CN105349504B - A method of transglutaminase is prepared using krill - Google Patents
A method of transglutaminase is prepared using krill Download PDFInfo
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- CN105349504B CN105349504B CN201510950511.4A CN201510950511A CN105349504B CN 105349504 B CN105349504 B CN 105349504B CN 201510950511 A CN201510950511 A CN 201510950511A CN 105349504 B CN105349504 B CN 105349504B
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- transglutaminase
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- 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
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- 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
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Abstract
The invention discloses a kind of methods using krill preparation transglutaminase.It is that the krill cleaned is shredded to addition buffer to be homogenized, centrifugation, take clear liquid cellulose acetate film filtering removal upper layer turbid, obtained filtrate dialyses to it with sodium acetate dialyzate, ultrafiltration is carried out using ultrafiltration apparatus again, after purification, obtained refined solution is concentrated by ultrafiltration by ion exchange chromatography for filtered solution, it is freeze-dried the transglutaminase to get purifying.Method and process of the invention is reasonable, easy to operate, and preparation speed is fast, and the transglutaminase purity and the enzyme activity rate of recovery extracted are high.
Description
Technical field
The present invention relates to transferase, especially a kind of method using krill preparation transglutaminase.
Background technique
Transglutaminase is that one kind is widely present in animal tissue and the intracorporal class of enzymes of human body fluid, it can be catalyzed egg
Between white matter molecule, the polymerization of covalent cross-linking between polypeptide and various primary amine, it and blood clotting, wound healing, epidermal keratinocytes
A variety of biological phenomenons such as change, erythrocyte membrane hardening are related.In recent years, transglutaminase has caused numerous technical staff's
Interest has very big application potential in food service industry, enzyme immobilizatio and textile industry.
It extracts from animal vegetable tissue and is given birth to by microbe fermentation method currently, the extracting method of transglutaminase mainly has
It produces.Wherein, the most extensive by Production by Microorganism Fermentation, but condition is difficult to control, and turns paddy by Production by Microorganism Fermentation
Transglutaminase is unable to reach the intensity for the bioactivity extracted from animal vegetable tissue.Have in the prior art from cavy and fish tissues
Middle preparation transglutaminase, but its research is still in the primary stage.
Krill it is full of nutrition, be high protein foods.Up to the present, it has not been found that being made from krill
The report of standby transglutaminase.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods rationally, preparation speed is fast, benefit easy to operate, obtaining object purity is high
With the method for krill preparation transglutaminase.
The technical solution adopted by the present invention to solve the technical problems are as follows: a kind of to prepare transglutamin-ase 9 using krill
The method of enzyme, it is characterised in that: it passes through following process steps:
(1) feedstock processing: selection krill is raw material, is cleaned with clear water, and chopping is added buffer, is homogenized, from
The heart collects clear liquid；Wherein, the ratio of the buffer and krill is 1 ︰ 2~3 (m/v)；
(2) it filters: clear liquid obtained in step (1) being filtered using cellulose acetate film, collects filtrate；
(3) it dialyses: filtrate obtained in step (2) being dialysed using sodium acetate dialyzate, wherein filtrate and acetic acid
Sodium dialysate volumes ratio is 1:20~30, and 4 DEG C of dialysed overnights obtain krill protein crude extract；
(4) ultrafiltration: use molecule interception for 10000 dalton krill crude extract obtained in step (3)
Ultrafiltration apparatus carries out ultra-filtration and separation, retains the impurity including small protein, obtains krill protein filtered solution；
(5) krill protein filtered solution obtained in step (4) cation exchange chromatography: is used into strong cation
Exchanger resin carries out ion-exchange chromatography, using elution, collects absorption peak, obtains transglutaminase refined solution；
(6) be concentrated by ultrafiltration: it is 10000 that transglutaminase refined solution obtained in step (5), which is passed through molecule interception,
The ultrafiltration apparatus of dalton is concentrated by ultrafiltration, and transglutaminase concentrate is obtained；
(7) it is freeze-dried: 80~90% ethyl alcohol of transglutaminase concentrate obtained in step (6) is settled, from
The heart collects precipitating；Pellet frozen is dry, obtain the transglutaminase of purifying.
