CN110055238A - A kind of extracting method of sea cucumber alkaline phosphatase - Google Patents

A kind of extracting method of sea cucumber alkaline phosphatase Download PDF

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Publication number
CN110055238A
CN110055238A CN201910395291.1A CN201910395291A CN110055238A CN 110055238 A CN110055238 A CN 110055238A CN 201910395291 A CN201910395291 A CN 201910395291A CN 110055238 A CN110055238 A CN 110055238A
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sea cucumber
tris
alkaline phosphatase
hcl
freeze
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谢智鑫
王宇聪
王浩
杨红
李志如
刘容旭
王子欢
刘丹怡
王钰堡
韩建春
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Northeast Agricultural University
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Northeast Agricultural University
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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
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  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
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  • Enzymes And Modification Thereof (AREA)

Abstract

A kind of extracting method of sea cucumber alkaline phosphatase, belongs to zymetology field.The method is as follows: taking sea cucumber, cleans up, drains away the water；Sea cucumber is uniformly coated on plate and is lyophilized；Sea cucumber freeze-dried powder is packed into supercritical CO2Extraction and separation in the feed chamber of extraction equipment；The centrifugation of Tris-HCl buffer homogeneous is added into sea cucumber freeze-dried powder, obtains sea cucumber ALP crude enzyme liquid；Sea cucumber ALP crude enzyme liquid is subjected to anion-exchange chromatography, filler is DEAE- anion chromatography medium.The invention has the advantages that sea cucumber gets rid of extra moisture, then through supercritical CO after freeze-drying2It extracts, gets rid of extra fat, while realizing the interference for excluding moisture and fat, and also there is certain bactericidal effect, in solution traditional extraction technique the problems such as organic solvent residual, extraction and purification effect with better alkaline phosphatase.

