CN109735517A - Using the method for immobilization horse antibody extraction purification lipase - Google Patents
Using the method for immobilization horse antibody extraction purification lipase Download PDFInfo
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Abstract
The invention discloses a kind of methods using immobilization horse antibody extraction purification lipase to obtain fermentation liquid comprising steps of the pseudomonad of yielding lipase is used to ferment for fermenting microbe；Fermentation liquid is centrifuged, supernatant is collected, obtains lipase crude enzyme liquid；By immobilization horse antibody column on lipase crude enzyme liquid, column is washed with cleaning solution later, then using disodium hydrogen phosphate-citrate buffer solution elution, collect eluent, obtain lipase after purification；Polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；Drying will be precipitated, obtains lipase.The present invention utilizes antigen-antibody specific bond extraction purification lipase, impurity can be effectively removed, high specificity, gained lipase is with high purity, high-quality, high income, loss of activity are few, it can also be purified when lipase concentration is lower, and production cost is low, is a kind of efficient lipase method for extraction and purification.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of side using immobilization horse antibody extraction purification lipase
Method.
Background technique
Lipase, that is, Lipase, it be catalyzed natural substrate grease hydrolysis, generate fatty acid, glycerol and
Monoglyceride or diester.Lipase basic composition unit is only amino acid, usually only a polypeptide chain.Its catalytic activity is only
Only it is decided by its protein structure.Lipase is a kind of enzyme with a variety of catalytic capabilities, can be catalyzed triglyceride and
Hydrolysis, alcoholysis, esterification, the reaction of transesterification and esters reverse reaction of some other water-insoluble esters, in addition to this go back table
Reveal the activity of some other enzyme, such as phosphatidase, lysophospholipase, cholesterol esterase, acylpetide hydrolase activity.Lipase is not
With active the characteristics of playing dependent on reaction system, such as promote ester hydrolysis in oil-water interfaces, and can be with enzymatic in organic phase
Synthesis and transesterification.
Lipase widely exists in animals and plants and microorganism.What fatty enzyme was more is the kind of oil crops in plant
Son, such as castor bean, rapeseed, when oilseed germination, lipase can cooperate with to play a role with other enzymes is catalytically decomposed oil
Lipid material generates carbohydrate, provides nutriment and energy necessary to seed roots；Animal body include lipase it is more be
The pancreas and adipose tissue of higher mammal contain a small amount of lipase, for supplementing pancreatic lipase to fat digestion in intestinal juice
Deficiency, in the gastric juice of carnivore contain a small amount of tributyrinase.In animal body, each quasi-lipase, which controls, disappears
Change, absorb, fat is rebuild and the processes such as lipoprotein metabolism；Lipase content more horn of plenty in bacterium, fungi and yeast.Due to
Microbe species are more, breeding is fast, Yi Fasheng hereditary variation, have action pH more wider array of than animals and plants, operative temperature range and
Substratspezifitaet, and microbe-derived lipase is typically all the ectoenzyme of secretory, main fermentative microorganism has black song
Mould, Candida etc. is suitable for industrialized production and obtains high-purity sample, therefore microbial lipase is technical tallow
The important sources of fat enzyme.However, the microbial fermentation and extraction purification technology of existing lipase are still immature, product exists
Purity is low, if foreign protein is more, to obtain the product of purity is high, the problems such as but bringing complex process at high cost.
Summary of the invention
For the defects in the prior art, the present invention provides a kind of side using immobilization horse antibody extraction purification lipase
Method can effectively remove impurity, gained lipase purity is high, matter to utilize antigen-antibody specific bond extraction purification lipase
It measures, high income, it can also be purified when lipase concentration is lower, and production cost is low.
To achieve the above object, the present invention provides a kind of method using immobilization horse antibody extraction purification lipase,
Comprising steps of S1: using the pseudomonad of yielding lipase to ferment for fermenting microbe, obtain fermentation liquid；S2: by fermentation liquid from
The heart collects supernatant, obtains lipase crude enzyme liquid；S3: by immobilization horse antibody column on lipase crude enzyme liquid, cleaning solution is used later
Column is washed, then the disodium hydrogen phosphate used-citrate buffer solution elution, collects eluent, obtain lipase after purification；S4:
Polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will be lyophilized after precipitating dialysis
It is dry, obtain lipase.It should be noted that after collecting lipase precipitating, the phosphoric acid buffer for the 0.1M for being preferably 7.2 with pH value
Liquid is dialysed, then is lyophilized.
