protein peak of the peak expression not in conjunction with anion chromatography column；It washes
De- peak 1 indicates destination protein peak；Eluting peak 2 indicates the foreign protein peak with the firm connection of anion chromatography column.
Fig. 4 is glutamine of microbe transaminase electrophoretogram in purification process.M represents protein standard maker；1 generation of swimming lane
Table fermentation liquid electrophoretogram；Swimming lane 2 represents alcohol precipitation product electrophoretogram；Swimming lane 3 represents purpose peak electricity after anion-exchange chromatography
Swimming figure；Swimming lane 4 represents MTGase electrophoretogram after desalination；Swimming lane 5 represents MTGase electrophoretogram after freeze-drying.
Fig. 5 is to be constructed by neighbor-joining adjacent method based on bacterial strain proposed by the present invention and related strain
The systematic evolution tree of 16S rDNA sequence.
Specific embodiment
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection scope.Implement mistake of the invention
Journey, condition, reagent, experimental method etc. are in addition to what is specifically mentioned below the universal knowledege of this field and known
Common sense, there are no special restrictions to content by the present invention.
1 alcohol precipitation of embodiment obtains MTGase crude extract
(1) fermentation liquid 8000g, the 4 DEG C of centrifugation 10min for obtaining fermentation, removal precipitating；PH to 5.0 is slowly adjusted with phosphoric acid,
8000g, 4 DEG C of centrifugation 10min, removal precipitating；The dehydrated alcohol being pre-chilled in advance is slowly added to 1:1, is placed in ice chest placement
1h, 8000g, 4 DEG C of centrifugation 10min remove supernatant.
(2) for SDS-PAGE testing result as shown in swimming lane 2 in Fig. 4, MTGase molecular weight is about 38KDa, can from swimming lane 2
Contain a large amount of foreign protein in crude extract that alcohol precipitation obtains to find out.Alcohol precipitation primary fermentation liquid total enzyme activity is 2760.6U,
Total enzyme activity is 2484.5U after alcohol precipitation, and concrete outcome is as shown in table 1.Enzyme activity determination living as the result is shown return by this step total enzyme activity
Yield is 90%.
The stable detection of embodiment 2MTGase crude extract temperature
(1) phosphorus of pH6.0 is dissolved in after the MTGase (experimental group) that 1 alcohol precipitation of embodiment obtains being lyophilized with 3U/mL
In acid buffer, at the same choose two currently reported any two kinds of MTGase (control group 1:40587, control group 2:
40847 are all from Taixing Dongsheng Food Science & Technology Co., Ltd.) as control, the phosphoric acid of pH6.0 is equally dissolved in 3U/mL
In buffer.
(2) three kinds of MTGase are respectively placed in 40 DEG C, 50 DEG C, 60 DEG C and 70 DEG C of water-bath, as shown in Figure 2 each
Time interval under the conditions of temperature saves MTGase, unifies the enzyme activity for surveying MTGase after a certain period of time.A be 70 DEG C under the conditions of not
With MTGase stability as a result, B be 60 DEG C under the conditions of difference MTGase stability as a result, C be 50 DEG C under the conditions of it is different
MTGase stability as a result, D be 40 DEG C under the conditions of difference MTGase stability result.
As shown in Fig. 2, when placing 75s, the stability of heretofore described MTGase is than control under the conditions of 70 DEG C
The 40587 of group 1 are high by 23%, and 40847 than control group 2 are high by 19%；Under the conditions of 60 DEG C, the institute in the present invention when placing 5min
The stability of the MTGase stated is higher than 40587 by 22%, and 40847 than control group 2 are high by 25%；Under the conditions of 50 DEG C, placing
The stability of heretofore described MTGase is 40587 higher by 18% than control group 1 when 120min, than the 40847 of control group 2
It is high by 21%；Under the conditions of 40 DEG C, when placing 4d, the stability of heretofore described MTGase is 40587 higher than control group 1
14%, 40847 than control group 2 are high by 15%.In conclusion under the conditions of certain temperature, heretofore described MTGase's
Stability is above other two kinds of enzyme powders.
3 anion-exchange chromatography method of embodiment obtains high-purity MTGase after purification
(1) the purifying instrument selected is AM General companyAvant 25 purifies chromatographic column used as purchase
From the prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) of AM General company.