Description

A kind of extracting method of sea cucumber alkaline phosphatase
Technical field
The invention belongs to zymetology fields, and in particular to a kind of extracting method of sea cucumber alkaline phosphatase.
Background technique
Alkaline phosphatase (Alkaline phosphatase, ALP), is a kind of hydrolase, in alkaline condition (pH 9.0- 10.0) there is high catalytic activity under, phosphoric acid monoester key can be catalyzed and generate inorganic orthophosphate and corresponding phenol, pure and mild sugar etc..It should Fermentoid energy catalytic nucleic acid molecule takes off 5 ' phosphate groups, so that the 5 ' ends-P of DNA or RNA segment is made to be converted into the 5 ' ends-OH, But its not single enzyme, one group of isoenzymes, i.e. tissue non-specific alkaline phosphatase (TNAP) (be mainly expressed in liver, Bone and kidney), P-ALP (PLAP), reproduction cell alkaline phosphatase (GCAP) and intestinal alkaline phosphatase (IAP). ALP is widely present in animal, plant, in microorganism, and the ALP content under different living conditions is different, wherein ALP in marine organisms Content is generally higher.
Sea cucumber (Sea cucumber), belongs to Echinodermata (Echiondermate), Holothuroidea on taxology (Holothuroidea), it can live by the sea to the seabed of 8000 meters of depths.The type of whole world sea cucumber there are about more than 1200 kinds, But can be edible sea cucumber type it is seldom, about more than 40.About 140 kinds of China's sea cucumber type, wherein edible Less than the 15% of sum, the wherein quality most preferably yellow tidal sea sea area stichopus japonicus (Stichopus Japonicus) in the north.Stichopus japonicus tool There are very high nutrition and medical value, is listed in one of marine products " eight delicacies ".Wherein containing abundant in the by-product sea cucumber of sea cucumber Enzyme, this is related with the living environment of sea cucumber.
With the development of bioengineering, enzymic catalytic reaction is increasingly valued by people.However, due in exoenzyme Activity reduce and its unstability, significantly limit its application industrially.To find out its cause, mainly enzyme extract and The change of structure in purification process.Studies have shown that the performance of enzymatic activity is largely dependent upon its molecular conformation, conformation is risen Variation, activity or function also become therewith.Therefore people in depth inquire into enzyme kinetics and influence enzymatic reaction Factor, and by the way that the external environment of enzyme reaction is modified or changed to enzyme molecule to improve the activity and stabilization of enzyme in vitro Property.
Supercritical fluid (supercritical fluid, SCF) refers to that temperature and pressure are in critical-temperature and critical pressure Fluid more than power is the fluid during a kind of liquid gaseous state is in homogeneous.The advantages of this fluid has liquids and gases concurrently, viscosity It is small, diffusion coefficient is big, density is big, there are good dissolubility and mass transfer performances, and in Near The Critical Point fluid to pressure and temperature Variation it is very sensitive, be both a kind of good separating medium and a kind of good reaction medium.Further, since SCF has The physical property of consecutive variations can control reactivity and selectivity by influencing its local action.As a kind of non-aqueous reaction Medium, SCF is with unique property by more and more extensive concern in enzyme catalysis field.So enzyme is placed in supercriticality Under its catalytic kinetics can also be made to be improved.With supercritical CO2For, CO under high pressure2Change the pH in enzyme microenvironment Value, the free amino of covalent modification protein surface simultaneously form carbaminate, these carbaminates change lysine residue Charge, to change the activity of enzyme.
Sea cucumber alkaline phosphatase extractive technique route more commonly used at present is as follows: fresh sea cucumber intestine → plus 50mM/L Tris-HCl buffer solution → homogeneous → centrifugation (4 DEG C of 9000r/min, 20min) → stratification takes supernatant → plus just Butanol 1:4(V/V), homogeneous → centrifugation (4 DEG C of 9000r/min, 20min) → stratification, fetch water phase supernatant → addition sulfuric acid Ammonium → centrifugation (4 DEG C of 9000r/min, 20min) → is collected precipitating → buffer solution → dialysis → -80 DEG C and is saved.This method A large amount of organic solvent is needed, alkaline phosphatase can also partially remain in organic molten during extracting fat using n-butanol In agent, extraction effect is not good enough.
Summary of the invention
The purpose of the present invention is to solve the method extraction effect for extracting alkaline phosphatase from sea cucumber at present is poor The problem of, a kind of extracting method of sea cucumber alkaline phosphatase, i.e. supercritical CO are provided2Fluids extraction.This method first will be extra large Ginseng flower carries out cleaning freeze-drying, then passes through supercritical CO2Extraction removes the fat in sea cucumber, effectively eliminates extra water Divide the interference with fat, and supercritical CO2Extraction also has certain bactericidal effect, is more advantageous to subsequent to alkaline phosphatase The extraction of enzyme.
To achieve the above object, the technical solution adopted by the present invention is as follows:
A kind of extracting method of sea cucumber alkaline phosphatase, specific step is as follows for the method:
Step 1: fresh sea cucumber is taken, is cleaned up, is drained away the water；
Step 2: the sea cucumber that step 1 obtains uniformly being coated on plate and is lyophilized, and with a thickness of 3 ~ 5mm, makes its moisture Lower than 5%, milling is put into -80 DEG C of refrigerators and saves for use；
Step 3: sea cucumber freeze-dried powder is packed into supercritical CO2In the feed chamber of extraction equipment, setting extracting pressure is 20MPa, Separating pressure is 8MPa, and extraction and separation carry out simultaneously, and total time is 1.5 ~ 2h, retains the sea cucumber freeze-dried powder in feed chamber；