S1 specifically includes step: the single bacterium colony of the pseudomonad of yielding lipase being placed in shake flask culture, in 20-30
DEG C, cultivate 16-20h under the conditions of 200-300rpm, be then transferred in fermentation culture by 10% inoculum concentration, 20-29 DEG C,
60-70h is cultivated under the conditions of 200-300rpm, obtains the fermentation liquid.
It preferably, include: yeast extract 5g, peptone 5g, vegetable oil 10g, surplus in every liter of shake flask culture in S1
For water；PH value is 7.0.
Preferably, in S1, include: yeast extract 10g in every liter of fermentation culture, peptone 5g, NaCl 0.5g, plant
Object oil 10g, surplus are water；PH value is 7.0.
In S2, centrifugation is to be centrifuged 14-16min under the conditions of 4 DEG C, 10000-11000g using high speed freezing centrifuge.
In S3, the preparation method of immobilization horse antibody column carries out 10-16 comprising steps of horse is immunized every two weeks primary altogether
Immune, each 2mL in week, dosage are respectively the Freund's complete adjuvant that 3-21mg lipase adds equivalent, multiple spot subcutaneous inoculation,
ELISA detection antibody titer collects antiserum after being greater than 1:10000；Antiserum is centrifuged off cell component and other precipitatings
Object uses proteinA or proteinG affinity column to be purified into polyclonal antibody after collecting supernatant；By polyclonal antibody
It is connect with the Sepharose2B of activation or Sepharose4B, obtains immobilization horse antibody column.
Preferably, in S3, the weight of horse is 210-370 kilograms, and the age of horse is 2-12 years old, and the kind of horse is Mongolian horse.
In S3, disodium hydrogen phosphate-potassium phosphate buffer that cleaning solution is the 0.1M that pH value is 7.2；Eluent is
0.1M disodium hydrogen phosphate-citrate buffer solution pH value is 3.
It further include that pH value is used to clean column to neutrality for disodium hydrogen phosphate-potassium phosphate buffer of 8 1M in S3
Step.Preferably, the buffer can be used to adjust eluent to pH value as 7.0.Explanation need to be used, this can make affinity column
Recycling, and keep lipase active.
In S4, centrifugation is to be centrifuged 5-10min under the conditions of 4 DEG C, 3000-5000g.
Technical solution provided by the invention, with following the utility model has the advantages that (1) is provided by the invention anti-using immobilization horse
The method of body extraction purification lipase can effectively remove impurity using antigen-antibody specific bond extraction purification lipase, special
Anisotropic strong, gained lipase is with high purity, high-quality, high income, activity height, can also carry out to it when lipase concentration is lower
Purification, it is high-efficient；It is monitored by antibody titer, selects high titre to resist for making affinity column more, lipase is effectively ensured
Yield and purity；(2) method provided by the invention using immobilization horse antibody extraction purification lipase, simple production process,
Production cost is low；Be easy to get polyclonal antibody, immune affinity chromatographic column can Reusability, discharge of wastewater and ring can be effectively reduced
Border pollution, is a kind of efficient lipase extracting method.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described.Implement below
Example is only used for clearly illustrating technical solution of the present invention, therefore is intended only as example, and cannot be used as a limitation and limit this hair
Bright protection scope.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantitative test in following embodiment,
Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.The vacation that the present invention uses
Monad is aeruginosa atcc 21808.
