CN109136201A - A kind of purification technique of enzyme biochemical reagents - Google Patents
A kind of purification technique of enzyme biochemical reagents Download PDFInfo
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- CN109136201A CN109136201A CN201810773625.XA CN201810773625A CN109136201A CN 109136201 A CN109136201 A CN 109136201A CN 201810773625 A CN201810773625 A CN 201810773625A CN 109136201 A CN109136201 A CN 109136201A
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- silica gel
- biochemical reagents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
The present invention relates to a kind of purification techniques of enzyme biochemical reagents, which comprises the steps of: (1) enzyme biochemical reagents pre-process；(2) microsphere silica gel pre-processes；(3) it adsorbs: pretreated microsphere silica gel, standing adsorption after stirring is added after adjusting enzyme biochemical reagents filtrate pH value；(4) it elutes: being stirred and eluted with Klorvess Liquid, filter out eluent, repetition elutes and merges eluent；(5) it is centrifuged: eluent centrifuge tube being centrifuged removing protein, desalination, obtains centrifugate；By centrifugate it is dry after to get arriving the enzyme biochemical reagents；Present invention process is simple, and cost is relatively low, and enzyme biochemical reagents purity is high, the activity prepared is high.
Description
Technical field
The present invention relates to field of biology, and in particular to a kind of purification technique of enzyme biochemical reagents.
Background technique
With the development of life science, enzyme biochemical reagents have evolved into a major class of chemical reagent, there is commodity 10000
It is a variety of.The enzyme biochemical reagents kind of China's sale has 2500 kinds.Enzyme biochemical reagents are heated, make moist, easily lose and live after light
Power, storage life is short, therefore it is harsher to store and transport condition.For example, To Be Protected from Heat for most enzymatic reagents, need to be saved at 0-6 DEG C, some
Enzymatic reagent as genetic engineering need to then save at -20 DEG C.Enzyme biochemical reagents by contained in bio-tissue or
Be in the metabolic process caused by substance can be divided into amino acid, polypeptide, protein, nucleotide, nucleic acid, enzyme, coenzyme, carbohydrate,
Esters, hormone etc.；Electrophoresis reagents, chromatorgaphy reagent, immunoreagent, labelled reagent, tissue can be divided by the needs of biological study
Chemical reagent etc..
Enzyme biochemical reagents are different depending on the application to have certain requirement to its purity and technology.Such as enzymatic reagent, have thick
Enzyme, crystalline enzyme, multiple crystalline enzyme and the enzyme preparation without certain miscellaneous enzymes processed etc. are a variety of.There are three types of producers for enzyme biochemical reagents
Method: it 1. separates, purify from organism；2. chemical synthesis；3. fermenting.Have to the technical requirements of enzyme biochemical reagents product: containing
Amount, fusing point, freezing point, optical activity, water content, spectral signature, refractive power, density and bioactivity etc..
Type and quantity difference in various animal organs, tissue and body fluid containing enzyme is very big.The total amount of usually contained enzyme
Not seldom, but the content of each enzyme is seldom, often in a few millionths to a few percent.Therefore the extracting and developing, pure of enzyme
Change and confirmation will lean on the Measurement for Biochemistry of higher level.
The country to the isolating and purifying of enzyme biochemical reagents, extract preparation method and once used crystallisation, ion-exchange chromatography
Method, affinity chromatography etc., but have certain drawback.Even current industrial production mainly uses base exchange method, passes through sun
The absorption of microsphere silica gel described in ion, high eluting salt, desalting and dewatering and atomization drying and etc. obtain enzyme biochemical reagents, but due to
Enzyme biochemical reagents eluate solution amount is big, salt content is high, common centrifugation apparatus burden weight, centrifugation film because work long hours vulnerable to
Pollution, the service life of film are greatly affected；The selected isolation and purification method of above-mentioned technology, eluent, centrifugation opportunity
Etc. conditions be not suitable for, cause to isolate and purify at high cost, the enzyme biochemical reagents purity after separation is not high, and extraction process backwardness makes
Obtain the problems such as enzyme biochemical reagents loss of activity is big.