Step 4: Tris-HCl buffer being added into the sea cucumber freeze-dried powder that step 3 obtains, with homogenizer homogeneous 15min, It is put into ultrasonic equipment after homogeneous, is dissolved after ultrasound with magnetic stirrer, the sea cucumber mixed liquor dissolved is centrifuged, is obtained The supernatant arrived carries out protein precipitation with the ammonium sulfate that saturation degree is 70%, and precipitating 50mM/L Tris-HCl pH is taken after centrifugation 8.9 buffer solutions obtain sea cucumber ALP crude enzyme liquid after desalting processing；
Step 5: sea cucumber ALP crude enzyme liquid is subjected to anion-exchange chromatography, filler is DEAE- anion chromatography medium, specifically Are as follows:
1. balancing chromatographic column using equilibrium liquid, then sample upper prop uses equilibrium liquid rebalancing；The equilibrium liquid is 50Mm/L Tris-HCl pH 8.9；
2. carrying out gradient elution, buffer solution A is 50 mM/L Tris-HCl pH 8.9, and buffer solution B is 50mM/L Tris-HCl PH 8.9+0.8M/L NaCl, setting eluent gradient timetable are 60min, elution flow rate 5ml/min；Collect eluent, detection The eluent that enzyme activity is 50U/mL is merged, is concentrated into 6mL and crosses Chromdex S-200 PG chromatographic column, by vigor peak by enzyme activity - 80 DEG C of freezings are concentrated after collection.
The beneficial effect of the present invention compared with the existing technology is: sea cucumber gets rid of extra water after freeze-drying Point, then through supercritical CO2Extraction, gets rid of extra fat, while realizing the interference for excluding moisture and fat, and also have The problems such as having certain bactericidal effect, solving organic solvent residual in traditional extraction technique, with better alkaline phosphatase Extraction and purification effect.
Specific embodiment
Further description of the technical solution of the present invention below, and however, it is not limited to this, all to the technology of the present invention Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in the present invention Protection scope in.
Specific embodiment 1: present embodiment record be a kind of sea cucumber alkaline phosphatase extracting method, it is described Specific step is as follows for method:
Step 1: fresh sea cucumber is taken, is cleaned up, is drained away the water；
Step 2: the sea cucumber that step 1 obtains uniformly being coated on plate and is lyophilized, and with a thickness of 3 ~ 5mm, makes its moisture Lower than 5%, milling is put into -80 DEG C of refrigerators and saves for use；
Step 3: sea cucumber freeze-dried powder is packed into supercritical CO2In the feed chamber of extraction equipment, setting extracting pressure is 20MPa, Separating pressure is 8MPa, and extraction and separation carry out simultaneously, and total time is 1.5 ~ 2h, retains the sea cucumber freeze-dried powder in feed chamber；
Step 4: Tris-HCl buffer being added into the sea cucumber freeze-dried powder that step 3 obtains, with homogenizer homogeneous 15min, It is put into ultrasonic equipment after homogeneous, is dissolved after ultrasound with magnetic stirrer, the sea cucumber mixed liquor dissolved is centrifuged, is obtained The supernatant arrived carries out protein precipitation with the ammonium sulfate that saturation degree is 70%, and precipitating 50mM/L Tris-HCl pH is taken after centrifugation 8.9 buffer solutions obtain sea cucumber ALP crude enzyme liquid after desalting processing；
Step 5: sea cucumber ALP crude enzyme liquid is subjected to anion-exchange chromatography, filler is DEAE- anion chromatography medium, specifically Are as follows:
1. balancing chromatographic column using equilibrium liquid, then sample upper prop uses equilibrium liquid rebalancing；The equilibrium liquid is 50Mm/L Tris-HCl pH 8.9；All solution of chromatography of the present invention will cross 0.45 μm of filter membrane, including sample；
2. carrying out gradient elution, buffer solution A is 50 mM/L Tris-HCl pH 8.9, and buffer solution B is 50mM/L Tris-HCl PH 8.9+0.8M/L NaCl, setting eluent gradient timetable are 60min, elution flow rate 5ml/min；Collect eluent, detection The eluent that enzyme activity is 50U/mL is merged, is concentrated into 6mL and crosses Chromdex S-200 PG chromatographic column, by vigor peak by enzyme activity (it will appear different absorbing proteins peaks after S-200 elution, the Activity determination of alkaline phosphatase carried out to each peak, it is active Save, no gives up) collect after be concentrated -80 DEG C of freezings.
The method of Conventional chromatography are as follows: balance-loading-balance-elution, DEAE anion chromatography operating method: less salt balance- Less salt loading-less salt balance-high eluting salt.Balancing chromatographic column with equilibrium liquid before loading is that the filler state in chromatographic column is allowed to reach Stable environment is convenient for loading, and equilibrium liquid balance is to allow the protein stabilization being adsorbed on chromatography column packing after loading.It uses Equilibrium liquid the same buffer of effect.
Specific embodiment 2: a kind of extracting method of sea cucumber alkaline phosphatase described in specific embodiment one, step In rapid four, the mass volume ratio of the sea cucumber freeze-dried powder and Tris-HCl buffer is 2g:200mL, and Tris-HCl is slow The concentration of fliud flushing is 50mM/L, pH 8.9, in Tris-HCl buffer containing percent 0.2 Triton X-100, Triton X-100 is protective agent.
Specific embodiment 3: a kind of extracting method of sea cucumber alkaline phosphatase described in specific embodiment one, step In rapid four, the ultrasound condition specifically: power 200W, time 15min.
Specific embodiment 4: a kind of extracting method of sea cucumber alkaline phosphatase described in specific embodiment one, step In rapid four, the actual conditions of the centrifugation are as follows: 10000r/min, 20min, 4 DEG C.