The present invention provides a kind of method using immobilization horse antibody extraction purification lipase, comprising steps of
S1: the single bacterium colony of the aeruginosa atcc 21808 of yielding lipase is placed in equipped with 25mL shake flask culture
In 250mL shaking flask, 16-20h is cultivated under the conditions of 20-30 DEG C, 200-300rpm, is then transferred to fermentation training by 10% inoculum concentration
In nutrient solution, 60-70h is cultivated under the conditions of 20-29 DEG C, 200-300rpm, obtains fermentation liquid；Wherein, in every liter of shake flask culture
It include: yeast extract 5g, peptone 5g, vegetable oil 10g, surplus is water；PH value is 7.0；Include: in every liter of fermentation culture
Yeast extract 10g, peptone 5g, NaCl 0.5g, vegetable oil 10g, surplus is water；PH value is 7.0；
S2: fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；Wherein, centrifugation be using high speed freeze from
Scheming is centrifuged 14-16min under the conditions of 4 DEG C, 10000-11000g；
S3: by immobilization horse antibody column on lipase crude enzyme liquid, during upper prop, the lipase active of efflux is detected, directly
To there is lipase active outflow, stop upper liquid；The disodium hydrogen phosphate for the 0.1M for being later 7.2 with pH value-potassium dihydrogen phosphate buffering
Liquid washes column, then uses pH value for 3 0.1M disodium hydrogen phosphate-citrate buffer solution elution, collects eluent, obtains after purification
Lipase；And after enzyme outflow, the disodium hydrogen phosphate-potassium phosphate buffer for the 1M for being immediately 8 with pH value washes column into
Property, also with solution tune eluent to pH value to 7；Wherein, the preparation method of immobilization horse antibody column is comprising steps of select 4 weights
Amount is immunized once every two weeks in the 210-370 kilograms of age 2-12 years old Mongolian horse, carries out 10-16 weeks immune, each 2mL altogether,
Dosage is respectively the Freund's complete adjuvant that 3-21mg lipase adds equivalent, multiple spot subcutaneous inoculation, and ELISA detection antibody titer is greater than
Antiserum is collected after 1:10000；Antiserum is centrifuged off cell component and other sediments, is used after collecting supernatant
ProteinA or proteinG affinity column is purified into polyclonal antibody；By the Sepharose2B of polyclonal antibody and activation
Or Sepharose4B connection, obtain immobilization horse antibody column；
S4: polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will sink
Form sediment lyophilization after dialysing, and obtains lipase；Wherein, centrifugation is to be centrifuged 5-10min under the conditions of 4 DEG C, 3000-5000g.
Combined with specific embodiments below to the method provided by the invention using immobilization horse antibody extraction purification lipase
It is described further.
Embodiment 1
The present embodiment provides a kind of methods using immobilization horse antibody extraction purification lipase, comprising steps of
S1: the single bacterium colony of the aeruginosa atcc 21808 of yielding lipase is placed in equipped with 25mL shake flask culture
In 250mL shaking flask, 20h is cultivated under the conditions of 28 DEG C, 200rpm, is then transferred in fermentation culture by 10% inoculum concentration,
28 DEG C, cultivate 68h under the conditions of 200rpm, obtain fermentation liquid；It wherein, include: yeast extract 5g, egg in every liter of shake flask culture
White peptone 5g, vegetable oil 10g, surplus are water；PH value is 7.0；It include: yeast extract 10g, peptone in every liter of fermentation culture
5g, NaCl 0.5g, vegetable oil 10g, surplus are water；PH value is 7.0；
S2: fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；Wherein, centrifugation be using high speed freeze from
Scheming is centrifuged 15min under the conditions of 4 DEG C, 10800g；
S3: by immobilization horse antibody column on lipase crude enzyme liquid, during upper prop, the lipase active of efflux is detected, directly
To there is lipase active outflow, stop upper liquid；The disodium hydrogen phosphate for the 0.1M for being later 7.2 with pH value-potassium dihydrogen phosphate buffering
Liquid washes column, then uses pH value for 3 0.1M disodium hydrogen phosphate-citrate buffer solution elution, collects eluent, obtains after purification
Lipase；And after enzyme outflow, the disodium hydrogen phosphate-potassium phosphate buffer for the 1M for being immediately 8 with pH value washes column into
Property, also with solution tune eluent to pH value to 7；Wherein, the preparation method of immobilization horse antibody column is comprising steps of select 4 illiteracies
(weight and corresponding age are respectively as follows: that weight is 240 kilograms and the age is 3 years old, weight is 290 kilograms and the age is 5 for ancient horse
Year, weight is 320 kilograms and the age is 7 years old, weight is 360 kilograms and the age is 10 years old), primary, progress altogether is immunized every two weeks
12 weeks immune, each 2mL, dosage are respectively the Freund's complete adjuvant that 15mg lipase adds equivalent, multiple spot subcutaneous inoculation,
ELISA detection antibody titer collects antiserum after being greater than 1:10000；Antiserum is centrifuged off cell component and other precipitatings
Object uses proteinA or proteinG affinity column to be purified into polyclonal antibody after collecting supernatant；By polyclonal antibody
It is connect with the Sepharose2B of activation, obtains immobilization horse antibody column；
S4: polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will sink
Form sediment lyophilization after dialysing, and obtains lipase, enzyme activity reaches ten thousand units/gram of 15-25 after measured；Wherein, centrifugation is in 4 DEG C, 000g
Under the conditions of be centrifuged 8min.