(2) sodium dihydrogen phosphate of suitable 20mM and the disodium hydrogen phosphate of 20mM are prepared respectively, by two kinds of buffers press than
Example mixing is finally formulated as the phosphate buffer of pH6.5, conductance about 2.5mS/cm.The membrane filtration of 0.22um is used after preparing,
Then deaerate 20-30min in Ultrasound Instrument.4 DEG C of refrigerators save backup.
(3) 1M sodium chloride is added on the basis of equilibration buffer, its pH is adjusted to 6.5 with phosphoric acid again after completely dissolution,
Conductance about 75.0mS/cm.The membrane filtration of 0.22um is used after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of ice
Case saves backup.
(4) the MTGase crude extract that alcohol precipitation obtains is dissolved in the 20mM phosphate buffer of pH6.5, is sufficiently dissolved
4 DEG C of centrifugation 10min of 8000r/min afterwards, are then filtered enzyme solution with the filter membrane of 0.22um, then deaerate in Ultrasound Instrument
20-30min.4 DEG C of refrigerators save backup.Preparing obtained sample protein concentration is 0.36mg/mL, and unit enzyme activity is 4.81U/
mL。
(5) first anion chromatographic column is balanced with the equilibration buffer of 20CV, takes pre-prepared enzyme solution 30mL
Then loading will be got off with the equilibration buffer of 10CV not in conjunction with chromatographic column or in conjunction with unstable albumen wash-out, such as Fig. 3
Shown in penetrate peak, then eluted with the elution buffer of 25CV in the method for linear elution, at this time sodium chloride ionic strength
Increase to 0.2M from 0, obtained eluting peak eluting peak 1 as shown in Figure 3, finally with the sodium chloride elution of 1M and chromatographic column jail
Consolidate the foreign protein closed, obtained eluting peak eluting peak 2 as shown in Figure 3.Flow velocity during all tests is all 1mL/
min.Collecting eluting peak 1 is MTGase after purification.
(6) as shown in the swimming lane 3 in Fig. 4, the sample purified is SDS-PAGE testing result through gray scale scanning purity
92%, all samples obtained after alcohol precipitation total enzyme activity rate of recovery after this step is 89.6%, specific enzyme activity 26.34U/
mg。
4 anion-exchange chromatography method of embodiment obtains high-purity MTGase after purification
(1) the purifying instrument selected is AM General companyAvant 25 purifies chromatographic column used as purchase
From the prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) of AM General company.
(2) sodium dihydrogen phosphate of suitable 20mM and the disodium hydrogen phosphate of 20mM are prepared respectively, by two kinds of buffers press than
Example mixing is finally formulated as the phosphate buffer of pH7.0, conductance about 2.5mS/cm.The membrane filtration of 0.22um is used after preparing,
Then deaerate 20-30min in Ultrasound Instrument.4 DEG C of refrigerators save backup.
(3) 1M sodium chloride is added on the basis of equilibration buffer, its pH is adjusted to 7.0 with phosphoric acid again after completely dissolution,
Conductance about 75.0mS/cm.The membrane filtration of 0.22um is used after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of ice
Case saves backup.
(4) the MTGase crude extract that alcohol precipitation obtains is dissolved in the 20mM phosphate buffer of pH7.0, is sufficiently dissolved
4 DEG C of centrifugation 10min of 8000r/min afterwards, are then filtered enzyme solution with the filter membrane of 0.22um, then deaerate in Ultrasound Instrument
20-30min.4 DEG C of refrigerators save backup.Preparing obtained sample protein concentration is 0.42mg/mL, and unit enzyme activity is 5.09U/
mL。
(5) first anion chromatographic column is balanced with the equilibration buffer of 20CV, takes pre-prepared enzyme solution 30mL
Then loading will be got off with the equilibration buffer of 10CV not in conjunction with chromatographic column or in conjunction with unstable albumen wash-out, such as Fig. 3
Shown in penetrate peak, then eluted with the elution buffer of 25CV in the method for linear elution, at this time sodium chloride ionic strength
Increase to 0.2M from 0, obtained eluting peak eluting peak 1 as shown in Figure 3, finally with the sodium chloride elution of 1M and chromatographic column jail
Consolidate the foreign protein closed, obtained eluting peak eluting peak 2 as shown in Figure 3.Flow velocity during all tests is all 1mL/
min.Collecting eluting peak 1 is MTGase after purification.