Claims (4)

1. a kind of extracting method of sea cucumber alkaline phosphatase, it is characterised in that: specific step is as follows for the method:
Step 1: fresh sea cucumber is taken, is cleaned up, is drained away the water；
Step 2: the sea cucumber that step 1 obtains uniformly being coated on plate and is lyophilized, and with a thickness of 3 ~ 5mm, makes its moisture Lower than 5%, milling is put into -80 DEG C of refrigerators and saves for use；
Step 3: sea cucumber freeze-dried powder is packed into supercritical CO2In the feed chamber of extraction equipment, setting extracting pressure is 20MPa, Separating pressure is 8MPa, and extraction and separation carry out simultaneously, and total time is 1.5 ~ 2h, retains the sea cucumber freeze-dried powder in feed chamber；
Step 4: Tris-HCl buffer being added into the sea cucumber freeze-dried powder that step 3 obtains, with homogenizer homogeneous 15min, It is put into ultrasonic equipment after homogeneous, is dissolved after ultrasound with magnetic stirrer, the sea cucumber mixed liquor dissolved is centrifuged, is obtained The supernatant arrived carries out protein precipitation with the ammonium sulfate that saturation degree is 70%, and precipitating 50mM/L Tris-HCl pH is taken after centrifugation 8.9 buffer solutions obtain sea cucumber ALP crude enzyme liquid after desalting processing；
Step 5: sea cucumber ALP crude enzyme liquid is subjected to anion-exchange chromatography, filler is DEAE- anion chromatography medium, specifically Are as follows:
1. balancing chromatographic column using equilibrium liquid, then sample upper prop uses equilibrium liquid rebalancing；The equilibrium liquid is 50Mm/L Tris-HCl pH 8.9；
2. carrying out gradient elution, buffer solution A is 50 mM/L Tris-HCl pH 8.9, and buffer solution B is 50mM/L Tris-HCl PH 8.9+0.8M/L NaCl, setting eluent gradient timetable are 60min, elution flow rate 5ml/min；Collect eluent, detection The eluent that enzyme activity is 50U/mL is merged, is concentrated into 6mL and crosses Chromdex S-200 PG chromatographic column, by vigor peak by enzyme activity - 80 DEG C of freezings are concentrated after collection.
2. a kind of extracting method of sea cucumber alkaline phosphatase according to claim 1, it is characterised in that: in step 4, The mass volume ratio of the sea cucumber freeze-dried powder and Tris-HCl buffer is 2g:200mL, Tris-HCl buffer it is dense Degree is 50mM/L, pH 8.9, in Tris-HCl buffer containing 0.2% Triton X-100.
3. a kind of extracting method of sea cucumber alkaline phosphatase according to claim 1, it is characterised in that: in step 4, The ultrasound condition specifically: power 200W, time 15min.
4. a kind of extracting method of sea cucumber alkaline phosphatase according to claim 1, it is characterised in that: in step 4, The actual conditions of the centrifugation are as follows: 10000r/min, 20min, 4 DEG C.
CN201910395291.1A 2019-05-13 2019-05-13 A kind of extracting method of sea cucumber alkaline phosphatase Pending CN110055238A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050065063A1 (en) * 2001-10-22 2005-03-24 Marco Gentile Supercritical fluids processing: preparation of protein microparticles and their stabilisation
CN102839162A (en) * 2012-09-26 2012-12-26 西藏俪阳科技有限公司 Preparation method for alkaline phosphatase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050065063A1 (en) * 2001-10-22 2005-03-24 Marco Gentile Supercritical fluids processing: preparation of protein microparticles and their stabilisation
CN102839162A (en) * 2012-09-26 2012-12-26 西藏俪阳科技有限公司 Preparation method for alkaline phosphatase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
QINGFENG TANG等: "Immunomodulatory effects of supercritical fluid CO2 extracts from freeze-dried powder of Tenebrio molitor larvae (yellow mealworm)", 《FOOD SCIENCE AND TECHNOLOGY》 *
揭广川等: "《食品工业新技术及应用》", 31 October 1995, 中国轻工业出版社 *
程菁恒: "海参肠碱性磷酸酶的提取、纯化及其特性研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 *
谢莉萍等: "合浦珠母贝碱性磷酸酶的分离纯化与性质研究", 《海洋科学》 *

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Application publication date: 20190726