Embodiment 2
The present embodiment provides a kind of methods using immobilization horse antibody extraction purification lipase, comprising steps of
S1: the single bacterium colony of the aeruginosa atcc 21808 of yielding lipase is placed in equipped with 25mL shake flask culture
In 250mL shaking flask, 16h is cultivated under the conditions of 28 DEG C, 200rpm, is then transferred in fermentation culture by 10% inoculum concentration,
28 DEG C, cultivate 60h under the conditions of 200rpm, obtain fermentation liquid；It wherein, include: yeast extract 5g, egg in every liter of shake flask culture
White peptone 5g, vegetable oil 10g, surplus are water；PH value is 7.0；It include: yeast extract 10g, peptone in every liter of fermentation culture
5g, NaCl 0.5g, vegetable oil 10g, surplus are water；PH value is 7.0；
S2: fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；Wherein, centrifugation be using high speed freeze from
Scheming is centrifuged 14min under the conditions of 4 DEG C, 10000g；
S3: by immobilization horse antibody column on lipase crude enzyme liquid, during upper prop, the lipase active of efflux is detected, directly
To there is lipase active outflow, stop upper liquid；The disodium hydrogen phosphate for the 0.1M for being later 7.2 with pH value-potassium dihydrogen phosphate buffering
Liquid washes column, then uses pH value for 3 0.1M disodium hydrogen phosphate-citrate buffer solution elution, collects eluent, obtains after purification
Lipase；And after enzyme outflow, the disodium hydrogen phosphate-potassium phosphate buffer for the 1M for being immediately 8 with pH value washes column into
Property, also with solution tune eluent to pH value to 7；Wherein, the preparation method of immobilization horse antibody column is comprising steps of select 4 illiteracies
(weight and corresponding age are respectively as follows: that weight is 220 kilograms and the age is 2 years old, weight is 270 kilograms and the age is 4 for ancient horse
Year, weight is 330 kilograms and the age is 8 years old, weight is 345 kilograms and the age is 9 years old), it is immunized once every two weeks, carries out 10 altogether
Immune, each 2mL in week, dosage are respectively the Freund's complete adjuvant that 3mg lipase adds equivalent, multiple spot subcutaneous inoculation, ELISA inspection
It surveys after antibody titer is greater than 1:10000 and collects antiserum；Antiserum is centrifuged off cell component and other sediments, in collection
Polyclonal antibody is purified into using proteinA or proteinG affinity column after clear liquid；By polyclonal antibody and activation
Sepharose2B connection obtains immobilization horse antibody column；
S4: polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will sink
Form sediment lyophilization after dialysing, and obtains lipase, enzyme activity reaches ten thousand units/gram of 15-25 after measured；Wherein, centrifugation is in 4 DEG C, 3000g
Under the conditions of be centrifuged 5min.