(6) as shown in the swimming lane 3 in Fig. 4, the sample purified is SDS-PAGE testing result through gray scale scanning purity
90%, all samples obtained after alcohol precipitation total enzyme activity rate of recovery after this step is 92.0%, specific enzyme activity 28.61U/
Mg, concrete outcome are as shown in table 1.
5 anion-exchange chromatography method of embodiment obtains high-purity MTGase after purification
(1) the purifying instrument selected is AM General companyAvant 25 purifies chromatographic column used as purchase
From the prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) of AM General company.
(2) sodium dihydrogen phosphate of suitable 20mM and the disodium hydrogen phosphate of 20mM are prepared respectively, by two kinds of buffers press than
Example mixing is finally formulated as the phosphate buffer of pH7.5, conductance about 2.5mS/cm.The membrane filtration of 0.22um is used after preparing,
Then deaerate 20-30min in Ultrasound Instrument.4 DEG C of refrigerators save backup.
(3) 1M sodium chloride is added on the basis of equilibration buffer, its pH is adjusted to 7.5 with phosphoric acid again after completely dissolution,
Conductance about 75.0mS/cm.The membrane filtration of 0.22um is used after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of ice
Case saves backup.
(4) the MTGase crude extract that alcohol precipitation obtains is dissolved in the 20mM phosphate buffer of pH7.5, is sufficiently dissolved
4 DEG C of centrifugation 10min of 8000r/min afterwards, are then filtered enzyme solution with the filter membrane of 0.22um, then deaerate in Ultrasound Instrument
20-30min.4 DEG C of refrigerators save backup.Preparing obtained sample protein concentration is 0.4mg/mL, and unit enzyme activity is 5.19U/mL.
(5) first anion chromatographic column is balanced with the equilibration buffer of 20CV, takes pre-prepared enzyme solution 30mL
Then loading will be got off with the equilibration buffer of 10CV not in conjunction with chromatographic column or in conjunction with unstable albumen wash-out, such as Fig. 3
Shown in penetrate peak, then eluted with the elution buffer of 25CV in the method for linear elution, at this time sodium chloride ionic strength
Increase to 0.2M from 0, obtained eluting peak eluting peak 1 as shown in Figure 3, finally with the sodium chloride elution of 1M and chromatographic column jail
Consolidate the foreign protein closed, obtained eluting peak eluting peak 2 as shown in Figure 3.Flow velocity during all tests is all 1mL/
min.Collecting eluting peak 1 is MTGase after purification.
(6) as shown in the swimming lane 3 in Fig. 4, the sample purified is SDS-PAGE testing result through gray scale scanning purity
93%, all samples obtained after alcohol precipitation total enzyme activity rate of recovery after this step is 91.4%, specific enzyme activity 28.21U/
mg。
6 anion-exchange chromatography method of embodiment obtains high-purity MTGase after purification
(1) the purifying instrument selected is AM General companyAvant 25 purifies chromatographic column used as purchase
From the prepackage anion-exchange column Hiprap Q Sepharose FF (1mL) of AM General company.
(2) sodium dihydrogen phosphate of suitable 20mM and the disodium hydrogen phosphate of 20mM are prepared respectively, by two kinds of buffers press than
Example mixing is finally formulated as the phosphate buffer of pH8.0, conductance about 2.5mS/cm.The membrane filtration of 0.22um is used after preparing,
Then deaerate 20-30min in Ultrasound Instrument.4 DEG C of refrigerators save backup.
(3) 1M sodium chloride is added on the basis of equilibration buffer, its pH is adjusted to 8.0 with phosphoric acid again after completely dissolution,
Conductance about 75.0mS/cm.The membrane filtration of 0.22um is used after preparing, then deaerate in Ultrasound Instrument 20-30min.4 DEG C of ice
Case saves backup.