The homogenate is the krill feeding refiner that buffer will be added, and controls 4000~6000r/min of revolving speed,
It is homogenized 10~30min.
The centrifugation is that the krill after being homogenized is sent into centrifuge, controls 6000~8000r/min of revolving speed, centrifugation
10~15min.
The buffer is that 8mM NaCl and 15mM Tris-HCl is dissolved in 1000ml deionized water to be made.
The sodium acetate dialyzate is to be dissolved in 20mM sodium acetate, 45mM NaCl, 2mM EDTA and 0.5mM DTT
In 1000ml deionized water, adjusts pH value and be made for 6.5.
The eluent is the Tris-HCl of 0.2mol/L.
The present invention is added buffer to the krill chopping cleaned and is homogenized, and is centrifuged, takes clear liquid acetate fiber
Film filtering removal upper layer turbid, obtained filtrate dialyses to it with sodium acetate dialyzate, then is surpassed using ultrafiltration apparatus
After purification, obtained refined solution is concentrated by ultrafiltration by ion exchange chromatography, is freeze-dried to get purifying for filter, filtered solution
Transglutaminase.The present invention uses cellulose acetate film to filter during the preparation process, can guarantee that adsorbance is minimum, will not inhale
Attached destination protein；Using ion-exchange chromatography, high sensitivity, repeated, selective good, analysis speed is fast；Using twice
Ultrafiltration can make sample be concentrated and be removed impurity.Side using krill preparation transglutaminase of the invention
Method, preparation process is reasonable, easy to operate, and preparation speed is fast, and the transglutaminase purity extracted is higher, after measured,
Its enzyme activity rate of recovery is 75~85%.
Specific embodiment
Below with reference to embodiment, the present invention will be further described.
Embodiment 1
A method of transglutaminase being prepared using krill, passes through following process steps:
(1) feedstock processing: selection krill is raw material, is cleaned with clear water, and chopping is added buffer, is sent to homogenate
In machine, revolving speed 6000r/min is controlled, is homogenized 20min；Krill after homogenate is sent into centrifuge, controls revolving speed 8000r/
Min is centrifuged 10min；Impurity is removed, clear liquid is collected；Wherein, buffer is to be dissolved in 8mM NaCl and 15mM Tris-HCl
It is prepared in 1000ml deionized water, the ratio of buffer and krill is 1 ︰ 2.5 (m/v)；
(2) it filters: clear liquid obtained in step (1) being filtered using cellulose acetate film, upper layer turbid is removed, collects filter
Liquid；
(3) it dialyses: filtrate obtained in step (2) being dialysed using sodium acetate dialyzate, wherein sodium acetate dialysis
Liquid is that 20mM sodium acetate, 45mM NaCl, 2mM EDTA and 0.5mM DTT are dissolved in 1000ml deionized water, adjusts pH value and is
6.5 being prepared；Filtrate and sodium acetate dialysate volumes ratio are 1:25, and 4 DEG C of dialysed overnights obtain krill protein and slightly mention
Liquid；
(4) ultrafiltration: use molecule interception for 10000 dalton krill crude extract obtained in step (3)
Ultrafiltration apparatus carries out ultra-filtration and separation, retains the impurity including small protein, obtains krill protein filtered solution；
(5) cation exchange chromatography: by krill protein filtered solution obtained in step (4) using GE company
Strong cation-exchanging resin carries out ion-exchange chromatography, is eluted using the Tris-HCl of 0.2mol/L as eluent, receives
Collect absorption peak, obtains transglutaminase refined solution；
(6) be concentrated by ultrafiltration: it is 10000 that transglutaminase refined solution obtained in step (5), which is passed through molecule interception,
The ultrafiltration apparatus of dalton is concentrated by ultrafiltration, and transglutaminase concentrate is obtained；
(7) it is freeze-dried: 85% ethyl alcohol of transglutaminase concentrate obtained in step (6) is settled, be centrifuged, receive
Collection precipitating；Pellet frozen is dry, obtain the transglutaminase of purifying.
The method of krill preparation transglutaminase, rational technology, operation letter are utilized provided by the present embodiment
It is single, isolate and purify that speed is fast, the transglutaminase purity extracted is higher.After measured, the enzyme activity rate of recovery is 82.5%.