Embodiment 3
The present embodiment provides a kind of methods using immobilization horse antibody extraction purification lipase, comprising steps of
S1: the single bacterium colony of the aeruginosa atcc 21808 of yielding lipase is placed in equipped with 25mL shake flask culture
In 250mL shaking flask, 18h is cultivated under the conditions of 30 DEG C, 300rpm, is then transferred in fermentation culture by 10% inoculum concentration,
29 DEG C, cultivate 70h under the conditions of 300rpm, obtain fermentation liquid；It wherein, include: yeast extract 5g, egg in every liter of shake flask culture
White peptone 5g, vegetable oil 10g, surplus are water；PH value is 7.0；It include: yeast extract 10g, peptone in every liter of fermentation culture
5g, NaCl 0.5g, vegetable oil 10g, surplus are water；PH value is 7.0；
S2: fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；Wherein, centrifugation be using high speed freeze from
Scheming is centrifuged 16min under the conditions of 4 DEG C, 11000g；
S3: by immobilization horse antibody column on lipase crude enzyme liquid, during upper prop, the lipase active of efflux is detected, directly
To there is lipase active outflow, stop upper liquid；The disodium hydrogen phosphate for the 0.1M for being later 7.2 with pH value-potassium dihydrogen phosphate buffering
Liquid washes column, then uses pH value for 3 0.1M disodium hydrogen phosphate-citrate buffer solution elution, collects eluent, obtains after purification
Lipase；And after enzyme outflow, the disodium hydrogen phosphate-potassium phosphate buffer for the 1M for being immediately 8 with pH value washes column into
Property, also with solution tune eluent to pH value to 7；Wherein, the preparation method of immobilization horse antibody column is comprising steps of select 4 illiteracies
(weight and corresponding age are respectively as follows: that weight is 232 kilograms and the age is 3 years old, weight is 268 kilograms and the age is 6 for ancient horse
Year, weight is 341 kilograms and the age is 9 years old, weight is 366 kilograms and the age is 11 years old) it is immunized once every two weeks, 10 are carried out altogether
Week is immunized, and each 2mL, dosage is respectively the Freund's complete adjuvant that 21mg lipase adds equivalent, multiple spot subcutaneous inoculation, ELISA
Detection antibody titer collects antiserum after being greater than 1:10000；Antiserum is centrifuged off cell component and other sediments, is collected
Polyclonal antibody is purified into using proteinA or proteinG affinity column after supernatant；By polyclonal antibody and activation
Sepharose4B connection obtains immobilization horse antibody column；
S4: polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will sink
Form sediment lyophilization after dialysing, and obtains lipase, enzyme activity reaches ten thousand units/gram of 15-25 after measured；Wherein, centrifugation is in 4 DEG C, 5000g
Under the conditions of be centrifuged 10min.
Comparative example 1
This comparative example provides a kind of method using immobilization horse antibody extraction purification lipase, comprising steps of
S1: the single bacterium colony of the aeruginosa atcc 21808 of yielding lipase is placed in equipped with 25mL shake flask culture
In 250mL shaking flask, 20h is cultivated under the conditions of 28 DEG C, 200rpm, is then transferred in fermentation culture by 10% inoculum concentration,
28 DEG C, cultivate 68h under the conditions of 200rpm, obtain fermentation liquid；It wherein, include: yeast extract 5g, egg in every liter of shake flask culture
White peptone 5g, vegetable oil 10g, surplus are water；PH value is 7.0；It include: yeast extract 10g, peptone in every liter of fermentation culture
5g, NaCl 0.5g, vegetable oil 10g, surplus are water；PH value is 7.0；
S2: fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；Wherein, centrifugation be using high speed freeze from
Scheming is centrifuged 15min under the conditions of 4 DEG C, 10800g；
S3: by immobilization horse antibody column on lipase crude enzyme liquid, during upper prop, the lipase active of efflux is detected, directly
To there is lipase active outflow, stop upper liquid；The disodium hydrogen phosphate for the 0.1M for being later 4.8 with pH value-potassium dihydrogen phosphate buffering
Liquid washes column, then uses pH value for 4 0.1M disodium hydrogen phosphate-citrate buffer solution elution, collects eluent, obtains after purification
Lipase；And after enzyme outflow, the disodium hydrogen phosphate-potassium phosphate buffer for the 1M for being immediately 8 with pH value washes column into
Property, also with the solution tune eluent to pH to 7；Wherein, the preparation method of immobilization horse antibody column is comprising steps of select 4 Mongolia
Horse (weight and corresponding age be respectively as follows: that weight is 240 kilograms and the age is 3 years old, weight is 290 kilograms and the age be 5 years old,
Weight is 320 kilograms and the age is 7 years old, weight is 360 kilograms and the age is 10 years old), it is immunized once, carries out 8 weeks altogether every two weeks
Immune, each 2mL, dosage is respectively the Freund's complete adjuvant that 15mg lipase adds equivalent, multiple spot subcutaneous inoculation, ELISA inspection
It surveys after antibody titer is greater than 1:10000 and collects antiserum；Antiserum is centrifuged off cell component and other sediments, in collection
Polyclonal antibody is purified into using proteinA affinity column after clear liquid；By the Sepharose2B of polyclonal antibody and activation
Connection, obtains immobilization horse antibody column；
S4: polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will sink
Form sediment lyophilization after dialysing, and obtains lipase；Wherein, centrifugation is to be centrifuged 8min under the conditions of 4 DEG C, 4000g.