(4) the MTGase crude extract that alcohol precipitation obtains is dissolved in the 20mM phosphate buffer of pH8.0, is sufficiently dissolved
4 DEG C of centrifugation 10min of 8000r/min afterwards, are then filtered enzyme solution with the filter membrane of 0.22um, then deaerate in Ultrasound Instrument
20-30min.4 DEG C of refrigerators save backup.Preparing obtained sample protein concentration is 0.42mg/mL, and unit enzyme activity is 5.09U/
mL。
(5) first anion chromatographic column is balanced with the equilibration buffer of 20CV, takes pre-prepared enzyme solution 30mL
Then loading will be got off with the equilibration buffer of 10CV not in conjunction with chromatographic column or in conjunction with unstable albumen wash-out, such as Fig. 3
Shown in penetrate peak, then eluted with the elution buffer of 25CV in the method for linear elution, at this time sodium chloride ionic strength
Increase to 0.2M from 0, obtained eluting peak eluting peak 1 as shown in Figure 3, finally with the sodium chloride elution of 1M and chromatographic column jail
Consolidate the foreign protein closed, obtained eluting peak eluting peak 2 as shown in Figure 3.Flow velocity during all tests is all 1mL/
min.Collecting eluting peak 1 is MTGase after purification.
(6) as shown in the swimming lane 3 in Fig. 4, the sample purified is SDS-PAGE testing result through gray scale scanning purity
87%, all samples obtained after alcohol precipitation total enzyme activity rate of recovery after this step is 94.7%, specific enzyme activity 27.81U/
mg。
The salt-free MTGase of high-purity is obtained after 7 desalination of embodiment
(1) the purifying instrument selected is AM General companyAvant 25, desalting column are purchased from AM General
The prepacked column Hiprep 26/10Desalting of company.
(2) first desalting column is balanced with the flow velocity of 10mL/min with ultrapure water, then hands over anion in embodiment 5
The direct loading of sample that chromatography obtains is changed, loading total volume is 12mL, and loading total enzyme activity is 100U, and loading total protein is 5mg.
(3) the corresponding sample of protein peak, sample of the SDS-PAGE testing result as shown in swimming lane 4 in Fig. 4, after desalination are collected
It is 92% through gray scale scanning purity, all samples obtained after anion-exchange chromatography total enzyme activity rate of recovery after this step is
95%, concrete outcome is as shown in table 1.
The glutamine transaminage rate of recovery during the present invention isolates and purifies, as shown in table 1:
1 MTGase purification result of table
Note: alcohol precipitation is the result in embodiment 1；Anion-exchange chromatography is the result in embodiment 4；Desalination is real
Apply the result in example 7.
In conclusion the isolation and purification method of the new glutamine of microbe transaminase of one kind proposed by the present invention has obviously
Beneficial effect.Firstly, it can be seen that, the MTGase stability proposed in the present invention is apparently higher than other types from embodiment 2
MTGase, different temperatures stability inferior is higher by 10-20%, and property is stablized, no matter medical in fermentation liquid post-processing or later period
There is higher stability in, meets pharmaceutical grade requirement.Secondly, by embodiment 2-6 it can be found that in pH6.5-8.0
Under the conditions of it is available than the MTGase living for being greater than 25U/mg with one step of method of anion-exchange chromatography, process is a series of
The ult rec that processing finally obtains the solid enzyme powder for meeting pharmaceutical grade requirement can achieve 70% or more, with existing literature
Report is compared, and has apparent advantage.Obtain the higher MTGase's of purity by different purification schemes in existing literature report
The rate of recovery 70% hereinafter, and this result is that no desalination and freeze-drying before as a result, and mentioned in the present invention
The rate of recovery that method obtains merely the MTGase of high-purity can achieve 80% or more, obtain even across desalination and freeze-drying
It obtaining dry MTGase enzyme powder ult rec and also reaches 70% or more, the rate of recovery is high, and method is simple, and it is at low cost, it can industry
Amplification is suitable for industrialized production.Finally, MTGase isoelectric point is 8.0, used ion-exchange chromatography in existing scheme
Method is all cation-exchange chromatography, and purification process mentioned in the present invention is to be handed under the conditions of pH6.5-8.0 with anion
Chromatography is changed, our conventional purification process and Purification: Principles have been broken, is provided for MTGase purifying and other protein purifications
One new approaches.
