CN1614012A - Method for separating L-amino-acid oxidase from venin - Google Patents
Method for separating L-amino-acid oxidase from venin Download PDFInfo
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- CN1614012A CN1614012A CN 200410052289 CN200410052289A CN1614012A CN 1614012 A CN1614012 A CN 1614012A CN 200410052289 CN200410052289 CN 200410052289 CN 200410052289 A CN200410052289 A CN 200410052289A CN 1614012 A CN1614012 A CN 1614012A
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Abstract
It was involved in separating L-amino acid oxidase from the snake venom of Agkistrodonhalysussuriensis. Through two steps of colum chromatography (i.e. heparin fast flow and qsepherose fast flow) L-amino acid oxidase was separated and purified from the snake toxin of Gloydius ussuriensis. Not including transfer between concentrating and buffer system. It could avoid the influence of freezing and the pH value change to enzyme activity. The method was found to be simple, rapid and convenient. The ratio of recovery was high.
Description
Technical field
The present invention relates to a kind of L-amino-acid oxidase, relate in particular to a kind of separation method of L-amino-acid oxidase.
Background technology
The amino acid whose deamination of L-amino-acid oxidase energy catalysis L-generates corresponding alpha-ketoacid, ammonia and hydrogen peroxide.On industrial production, the L-amino-acid oxidase is used to produce alpha-ketoacid; The L-amino acid of in L-amino acid and D-amino acid miscellany, degrading, thus isolate D-amino acid.The L-amino-acid oxidase also can be used to the amino acid whose enzyme electrodes of formation determination L-.Snake venom is one of main source of L-amino-acid oxidase.The yellow of snake venom is exactly that the L-amino-acid oxidase produces.A plurality of L-amino-acid oxidases from snake venom, have been separated at present, and their physico-chemical property and physiologically active carried out a large amount of research, find that the L-amino-acid oxidase has antitumor, antibiotic, antiviral, inhibition or induced platelet to assemble isoreactivity, these are directly active or relevant with hydrogen peroxide of its enzyme reaction generation indirectly.
Snake venom L-amino acid oxidase has many common traits: they all are to be made of two same subunit, and apparent molecular weight is about 120kD; Each subunit is about 60kD; All contain two non-covalent bonded prothetic group FAD or FMN.Studies show that freezing and pH changes reduction even the forfeiture that prothetic group is broken away from and cause enzymic activity.In the L-amino-acid oxidase separating technology of report, great majority comprise all and dialyse and change the step of buffer system that this must cause the influence to this enzymic activity concentrated at present, thereby influence is to the research of this enzyme zymologic property and physiologically active and in industrial application.Therefore, seek a kind of can within a short period of time, do not have to concentrate and L-amino-acid oxidase separation method that the buffering system changes has very important effect for the further investigation and the development and use of this enzyme.
Summary of the invention
The objective of the invention is to concentrate and buffer system is changed in dialysis at comprising in the existing L-amino-acid oxidase isolation technique, thereby to the problem that L-amino-acid oxidase enzymic activity impacts, providing a kind of does not have to concentrate and dialyse and change the L-amino-acid oxidase separation method of buffer system.
Technical scheme of the present invention is achieved in that a kind of method of separating the L-amino-acid oxidase from the agkistrodon halys ussuriensis snake venom, and its characteristics are to realize by affinity chromatography and ion exchange chromatography two-step chromatography.
Wherein, above-mentioned affinity chromatography is a heparin affinity chromatography; Above-mentioned ion exchange chromatography is an anion-exchange chromatography.
