CN104263715A - Method for immobilizing phospholipase A2 by calcium alginate-chitosan - Google Patents
Method for immobilizing phospholipase A2 by calcium alginate-chitosan Download PDFInfo
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- CN104263715A CN104263715A CN201410497999.5A CN201410497999A CN104263715A CN 104263715 A CN104263715 A CN 104263715A CN 201410497999 A CN201410497999 A CN 201410497999A CN 104263715 A CN104263715 A CN 104263715A
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Abstract
The invention provides a method for immobilizing phospholipase A2 by calcium alginate-chitosan. A physical embedding method is adopted for immobilizing lysomaxoil. According to the method provided by the invention, the influence of the concentration of calcium alginate, the concentration of chitosan, the concentration of calcium ions, the concentration of glutaraldehyde, the cross-linking time and other different immobilization conditions on the immobilization efficiency and catalytic effect of lysomaxoil is studied, and a response surface analysis method is adopted for optimizing the studied factors. Lysomaxoil is immobilized according to the embedding property of a composite carrier in the method provided by the invention, the activity and stability of an enzyme in a reaction system are improved, and the activity and selectivity of the enzyme are regulated and controlled, thereby being conductive to recovery of the enzyme and production of a product, enabling the recovery rate of activity of the finally obtained immobilized enzyme to be 80% and having important guide significance for an enzymatic degumming process in oil refining.
Description
Technical field
The present invention relates to one Ah-ACMS and fix phospholipase A
2method.
Background technology
Indication acyltransferase is a kind of phospholipase A herein
2(PLA
2).PLA
2be that a class can at the enzyme of the Sn-2 site selectivity fracture ester bond of phosphatide glycerol moiety, phospholipid hydrolysis can be become a series of biologically active substance by it; Sterol ester also can be turned to sterol ester by indication acyltransferase herein.PLA
2enzyme source is from pig pancreas, and material relative shortage, and there is the acceptance problem of the religion cultures such as Islamic, does not promote out; But the source of indication acyltransferase is gas list bag Pseudomonas herein, there are not the problems referred to above, be easy to promote.In the nineties in last century, Enzymax technique is used for enzymatic degumming industrial production by German Lurgi company the earliest, and the enzyme that this technique adopts is exactly stable PLA
2but the consumption of enzyme is very large, and exploitation is just badly in need of in the recycling of such enzyme.Domestic also have many relevant reports, now for the enzyme that comes unstuck the mainly Lecitase 10L (PLA that releases of Novozymes company
2), Lecitase Novo (PLA
1) and Lecitase Ultra (PLA
1).Wherein, the Phospholipid hydrolase Lecitase 10L in Pancreas Sus domestica source is not used in fat degumming, and is had more the microbe-derived Lysomax oil (PLA of advantage
2) replaced.
But, free Lysomax oil (PLA
2) expensive, consumption is 100ppm, is the twice of Lecitase Ultra consumption, not easily reclaims, and to reaction environment bad adaptability.And for immobilized phospholipase A
2, it has following advantage: uniform particles and naked eyes are visible, is easy to immobilized enzyme, substrate to separate with product, facilitates the purifying that product is follow-up; The temperature stability of enzyme improves, and optimum pH and optimum temperuture change, and increases temperature and pH value subject range, reduces inhibitor and protease sensitive; The yield of product can be increased, namely improve the output of oil yield and sterol ester; Can reuse repeatedly; Acyltransferase reaction conditions is more easy to get control; The service efficiency of enzyme is high, and use cost is low; Relative resolvase, is more suitable for industrialization, serialization, automatic production etc.
The method preparing immobilized enzyme mainly contains embedding, absorption, crosslinking and covalent bonds etc., and wherein entrapping method does not need the amino-acid residue of chemically modified zymoprotein, has reaction conditions gentleness, seldom changes enzyme molecular structure, enzyme activity loses the advantages such as little.Alginate calcium, chitosan are embedded materials the most common, therefore can be used as the carrier of immobilized enzyme with it.Alginate calcium, chitosan can form SchiffShi alkali with glutaraldehyde base, form the protective membrane of one deck densification in capsule surface, can increase the intensity of microcapsule ball.Take Ah-ACMS as embedded material, glutaraldehyde as cross linker, by acyltransferase immobilization, finally selects optimum fixing condition.