Claims (6)
1. a kind of cyclopentadienyl source streptomycete (Streptoverticillium mobaraense) bacterial strain, which is characterized in that be from soil
One plant of new streptomycete bacterial strain that middle Natural Selection obtains is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center, the deposit date is on May 13rd, 2015, deposit number was CGMCC No.10804.
2. a kind of luxuriant source streptomycete (Streptoverticillium mobaraense) bacterial strain paddy ammonia as described in claim 1
The isolation and purification method of amide transaminase, which is characterized in that the described method comprises the following steps:
(1) the luxuriant source streptomycete fermentation liquid supernatant is taken, is handled with the dehydrated alcohol 1:1 of pre-cooling, ice chest is placed in and places 1h,
8000g, 4 DEG C of centrifugation 10min collect precipitating, obtain the crude extract of MTGase；
(2) step (1) resulting crude extract is dissolved in the phosphate buffer of pH 6.5-8.0, after completely dissolution 8000r/min
4 DEG C of centrifugation 10min, are then filtered with 0.22 μm of filter membrane, and then deaerate 20-30min, use AKTAavant 25 at room temperature
Purification system carries out anion-exchange chromatography, the purification column model anion-exchange column Hitrap Q Sepharose of selection
Fast Flow 1mL, the flow velocity of equilibration buffer, sample and elution buffer are 1mL/min, with the mode of linear elution
Collect first eluting peak occurred in elution process；
Wherein, the specific steps that first eluting peak occurred in elution process is collected with the mode of linear elution are as follows:
First anion chromatographic column is balanced with the equilibration buffer of 20CV, then with the equilibration buffer of 10CV will not with color
Spectrum column combines or gets off in conjunction with unstable albumen wash-out, then is washed with the elution buffer of 25CV in the method for linear elution
De-, sodium chloride ionic strength increases to 0.2M from 0 at this time, finally with the miscellaneous egg of the sodium chloride elution and chromatographic column firm connection of 1M
White, collecting first eluting peak is MTGase after purification；
(3) sample that step (2) recycling obtains is subjected to desalination, the desalting column model Hiprep 26/10 of selection again
The flow velocity of Desalting, sample and ultrapure water is 10mL/min；
(4) sample that step (3) recycling obtains is freeze-dried, finally obtains the luxuriant source streptomycete glutamine of high-purity
Transaminase.
3. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute
0.22 μm of film filtration sterilization will be passed through by stating sample all in method and used buffer.
4. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute
State the sodium phosphate buffer for the 15-25mM that the equilibration buffer in step (2) is pH6.5-8.0, elution buffer pH6.5-
Sodium phosphate buffer+1M the sodium chloride of 8.0 15-25mM, elution process are linear elution, 0-20% elution buffer, elution
25 column volumes, the concentration of sample are 0.4-0.5mg/mL, and unit enzyme activity is 5-6U/mL, and the total protein of loading is 10-15mg,
Total enzyme activity is 100-300U；
Wherein, the preparation method of the equilibration buffer are as follows: prepare the sodium dihydrogen phosphate of 20mM and the phosphoric acid hydrogen two of 20mM respectively
Two kinds of buffers are mixed the sodium phosphate buffer for being finally formulated as the 15-25mM of the pH6.5-8.0 by sodium in proportion.
5. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute
Stating solution used in desalination in step (3) is ultrapure water, and sample loading volume is less than or equal to the 30% of column volume.
6. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute
It states in the freezing dry process in step (4), first time sublimation temperature is -10 DEG C, and second of sublimation temperature is 4 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610070332.6A CN105754892B (en) | 2016-02-01 | 2016-02-01 | A kind of isolation and purification method of glutamine of microbe transaminase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610070332.6A CN105754892B (en) | 2016-02-01 | 2016-02-01 | A kind of isolation and purification method of glutamine of microbe transaminase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105754892A CN105754892A (en) | 2016-07-13 |
| CN105754892B true CN105754892B (en) | 2019-09-06 |
Family
ID=56342997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610070332.6A Active CN105754892B (en) | 2016-02-01 | 2016-02-01 | A kind of isolation and purification method of glutamine of microbe transaminase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105754892B (en) |
Families Citing this family (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 |
| 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 |
| 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 |
| CN112553176B (en) * | 2020-12-29 | 2022-04-29 | 江南大学 | Glutamine transaminase with improved thermal stability |
| 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 |
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 |
|---|
| "Medium Design Based on Stoichiometric Analysis of Microbial Transglutaminase Production by Streptoverticillium mobaraense";Y. Zhu et al.,;《Biotechnology and Bioengineering》;19961231;第50卷;第291-298页 * |
| "响应面法优化茂源链霉菌产";汪燕等;《生物工程》;20151231;第36卷(第16期);第203-207页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105754892A (en) | 2016-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105754892B (en) | A kind of isolation and purification method of glutamine of microbe transaminase | |
| CN108220371B (en) | Preparation method of pea peptide with prebiotic effect and application of pea peptide in food | |
| JPH05500752A (en) | A thermostable purified endoglucanase from the thermophilic bacterium Acidothermus cellulolyticus | |
| Moreira et al. | Production of cellulases by Thermomonospora sp. | |
| JPS59156282A (en) | Preparation of novel heat-resistant lipase | |
| US20210095269A1 (en) | Method For Isolating DNA By Using Cas Protein System | |
| CN105542010A (en) | Lipoprotein-associated phospholipase A2 monoclonal antibodies, antibody pairs, preparation method and application | |
| CN116286560B (en) | Raoult bacterium HC6 and application thereof in low-temperature production of 2, 3-butanediol | |
| Gopinath et al. | Isolation, Screening and Purification of Cellulase from Cellulase Producing Klebsiella variicola RBEB3 (KF036184. 1). | |
| CN113956994B (en) | Two bacterial strains capable of breaking carbon glycoside bond of flavonoid carbon glycoside compound and application thereof | |
| CN105985939B (en) | A kind of its purposes of poly (hydroxyalkanoate) pellet degradation enzyme and (R) -3HB production method | |
| Osowiecki et al. | The mechanism of competence in the transformation of Streptococci of serological group H: Purification and some properties of the competence factor | |
| CN102994408B (en) | Carrageenan degrading bacterium and fermentation method and application thereof | |
| Eze et al. | Purification and Enzymatic Properties of β-galactosidase Produced from Lactobacillus acidophilus isolated from Dairy Waste-Water | |
| CN114574394B (en) | Pseudomonas for producing dye decolorized peroxidase and application thereof | |
| JP6997079B2 (en) | Composition for medium | |
| Subathra Devi et al. | Studies on growth kinetics of Serratia marcescens VITSD2 and optimization of fermentation conditions for serratiopeptidase production | |
| Dolia et al. | Aqueous two‐phase partitioning of glucose isomerase from actinoplanes missouriensis in the presence of PEG‐derivatives and its immobilization on chitosan beads | |
| CN116426500B (en) | Lipase mutant with high esterification capability and expression application thereof | |
| CN104099306A (en) | Alpha transglucosidase and recombinant expression bacterial strain thereof | |
| CN114807267B (en) | Simultaneous preparation of neokestose and 1 method for preparing F-fructo-oligosaccharide and special engineering strain thereof | |
| CN116515795B (en) | Application of Aspergillus tubingensis in preparing phytase and/or degrading phytic acid | |
| RU2141526C1 (en) | STRAIN OF BACTERIUM FLAVOBACTERIUM SPECIES - PRODUCER OF α-N-ACETYLGALACTOSE AMINIDASE | |
| Baskar et al. | Production and purification of fungal milk clotting enzyme from Aspergillus candidus | |
| JP2000078971A (en) | New enzyme for synthesizing oligopeptide from d- or l- aminoacid ester and microorganism producing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 200062 Putuo District, Zhongshan North Road, No. 3663, Applicant after: East China Normal University Applicant after: Taixing East Biological Technology Co., Ltd. Address before: 200062 Putuo District, Zhongshan North Road, No. 3663, Applicant before: East China Normal University Applicant before: Taixing Dongsheng Food Science and Technology Co., Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 200241 No. 500, Dongchuan Road, Shanghai, Minhang District Co-patentee after: TAIXING DONGSHENG BIO-TECH Co.,Ltd. Patentee after: EAST CHINA NORMAL University Address before: 200062 No. 3663, Putuo District, Shanghai, Zhongshan North Road Co-patentee before: TAIXING DONGSHENG BIO-TECH Co.,Ltd. Patentee before: EAST CHINA NORMAL University |