Above-mentioned heparin affinity chromatography comprises the steps:
(1) sample preparation: snake venom is dissolved in the Tris-HCl damping fluid, centrifugal, get supernatant liquor;
(2) dress post: mobile phase A liquid is the Tris-HCl damping fluid, and B liquid is the A liquid that contains NaCl; Filler is Heparin-Sepharose Fast Flow; Earlier with the abundant balance chromatography column of A liquid when electric conductivity value does not change, again on through the snake venom sample supernatant liquor of above-mentioned sample preparation;
(3) wash-out: with A liquid wash-out, use 0%B-100%B linear gradient wash-out more earlier, wash chromatography column with 100%B then; Flow velocity is 0.5～3mL/min;
(4) collect detection: collect elutriant, under the photoabsorption of 280nm, detect elutriant.Above-mentioned heparin affinity chromatography specifically comprises the steps:
(1) sample preparation: snake venom is dissolved in the 25mmol/L Tris-HCl damping fluid, pH=8.5, under 4 ℃ of conditions, the centrifugal 10min of 10000r/min gets supernatant liquor;
(2) dress post: chromatographic column volume 25ml, filler is Heparin-Sepharose FastFlow, mobile phase A liquid is 25mmol/L Tris-HCl damping fluid, pH=8.5, B liquid is the A liquid that contains the NaCl of 1mol/L, when earlier fully equilibrating to electric conductivity value and do not change, go up snake venom sample again through sample preparation with A liquid;
(3) wash-out: with behind the 50mL A liquid wash-out, with 0%B-100%B linear gradient wash-out 250mL, wash post 50mL with 100%B then more earlier, flow velocity is 2mL/min;
(4) collect detection: collect elutriant with the substep collector, under the photoabsorption of 280nm, detect protein content.
Above-mentioned anion-exchange chromatography comprises the steps:
(1) dress post: mobile phase A liquid is the Tris-HCl damping fluid, and B liquid is the A liquid that contains NaCl; Filler is Heparin-Sepharose Fast Flow; Earlier with the abundant balance chromatography column of A liquid when electric conductivity value does not change, again on through claim
(4) the snake venom sample of described affinity chromatography;
(2) wash-out: with A liquid wash-out, use 0%B-100%B linear gradient wash-out more earlier, wash chromatography column with 100%B then; Flow velocity is 0.5～3mL/min;
(3) detect collection: collect elutriant, under the photoabsorption of 280nm, detect elutriant, collect the elutriant that contains the L-amino-acid oxidase
Above-mentioned anion-exchange chromatography specifically comprises the steps:
(1) dress post: mobile phase A liquid is 25mmol/L Tris-HCl damping fluid, and pH=8.5, B liquid are the A liquid that contains 1mol/L NaCl, and filler is Heparin-SepharoseFast Flow; Earlier with the abundant balance chromatography column of A liquid when electric conductivity value does not change, again on through the snake venom sample of affinity chromatography;
(2) wash-out: with behind the 100mL A liquid wash-out, with 0%B-100%B linear gradient wash-out 500mL, wash post 100mL with 100%B then more earlier, flow velocity is 2mL/min;
(3) detect collection: collecting elutriant with Fraction Collector, is to detect elutriant under the 280nm at wavelength, collects the elutriant that contains the L-amino-acid oxidase.
Above-mentioned heparin affinity chromatography and anion-exchange chromatography all are to carry out on AKTA Prime equipment.
Beneficial effect of the present invention: the present invention isolates the L-amino-acid oxidase by heparin affinity chromatography and two step of anion-exchange chromatography column chromatography from snake venom.Isolating L-amino-acid oxidase presents a band in the SDS-PAGE electrophorogram, molecular weight is about 58000.Simple, the consuming time weak point of the inventive method, rate of recovery height, and do not have to concentrate and the step of buffering system conversion have effectively avoided freezing and pH to change influence to enzymic activity, are convenient to promote, and can linearly be amplified in the production technique.
Description of drawings
Fig. 1 is the separation graph of snake venom sample on Heparin-Sepharose Fast Flow;
Fig. 2 is the separating spectrum of the active peak of LAO on Q-Sepharose High Performace;
Fig. 3 is LAO electrophoretogram on the SDS-polyacrylamide gel.