Summary of the invention
In view of this, the present invention provides a kind of purification techniques of enzyme biochemical reagents, which is characterized in that including following step
It is rapid:
(1) biological cell pre-processes；
A. biological cell is ground into a powder under -5 DEG C of freezing conditions with mortar；
B. to every 10g step a treated biological cell, 0.5ml lysate is added, cracks 20-30 minute, will biology carefully
Cellular lysate liquid is collected into EP pipe；
By weight percentage, lysate includes EDTA, 0.05- of the sodium chloride of 0.1-0.15%, 1.0-1.5%
0.1% hydrogen peroxide and 0.2-0.5%TritoAx-100；Remaining part is deionized water；
C. it handles step b well cell suspension to be placed at 4 DEG C, with 3000r/min revolving speed centrifugation 5 minutes；Place 3-4 points
Clock avoids the lipid layer of upper layer floating, takes supernatant, obtain enzyme biochemical reagents pretreatment fluid until cell suspension becomes clear；It will
Enzyme biochemical reagents after mixing evenly, cross 100-150 mesh filter screen and obtain enzyme biochemical reagents filtrate；
(2) microsphere silica gel pre-processes；
Take microsphere silica gel to be washed with distilled water to without obvious impurity and be filtered dry, after first being impregnated with hydrochloric acid solution, then with distillation
Water rinses to obtain microsphere silica gel A；
Wherein, microsphere silica gel is selected from polysuccinimide and one of microballoon is made in 3- aminopropyl silica gel；
Microsphere silica gel pretreatment, the 0.8-1.2moC/C hydrochloric acid that microsphere silica gel is soaked in 2-4 times of microsphere silica gel volume are molten
Liquid, soaking time 10-12h are 5.5-6.5 with distilled water flushing to efflux pH value, obtain microsphere silica gel A；
Microsphere silica gel A is soaked in the 0.8-1.2moC/C sodium hydroxide solution of 2-4 times of microsphere silica gel volume again, when immersion
Between be 10-12h, be 9-10 with distilled water flushing to efflux pH value, obtain microsphere silica gel B；
(3) it adsorbs；
Step (2) thus obtained microsphere silica gel B is added into enzyme biochemical reagents filtrate, stirs and evenly mixs rear standing adsorption, discards
Supernatant is washed with distilled water, and obtains microsphere silica gel C；
Enzyme biochemical reagents filtrate pH value is 9.0-10；
The volume ratio of enzyme biochemical reagents filtrate and microsphere silica gel B are 10:3, and control enzyme biochemical reagents filtrate pH value is
8-10；
(4) it elutes；
Klorvess Liquid is added into step (3) thus obtained microsphere silica gel C, stirring elution filters out eluent, repeats elution and closes
And gained eluent, reservation filter out the microsphere silica gel after eluent；
The concentration of Klorvess Liquid is 0.5-2.5moC/C, and the frequency for stirring elution is 60-100r/min, time 45-
65min；
Klorvess Liquid is 1-1.5moC/C, and stirring elution time is 70-90min, and the dosage of Klorvess Liquid is step
(1) 0.5-1 times of enzyme biochemical reagents filtrate volume；
Microsphere silica gel after filtering out eluent continues step (2) as microsphere silica gel A and prepares microsphere silica gel B；
(5) it is centrifuged；
Eluent obtained by step (4) is first placed in the centrifuge tube that interception is 70KD, is centrifuged and takes subnatant；
Subnatant is placed in the centrifuge tube that interception is 5KD, is centrifuged and upper layer trapped fluid is taken to try to get to enzyme biochemistry
Agent；
Interception is the revolving speed of 70KD centrifuge tube when being 5000-6000r/min, centrifugation time 3-8min；
Interception is the revolving speed of 5KD centrifuge tube when being 7000-9000r/min, centrifugation time 30-50min；
Enzyme biochemical reagents after purification are arrived after centrifugate is dried.
Beneficial achievement of the invention are as follows: the present invention provides a kind of purification technique of enzyme biochemical reagents, simple process, at
This is lower, and enzyme biochemical reagents purity is high, the activity prepared is high.