Comparative example 2
This comparative example provides a kind of method using immobilization horse antibody extraction purification lipase, comprising steps of
S1: the single bacterium colony of the aeruginosa atcc 21808 of yielding lipase is placed in equipped with 25mL shake flask culture
In 250mL shaking flask, 20h is cultivated under the conditions of 28 DEG C, 200rpm, is then transferred in fermentation culture by 10% inoculum concentration,
28 DEG C, cultivate 68h under the conditions of 200rpm, obtain fermentation liquid；It wherein, include: yeast extract 5g, egg in every liter of shake flask culture
White peptone 5g, vegetable oil 10g, surplus are water；PH value is 7.0；It include: yeast extract 10g, peptone in every liter of fermentation culture
5g, NaCl 0.5g, vegetable oil 10g, surplus are water；PH value is 7.0；
S2: fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；Wherein, centrifugation be using high speed freeze from
Scheming is centrifuged 15min under the conditions of 4 DEG C, 10800g；
S3: by immobilization horse antibody column on lipase crude enzyme liquid, during upper prop, the lipase active of efflux is detected, directly
To there is lipase active outflow, stop upper liquid；The disodium hydrogen phosphate for the 0.1M for being later 7.2 with pH value-potassium dihydrogen phosphate buffering
Liquid washes column, then uses pH value for 3 disodium hydrogen phosphate-citrate buffer solution elution, collects eluent, obtains fat after purification
Enzyme；And after enzyme outflow, the disodium hydrogen phosphate-potassium phosphate buffer for the 1M for being immediately 8 with pH value washes column to neutrality,
With the solution tune eluent to pH to 7；Wherein, the preparation method of immobilization horse antibody column is comprising steps of select 4 Mongolian horses (weight
Amount and corresponding age are respectively as follows: that weight is 240 kilograms and the age is 3 years old, weight is 290 kilograms and the age is 5 years old, weight is
320 kilograms and the age is 7 years old, weight is 360 kilograms and the age is 10 years old), be immunized every two weeks primary, altogether carry out being immunized for 6 weeks,
Each 2mL, dosage are respectively the Freund's complete adjuvant that 15mg lipase adds equivalent, multiple spot subcutaneous inoculation, ELISA detection antibody drop
Degree collects antiserum after being greater than 1:10000；Antiserum is centrifuged off cell component and other sediments, is adopted after collecting supernatant
Polyclonal antibody is purified into proteinA affinity column；By polyclonal antibody and the Sepharose2B of activation connection, obtain
Immobilization horse antibody column；
S4: polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will sink
Form sediment lyophilization after dialysing, and obtains lipase；Wherein, centrifugation is to be centrifuged 8min under the conditions of 4 DEG C, 4000g.
Comparative example 3
This comparative example provides a kind of method of extraction purification lipase, comprising steps of
S1: the single bacterium colony of the aeruginosa atcc 21808 of yielding lipase is placed in equipped with 25mL shake flask culture
In 250mL shaking flask, 20h is cultivated under the conditions of 28 DEG C, 200rpm, is then transferred in fermentation culture by 10% inoculum concentration,
28 DEG C, cultivate 68h under the conditions of 200rpm, obtain fermentation liquid；It wherein, include: yeast extract 5g, egg in every liter of shake flask culture
White peptone 5g, vegetable oil 10g, surplus are water；PH value is 7.0；It include: yeast extract 10g, peptone in every liter of fermentation culture
5g, NaCl 0.5g, vegetable oil 10g, surplus are water；PH value is 7.0；
S2: fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；Wherein, centrifugation be using high speed freeze from
Scheming is centrifuged 15min under the conditions of 4 DEG C, 10800g；
S3: filling chromatographic column with Sephadexg-100 gel particle, with the neutral phosphate buffer for the 0.1M that pH value is 7
Liquid balances chromatographic column, and obtained lipase crude enzyme liquid is loaded on chromatographic column, is eluted with neutral phosphate buffer liquid, receives
Collect eluent corresponding to Peak Activity, resulting eluent is concentrated by ultrafiltration, gel filtration crude enzyme liquid is obtained；
It is coupled on Sepharose4B using lipase inhibitor as ligand by covalent bond, is filled with Sepharose4B
Affinity column is balanced chromatographic column with neutral phosphate buffer liquid, crude enzyme liquid is loaded on chromatographic column, is rinsed with deionized water
Chromatographic column, then eluted with the citrate buffer solution of 0.1M pH4, eluent is collected, lipase after purification is obtained；
S4: polyethylene glycol is added in lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；It will sink
Shallow lake drying, obtains lipase；Wherein, centrifugation is to be centrifuged 10min under the conditions of 4 DEG C, 6000g.