Wherein, among Fig. 1, L-amino-acid oxidase enzymic activity is in penetrating the peak; Among Fig. 3,1 is standard protein, and 2 is slightly poison of agkistrodon halys ussuriensis, and 3 for penetrating the peak under the Heparin-Sepharose Fast Flow, and 4 is the active peak after Q-Sepharose High Performace separates, and the SDS-PAGE gel strength is 12.5%.
Embodiment
Embodiment:
1, heparin affinity chromatography
(1) sample preparation: take by weighing agkistrodon halys ussuriensis snake venom 200mg, be dissolved in the 5mL 25mmol/LTris-HCl damping fluid in (pH8.5), under 4 ℃ of conditions, the centrifugal 10min of 10000r/min.Get supernatant liquor.
(2) dress post: chromatographic column volume 25ml, filler are Heparin-Sepharose Fast Flow.Mobile phase A liquid is 25mmol/L Tris-HCl damping fluid (pH8.5), and B liquid is the A liquid that contains the NaCl of 1mol/L.When fully equilibrating to electric conductivity value and not changing with A liquid, last snake venom sample.
(3) wash-out: behind the A liquid 50mL,, wash post 50mL with 100%B again with 0%B-100%B linear gradient wash-out 250mL; Flow velocity is 2mL/min.
(4) collect detection: collect elutriant with the substep collector, every pipe is collected 3mL, detects protein content under the photoabsorption of 280nm.Whole chromatography process is carried out on AKTA Prime equipment.
2, anion-exchange chromatography,
(1) dress post: the chromatographic column volume is 75ml, and mobile phase A liquid is 25mmol/L Tris-HCl damping fluid, and pH=8.5, B liquid are the A liquid that contains 1mol/L NaCl, and filler is Heparin-Sepharose Fast Flow; Earlier with the abundant balance chromatography column of A liquid when electric conductivity value does not change, again on through the snake venom sample of affinity chromatography;
(2) wash-out: with behind the 100mL A liquid wash-out, with 0%B-100%B linear gradient wash-out 500mL, wash post 100mL with 100%B then more earlier, flow velocity is 2mL/min;
(3) detect collection: collect elutriant with Fraction Collector, every pipe is collected 4ml, is to detect elutriant under the 280nm at wavelength, collects the elutriant that contains the L-amino-acid oxidase.Whole chromatography process is carried out on AKTA Prime equipment.
Claims (7)
1, a kind of method of separating the L-amino-acid oxidase from snake venom is characterized in that comprising the steps:
(1) affinity chromatography;
(2) ion exchange chromatography.
2, method of separating the L-amino-acid oxidase from snake venom according to claim 1 is characterized in that the described affinity chromatography of step (1) is a heparin affinity chromatography.
3, method of separating the L-amino-acid oxidase from snake venom according to claim 1 is characterized in that the described ion exchange chromatography of step (2) is an anion-exchange chromatography.
4, method of separating the L-amino-acid oxidase from snake venom according to claim 2 is characterized in that described heparin affinity chromatography specifically comprises the steps:
(1) sample preparation: snake venom is dissolved in the Tris-HCl damping fluid, centrifugal, get supernatant liquor;
(2) dress post: mobile phase A liquid is the Tris-HCl damping fluid, and B liquid is the A liquid that contains NaCl; Filler is Heparin-Sepharose Fast Flow; Earlier with the abundant balance chromatography column of A liquid when electric conductivity value does not change, again on through the snake venom sample supernatant liquor of above-mentioned sample preparation;
(3) wash-out: with A liquid wash-out, use 0%B-100%B linear gradient wash-out more earlier, wash chromatography column with 100%B then; Flow velocity is 0.5～3mL/min;
(4) collect detection: collect elutriant, under the photoabsorption of 280nm, detect elutriant.