Embodiment 2
A method of transglutaminase being prepared using krill, passes through following process steps:
(1) feedstock processing: selection krill is raw material, is cleaned with clear water, and chopping is added buffer, is sent to homogenate
In machine, revolving speed 5000r/min is controlled, is homogenized 10min；Krill after homogenate is sent into centrifuge, controls revolving speed 7000r/
Min is centrifuged 12min；Impurity is removed, clear liquid is collected；Wherein, buffer is to be dissolved in 8mM NaCl and 15mM Tris-HCl
It is prepared in 1000ml deionized water, the ratio of buffer and krill is 1 ︰ 3 (m/v)；
(2) it filters: clear liquid obtained in step (1) being filtered using cellulose acetate film, upper layer turbid is removed, collects filter
Liquid；
(3) it dialyses: filtrate obtained in step (2) being dialysed using sodium acetate dialyzate, wherein sodium acetate dialysis
Liquid is that 20mM sodium acetate, 45mM NaCl, 2mM EDTA and 0.5mM DTT are dissolved in 1000ml deionized water, adjusts pH value and is
6.5 being prepared；Filtrate and sodium acetate dialysate volumes ratio are 1:30, and 4 DEG C of dialysed overnights obtain krill protein and slightly mention
Liquid；
(4) ultrafiltration: use molecule interception for 10000 dalton krill crude extract obtained in step (3)
Ultrafiltration apparatus carries out ultra-filtration and separation, retains the impurity including small protein, obtains krill protein filtered solution；
(5) krill protein filtered solution obtained in step (4) cation exchange chromatography: is used into Dowex 50
Storng-acid cation exchange resin carries out ion-exchange chromatography, is washed using the Tris-HCl of 0.2mol/L as eluent
It is de-, absorption peak is collected, transglutaminase refined solution is obtained；
(6) be concentrated by ultrafiltration: it is 10000 that transglutaminase refined solution obtained in step (5), which is passed through molecule interception,
The ultrafiltration apparatus of dalton is concentrated by ultrafiltration, and transglutaminase concentrate is obtained；
(7) it is freeze-dried: 90% ethyl alcohol of transglutaminase concentrate obtained in step (6) is settled, be centrifuged, receive
Collection precipitating；Pellet frozen is dry, obtain the transglutaminase of purifying.
The method of krill preparation transglutaminase, rational technology, preparation speed are utilized provided by the present embodiment
Degree is fast.After measured, the enzyme activity rate of recovery is 78%.
Embodiment 3
A method of transglutaminase being prepared using krill, passes through following process steps:
(1) feedstock processing: selection krill is raw material, is cleaned with clear water, and chopping is added buffer, is sent to homogenate
In machine, revolving speed 4000r/min is controlled, is homogenized 30min；Krill after homogenate is sent into centrifuge, controls revolving speed 6000r/
Min is centrifuged 15min；Impurity is removed, clear liquid is collected；Wherein, buffer is to be dissolved in 8mM NaCl and 15mM Tris-HCl
It is prepared in 1000ml deionized water, the ratio of buffer and krill is 1 ︰ 2 (m/v)；
(2) it filters: clear liquid obtained in step (1) being filtered using cellulose acetate film, upper layer turbid is removed, collects filter
Liquid；
(3) it dialyses: filtrate obtained in step (2) being dialysed using sodium acetate dialyzate, wherein sodium acetate dialysis
Liquid is that 20mM sodium acetate, 45mM NaCl, 2mM EDTA and 0.5mM DTT are dissolved in 1000ml deionized water, adjusts pH value and is
6.5 being prepared；Filtrate and sodium acetate dialysate volumes ratio are 1:20, and 4 DEG C of dialysed overnights obtain krill protein and slightly mention
Liquid；
(4) ultrafiltration: use molecule interception for 10000 dalton krill crude extract obtained in step (3)
Ultrafiltration apparatus carries out ultra-filtration and separation, retains the impurity including small protein, obtains krill protein filtered solution；
(5) cation exchange chromatography: by krill protein filtered solution obtained in step (4) using commercially available strong
Cation exchange resin carries out ion-exchange chromatography, is eluted using the Tris-HCl of 0.2mol/L as eluent, collects
Absorption peak obtains transglutaminase refined solution；
(6) be concentrated by ultrafiltration: it is 10000 that transglutaminase refined solution obtained in step (5), which is passed through molecule interception,
The ultrafiltration apparatus of dalton is concentrated by ultrafiltration, and transglutaminase concentrate is obtained；
(7) it is freeze-dried: 80% ethyl alcohol of transglutaminase concentrate obtained in step (6) is settled, be centrifuged, receive
Collection precipitating；Pellet frozen is dry, obtain the transglutaminase of purifying.