Using the method extraction purification lipase of the embodiment of the present invention 1 to embodiment 3, the purity of obtained lipase is received
Rate, activity are as shown in table 1 below, and using the lipase that the method extraction purification using comparative example 1 to comparative example 3 obtains as pair
According to.In table 1, the data of embodiment 1 are set as 1, other organize the ratio for 1 corresponding data of data and embodiment that other data are each group
Value.
The lipase of 1 distinct methods extraction purification of table
| Group | Lipase purity | Lipase yield | Lipase active |
| Embodiment 1 | 1 | 1 | 1 |
| Embodiment 2 | 98.6% | 99.2% | 99.6% |
| Embodiment 3 | 99.0% | 99.4% | 99.3% |
| Comparative example 1 | 92.5% | 80.5% | 90.3% |
| Comparative example 2 | 93.3% | 85.3% | 90.5% |
| Comparative example 3 | 60.7% | 62.8% | 70.7% |
Unless otherwise indicated, technical term or scientific term used in this application should be fields technology of the present invention
The ordinary meaning that personnel are understood.Unless specifically stated otherwise, the phase of the component and step that otherwise illustrate in these embodiments
Step, numerical expression and numerical value are not limit the scope of the invention.In all examples being illustrated and described herein, unless
It states otherwise, any occurrence should be construed as merely illustratively, not as limitation, therefore, exemplary embodiment
Other examples can have different values.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover within the scope of the claims and the description of the invention.
Claims (10)
1. a kind of method using immobilization horse antibody extraction purification lipase, which is characterized in that comprising steps of
S1: it uses the pseudomonad of yielding lipase to ferment for fermenting microbe, obtains fermentation liquid；
S2: the fermentation liquid is centrifuged, and is collected supernatant, is obtained lipase crude enzyme liquid；
S3: by immobilization horse antibody column on the lipase crude enzyme liquid, column is washed with cleaning solution later, then use disodium hydrogen phosphate-
Citrate buffer solution elution, collects eluent, obtains lipase after purification；
S4: polyethylene glycol is added in the lipase after purification, precipitates lipase；It is centrifuged later, collects precipitating；By institute
Lyophilization after precipitating is dialysed is stated, the lipase is obtained.
2. the method according to claim 1 using immobilization horse antibody extraction purification lipase, which is characterized in that
The S1 specifically includes step:
The single bacterium colony of the pseudomonad of yielding lipase is placed in shake flask culture, under the conditions of 20-30 DEG C, 200-300rpm
16-20h is cultivated, is then transferred in fermentation culture by 10% inoculum concentration, is cultivated under the conditions of 20-29 DEG C, 200-300rpm
60-70h obtains the fermentation liquid.
3. the method according to claim 2 using immobilization horse antibody extraction purification lipase, it is characterised in that:
It include: yeast extract 5g, peptone 5g, vegetable oil 10g in every liter of shake flask culture in the S1, surplus is
Water；PH value is 7.0.
4. the method according to claim 2 using immobilization horse antibody extraction purification lipase, it is characterised in that:
It include: yeast extract 10g, peptone 5g, NaCl 0.5g, vegetable oil in every liter of fermentation culture in the S1
10g, surplus are water；PH value is 7.0.
5. the method according to claim 1 using immobilization horse antibody extraction purification lipase, it is characterised in that:
In the S2, the centrifugation is to be centrifuged 14- under the conditions of 4 DEG C, 10000-11000g using high speed freezing centrifuge
16min。
6. the method according to claim 1 using immobilization horse antibody extraction purification lipase, which is characterized in that
In the S3, the preparation method of the immobilization horse antibody column comprising steps of
Horse is immunized primary every two weeks, carries out being immunized for 10-16 week altogether, each 2mL, dosage is respectively that 3-21mg lipase adds
The Freund's complete adjuvant of amount, multiple spot subcutaneous inoculation, ELISA detection antibody titer collect antiserum after being greater than 1:10000；
The antiserum is centrifuged off cell component and other sediments, collect after supernatant using proteinA or
ProteinG affinity column is purified into polyclonal antibody；
By Sepharose2B or the Sepharose4B connection of the polyclonal antibody and activation, the immobilization horse antibody is obtained
Column.