Specific embodiment
In order to which technical problems, technical solutions and advantages to be solved are more clearly understood, tie below
Embodiment is closed, the present invention will be described in detail；It should be noted that specific embodiment described herein is only to explain
The present invention is not intended to limit the present invention, and the product for being able to achieve said function belongs to equivalent replacement and improvement, is all contained in this hair
Within bright protection scope；The specific method is as follows:
Embodiment: in view of this, the present invention provides a kind of purification techniques of enzyme biochemical reagents, which is characterized in that packet
It includes in view of this, the present invention provides a kind of purification techniques of enzyme biochemical reagents, which comprises the following steps:
(1) biological cell pre-processes；
A. the biological cell is ground into a powder under -5 DEG C of freezing conditions with mortar；
B. to every 10g step a treated biological cell, 0.5ml lysate is added, cracks 20-30 minutes, by institute
Biological cell lysate is stated to be collected into EP pipe；
By weight percentage, the lysate includes EDTA, 0.05- of the sodium chloride of 0.1-0.15%, 1.0-1.5%
0.1% hydrogen peroxide and 0.2-0.5%TritoAx-100；Remaining part is deionized water；
C. it handles step b well cell suspension to be placed at 4 DEG C, with 3000r/min revolving speed centrifugation 5 minutes；Place 3-4 points
Clock avoids the lipid layer of upper layer floating, takes supernatant until the cell suspension becomes clear, obtains the pretreatment of enzyme biochemical reagents
Liquid；After mixing evenly by the enzyme biochemical reagents, it crosses 100-150 mesh filter screen and obtains enzyme biochemical reagents filtrate；
By the separately sampled progress PAGE gel electrophoresis experiment of the eluent of embodiment and centrifugate, electrophoresis result table
Bright, eluent and centrifugate can see obvious band in 14.4KDa series, and eluent can be seen that impurity removes after centrifugation
Effect is preferable, the enzyme biochemical reagents purity is high of extraction.
Claims (1)
1. the present invention relates to a kind of purification techniques of enzyme biochemical reagents, which comprises the following steps:
(1) biological cell pre-processes；
A. the biological cell is ground into a powder under -5 DEG C of freezing conditions with mortar；
B. to every 10g step a treated biological cell, 0.5ml lysate is added, cracks 20-30 minutes, by the life
Object cell pyrolysis liquid is collected into EP pipe；
By weight percentage, the lysate includes EDTA, 0.05- of the sodium chloride of 0.1-0.15%, 1.0-1.5%
0.1% hydrogen peroxide and 0.2-0.5%TritoAx-100；Remaining part is deionized water；
C. it handles step b well cell suspension to be placed at 4 DEG C, with 3000r/min revolving speed centrifugation 5 minutes；Placement 3-4 minutes, directly
Become clear to the cell suspension, avoids the lipid layer of upper layer floating, take supernatant, obtain enzyme biochemical reagents pretreatment fluid；It will
The enzyme biochemical reagents after mixing evenly, cross 100-150 mesh filter screen and obtain enzyme biochemical reagents filtrate；
(2) the microsphere silica gel pretreatment；
Take the microsphere silica gel to be washed with distilled water to without obvious impurity and be filtered dry, after first being impregnated with hydrochloric acid solution, then with distillation
Water rinses to obtain the microsphere silica gel A；
Wherein, the microsphere silica gel is selected from polysuccinimide and one of microballoon is made in 3- aminopropyl silica gel；
The microsphere silica gel, is soaked in the 0.8-1.2moC/ of the 2-4 times of microsphere silica gel volume by the microsphere silica gel pretreatment
Celite acid solution, soaking time 10-12h are 5.5-6.5 with distilled water flushing to efflux pH value, obtain the microsphere silica gel A；