The method of krill preparation transglutaminase, rational technology, preparation speed are utilized provided by the present embodiment
Degree is fast.After measured, the enzyme activity rate of recovery is 81%.
Claims (3)
1. a kind of method using krill preparation transglutaminase, it is characterised in that: it passes through following process steps:
(1) feedstock processing: selection krill is raw material, is cleaned with clear water, and chopping is added buffer, is homogenized, and is centrifuged,
Collect clear liquid；Wherein, the ratio of the buffer and krill is 1 ︰ 2~3 (m/v)；
(2) it filters: clear liquid obtained in step (1) being filtered using cellulose acetate film, collects filtrate；
(3) it dialyses: filtrate obtained in step (2) being dialysed using sodium acetate dialyzate, wherein filtrate is saturating with sodium acetate
Analysis liquid volume ratio is 1:20~30, and 4 DEG C of dialysed overnights obtain krill protein crude extract；
(4) ultrafiltration: use molecule interception for the super of 10000 dalton krill crude extract obtained in step (3)
It filters equipment and carries out ultra-filtration and separation, retain the impurity including small protein, obtain krill protein filtered solution；
(5) krill protein filtered solution obtained in step (4) cation exchange chromatography: is used into strong cation exchange
Resin carries out ion-exchange chromatography, using elution, collects absorption peak, obtains transglutaminase refined solution；
(6) be concentrated by ultrafiltration: it is 10000 that transglutaminase refined solution obtained in step (5), which is passed through molecule interception,
The ultrafiltration apparatus that you pause is concentrated by ultrafiltration, and transglutaminase concentrate is obtained；
(7) it is freeze-dried: 80~90% ethyl alcohol of transglutaminase concentrate obtained in step (6) is settled, be centrifuged, receive
Collection precipitating；Pellet frozen is dry, obtain the transglutaminase of purifying；
The buffer is that 8mM NaCl and 15mM Tris-HCl is dissolved in 1000ml deionized water to be made；Institute
The sodium acetate dialyzate stated is that 20mM sodium acetate, 45mM NaCl, 2mM EDTA and 0.5mM DTT are dissolved in 1000ml
In deionized water, adjusts pH value and be made for 6.5；The eluent is the Tris-HCl of 0.2mol/L.
2. a kind of method using krill preparation transglutaminase according to claim 1, it is characterised in that:
The homogenate is the krill feeding refiner that buffer will be added, and controls 4000~6000r/min of revolving speed, homogenate 10
~30min.
3. a kind of method using krill preparation transglutaminase according to claim 1, it is characterised in that:
The centrifugation is that krill after being homogenized is sent into centrifuge, controls 6000~8000r/min of revolving speed, centrifugation 10~
15min。
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| CN201510950511.4A CN105349504B (en) | 2015-12-18 | 2015-12-18 | A method of transglutaminase is prepared using krill |
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| CN201510950511.4A CN105349504B (en) | 2015-12-18 | 2015-12-18 | A method of transglutaminase is prepared using krill |
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| CN105349504B true CN105349504B (en) | 2019-01-01 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109657A (en) * | 2014-07-07 | 2014-10-22 | 浙江万里学院 | Euphausia superba transglutaminase and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109657A (en) * | 2014-07-07 | 2014-10-22 | 浙江万里学院 | Euphausia superba transglutaminase and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 鳙鱼中转谷氨酰胺酶性质的研究及其对鱼糜凝胶化的影响;娄忠纬;《中国硕士学位论文全文数据库, 工程科技Ⅰ辑》;20150515;摘要和第3.3.1节 |
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