7. the method according to claim 6 using immobilization horse antibody extraction purification lipase, it is characterised in that:
In the S3, the weight of the horse is 210-370 kilograms, and the age of the horse is 2-12 years old, and the kind of the horse is to cover
Ancient horse.
8. the method according to claim 1 using immobilization horse antibody extraction purification lipase, it is characterised in that:
In the S3, disodium hydrogen phosphate-potassium phosphate buffer that the cleaning solution is the 0.1M that pH value is 7.2；
The concentration of the disodium hydrogen phosphate-citrate buffer solution is 0.1M, pH value 3.
9. the method according to claim 1 using immobilization horse antibody extraction purification lipase, it is characterised in that:
It further include that pH value is used to clean after enzyme outflow for disodium hydrogen phosphate-potassium phosphate buffer of 8 1M in the S3
Column is to neutral step；Preferably, the pH value is used to elute for disodium hydrogen phosphate-potassium phosphate buffer tune of 8 1M
Liquid is to pH value to 7.
10. the method according to claim 1 using immobilization horse antibody extraction purification lipase, it is characterised in that:
In the S4, the centrifugation is to be centrifuged 5-10min under the conditions of 4 DEG C, 3000-5000g.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
| CN114990091A (en) * | 2022-06-10 | 2022-09-02 | 中牛集团有限公司 | Preparation method and application of biological enzyme preparation for ecological leather |
| WO2024041562A1 (en) * | 2022-08-24 | 2024-02-29 | Novozymes A/S | Improved enzymatic splitting of oils and fats to produce free fatty acid |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101198695A (en) * | 2005-06-15 | 2008-06-11 | 巴斯福股份公司 | Lipase production method |
| CN101701211A (en) * | 2009-11-13 | 2010-05-05 | 江苏大学 | High-efficient extraction and purification method of rape lipase |
| CN102924586A (en) * | 2012-09-28 | 2013-02-13 | 内蒙古博格农牧业开发有限责任公司 | Method for producing pregnant mare serum gonactotropin by monoclonal antibody technology |
| CN104388404A (en) * | 2014-11-14 | 2015-03-04 | 安徽华亿农牧科技发展有限公司 | Microbial fermentation and extraction separation method of lipase |
| CN107488615A (en) * | 2017-09-18 | 2017-12-19 | 山东隆科特酶制剂有限公司 | The pseudomonad of one plant height yielding lipase and its enzymatic production method |
-
2019
- 2019-03-12 CN CN201910183987.8A patent/CN109735517A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101198695A (en) * | 2005-06-15 | 2008-06-11 | 巴斯福股份公司 | Lipase production method |
| CN101701211A (en) * | 2009-11-13 | 2010-05-05 | 江苏大学 | High-efficient extraction and purification method of rape lipase |
| CN102924586A (en) * | 2012-09-28 | 2013-02-13 | 内蒙古博格农牧业开发有限责任公司 | Method for producing pregnant mare serum gonactotropin by monoclonal antibody technology |
| CN104388404A (en) * | 2014-11-14 | 2015-03-04 | 安徽华亿农牧科技发展有限公司 | Microbial fermentation and extraction separation method of lipase |
| CN107488615A (en) * | 2017-09-18 | 2017-12-19 | 山东隆科特酶制剂有限公司 | The pseudomonad of one plant height yielding lipase and its enzymatic production method |
Non-Patent Citations (3)
| Title |
|---|
| BELGUITH H ET AL.: "Immunopurification and characterization of a rape", 《AFRICAN JOURNAL OF BIOTECHNOLOGY》 * |
| CHUNG HONG TAN ET AL.: "Novel lipase purification methods –a review of the latest developments", 《BIOTECHNOLOGY JOURNAL》 * |
| MARIANNE KORDEL ET AL.: "Extracellular Lipase of Pseudomonas sp. Strain ATCC 21808: Purification, Characterization, Crystallization, and Preliminary X-Ray Diffraction Data", 《JOURNAL OF BACTERIOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
| CN114990091A (en) * | 2022-06-10 | 2022-09-02 | 中牛集团有限公司 | Preparation method and application of biological enzyme preparation for ecological leather |
| WO2024041562A1 (en) * | 2022-08-24 | 2024-02-29 | Novozymes A/S | Improved enzymatic splitting of oils and fats to produce free fatty acid |
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