5, method of separating the L-amino-acid oxidase from snake venom according to claim 3 is characterized in that described anion-exchange chromatography specifically comprises the steps:
(1) dress post: mobile phase A liquid is the Tris-HCl damping fluid, and B liquid is the A liquid that contains NaCl; Filler is Heparin-Sepharose Fast Flow; Earlier with the abundant balance chromatography column of A liquid when electric conductivity value does not change, again on through the snake venom sample of affinity chromatography;
(2) wash-out: with A liquid wash-out, use 0%B-100%B linear gradient wash-out more earlier, wash chromatography column with 100%B then; Flow velocity is 0.5～3mL/min;
(3) detect collection: collect elutriant, under the photoabsorption of 280nm, detect elutriant, collect the elutriant that contains the L-amino-acid oxidase.
6, method of separating the L-amino-acid oxidase from snake venom according to claim 4 is characterized in that described heparin affinity chromatography specifically comprises the steps:
(1) sample preparation: snake venom is dissolved in the 25mmol/L Tris-HCl damping fluid, pH=8.5, under 4 ℃ of conditions, the centrifugal 10min of 10000r/min gets supernatant liquor;
(2) dress post: chromatographic column volume 25ml, filler is Heparin-Sepharose FastFlow, mobile phase A liquid is 25mmol/L Tris-HCl damping fluid, pH=8.5, B liquid is the A liquid that contains the NaCl of 1mol/L, when earlier fully equilibrating to electric conductivity value and do not change, go up snake venom sample again through sample preparation with A liquid;
(3) wash-out: with behind the 50mL A liquid wash-out, with 0%B-100%B linear gradient wash-out 250mL, wash post 50mL with 100%B then more earlier, flow velocity is 2mL/min;
(4) collect detection: collect elutriant with the substep collector, under the photoabsorption of 280nm, detect protein content.
7, method of separating the L-amino-acid oxidase from snake venom according to claim 5 is characterized in that described anion-exchange chromatography specifically comprises the steps:
(1) dress post: mobile phase A liquid is 25mmol/L Tris-HCl damping fluid, and pH=8.5, B liquid are the A liquid that contains 1mol/L NaCl, and filler is Heparin-SepharoseFast Flow; Earlier with the abundant balance chromatography column of A liquid when electric conductivity value does not change, again on through the snake venom sample of affinity chromatography;
(2) wash-out: with behind the 100mL A liquid wash-out, with 0%B-100%B linear gradient wash-out 500mL, wash post 100mL with 100%B then more earlier, flow velocity is 2mL/min;
(3) detect collection: collecting elutriant with Fraction Collector, is to detect elutriant under the 280nm at wavelength, collects to contain the active elutriant of L-amino-acid oxidase.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2004100522898A CN100348719C (en) | 2004-11-19 | 2004-11-19 | Method for separating L-amino-acid oxidase from venin |
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| CNB2004100522898A CN100348719C (en) | 2004-11-19 | 2004-11-19 | Method for separating L-amino-acid oxidase from venin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102121020A (en) * | 2010-12-07 | 2011-07-13 | 中山大学 | Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof |
| CN102604970A (en) * | 2012-03-20 | 2012-07-25 | 贵阳中医学院 | Preparation method and application of medoggreenpit-viper venom L-amino acid oxidase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526445A (en) * | 2003-03-04 | 2004-09-08 | 中国科学院昆明动物研究所 | Application of snake venom L-amino acid oxidase in preparing AIDS treating medicine |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102121020A (en) * | 2010-12-07 | 2011-07-13 | 中山大学 | Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof |
| CN102121020B (en) * | 2010-12-07 | 2012-09-26 | 中山大学 | Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof |
| CN102604970A (en) * | 2012-03-20 | 2012-07-25 | 贵阳中医学院 | Preparation method and application of medoggreenpit-viper venom L-amino acid oxidase |
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| CN100348719C (en) | 2007-11-14 |
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