The microsphere silica gel A is soaked in the 0.8-1.2moC/C sodium hydroxide solution of the 2-4 times of microsphere silica gel volume again, is soaked
The bubble time is 10-12h, is 9-10 with distilled water flushing to efflux pH value, obtains the microsphere silica gel B；
(3) it adsorbs；
The microsphere silica gel B obtained by step (2) is added into the enzyme biochemical reagents filtrate, stirs and evenly mixs rear standing adsorption,
Liquid is discarded supernatant, is washed with distilled water, the microsphere silica gel C is obtained；
The enzyme biochemical reagents filtrate pH value is 9.0-10；
The volume ratio of the enzyme biochemical reagents filtrate and the microsphere silica gel B are 10:3, control the enzyme biochemical reagents filter
Liquid pH value is 8-10；
(4) it elutes；
Klorvess Liquid is added into the microsphere silica gel C obtained by step (3), stirring elution filters out eluent, repeats elution and closes
And the gained eluent, reservation filter out the microsphere silica gel after the eluent；
The concentration of the Klorvess Liquid is 0.5-2.5moC/C, and the frequency of the stirring elution is 60-100r/min, and the time is
45-65min；
The Klorvess Liquid is 1-1.5moC/C, and stirring elution time is 70-90min, and the dosage of the Klorvess Liquid is
0.5-1 times of step (1) the enzyme biochemical reagents filtrate volume；
The microsphere silica gel filtered out after eluent as the microsphere silica gel A continue step (2) prepare it is described micro-
Ball silica gel B；
(5) it is centrifuged；
Eluent obtained by step (4) is first placed in the centrifuge tube that interception is 70KD, is centrifuged and takes subnatant；
Subnatant is placed in the centrifuge tube that interception is 5KD, is centrifuged and upper layer trapped fluid is taken to try to get to the enzyme biochemistry
Agent；
The interception is the revolving speed of 70KD centrifuge tube when being 5000-6000r/min, centrifugation time 3-8min；
The interception is the revolving speed of 5KD centrifuge tube when being 7000-9000r/min, centrifugation time 30-50min；
To get the enzyme biochemical reagents described in after purification after the centrifugate is dried.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810773625.XA CN109136201A (en) | 2018-07-15 | 2018-07-15 | A kind of purification technique of enzyme biochemical reagents |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810773625.XA CN109136201A (en) | 2018-07-15 | 2018-07-15 | A kind of purification technique of enzyme biochemical reagents |
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| CN109136201A true CN109136201A (en) | 2019-01-04 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1216776A (en) * | 1998-11-10 | 1999-05-19 | 同济医科大学 | Immobilized enzyme carrier |
| KR20080071841A (en) * | 2007-01-31 | 2008-08-05 | 에스케이에너지 주식회사 | Separating and purifying method of coenzyme q10 |
| CN105480985A (en) * | 2016-01-13 | 2016-04-13 | 江苏汉邦科技有限公司 | Preparation method of high-purity macroporous silica microspheres |
| CN108118044A (en) * | 2018-02-02 | 2018-06-05 | 华南理工大学 | A kind of method of separating-purifying egg white lysozyme |
| CN108250284A (en) * | 2018-01-24 | 2018-07-06 | 吉林大学 | A kind of method for producing melittin |
-
2018
- 2018-07-15 CN CN201810773625.XA patent/CN109136201A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1216776A (en) * | 1998-11-10 | 1999-05-19 | 同济医科大学 | Immobilized enzyme carrier |
| KR20080071841A (en) * | 2007-01-31 | 2008-08-05 | 에스케이에너지 주식회사 | Separating and purifying method of coenzyme q10 |
| CN105480985A (en) * | 2016-01-13 | 2016-04-13 | 江苏汉邦科技有限公司 | Preparation method of high-purity macroporous silica microspheres |
| CN108250284A (en) * | 2018-01-24 | 2018-07-06 | 吉林大学 | A kind of method for producing melittin |
| CN108118044A (en) * | 2018-02-02 | 2018-06-05 | 华南理工大学 | A kind of method of separating-purifying egg white lysozyme |
Non-Patent Citations (1)
| Title |
|---|
| 沈明才等: "交联壳聚糖微球纯化抑肽酶研究", 《广州化工》 * |
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