Summary of the invention
The present invention is to solve in enzymatic degumming process, free phospholipase A
2consumption greatly and the problem not easily reclaimed; and the method with Ah-ACMS immobilized liposome acyltransferase (Lysomax oil) proposed, and acyltransferase (Lysomax oil) immobilized method and application thereof have not yet to see report so far.Realized by following steps by the method for the composite carrier immobilized acyltransferase of Ah-ACMS (Lysomax oil): one, the immobilization of acyltransferase (Lysomax oil); Two, the mensuration of immobilized enzyme enzyme activity; Three, measure enzyme activity and then calculate the enzyme rate of recovery alive; Four, the optimization of fixing condition: respectively in alginate calcium concentration 0.1% ~ 5.0%, chitosan concentration is 0.1% ~ 5.0%, calcium chloride concentration is 0.1mol/L ~ 2.0 mol/L, glutaraldehyde concentration is 0.1% ~ 1.0%, crosslinking time is under the condition of 3h ~ 10h, obtains best fixing condition by measuring the immobilized enzyme rate of recovery; Five, adopt response phase method to carry out analysis discussion and finally determine best fixing condition.
Adopt Ah-ACMS as carrier immobilized acyltransferase (Lysomax oil), Ah-ACMS is as carrier, do not need the amino-acid residue of chemically modified zymoprotein, there is reaction conditions gentleness, seldom change enzyme molecular structure, enzyme activity loses the advantages such as little; In addition, the immobilized enzyme microballoon physical strength that Ah-ACMS immobilized liposome acyltransferase (Lysomax oil) finally obtains is good, and the rate of recovery of enzyme can reach 80%, has great importance for the enzymatic degumming technique in oil and fat refining.
Embodiment
Embodiment one: realized by following steps by the method for Ah-ACMS immobilized liposome acyltransferase (Lysomax oil): one, the preparation of immobilized liposome acyltransferase (Lysomax oil): take a certain amount of alginate calcium and be incubated dissolving in 40 DEG C of water-baths; take simultaneously a certain amount of chitosan 5% acetum in 40 DEG C of water-baths, be incubated dissolving, then add a certain amount of CaCl in consoluet chitosan-acetic acid solution
2solution, fully mixes.Accurately pipette 1ml with liquid-transfering gun and dilute the enzyme liquid of 10 times in certain density 10mL alginate calcium solution, stir.After static for some time, suck above-mentioned mixed solution with the 10mL syringe after sterilizing, inject certain density 10mL chitosan-acetic acid solution, CaCl with about 5/s
2in mixing solutions, stir with rotating speed 180r/min, form smooth microcapsule ball, then add finite concentration glutaraldehyde, rapid stirring, is placed on 4 DEG C of environment immobilization certain hours, by microcapsule with pH 8.0 NaHCO simultaneously
3, 0.1mo1/L NaCl, 0.l mol/L pH5.5 NaAc, pH be after 8.0 Tris-HCl sequential purge, then carries out lyophilize; Two, the mensuration of immobilized enzyme enzyme activity; Three, measure enzyme activity and then calculate the enzyme rate of recovery alive; Four, the optimization of fixing condition: respectively in alginate calcium concentration 0.1% ~ 5.0%, chitosan concentration is 0.1% ~ 5.0%, calcium chloride concentration is 0.1mol/L ~ 2.0 mol/L, glutaraldehyde concentration is 0.1% ~ 1.0%, crosslinking time is under the condition of 3h ~ 10h, obtains best fixing condition by measuring the immobilized enzyme rate of recovery; Five, adopt response phase method to carry out analysis discussion and finally determine best fixing condition.
Embodiment two: the difference of present embodiment and embodiment one be in step 4 by concentration be 1.5% ~ 2.5% alginate calcium in 40 DEG C of water-baths, be incubated the condition of dissolving under carry out the immobilization of acyltransferase (Lysomax oil).Other step is identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one be in step 4 by concentration be 1.5% ~ 2.5% chitosan 5.0% acetum in 40 DEG C of water-baths, be incubated dissolving condition under carry out the immobilization of acyltransferase (Lysomax oil).Other step is identical with embodiment one.
Embodiment four: the difference of present embodiment and embodiment one is to add the CaCl that concentration is 0.1mol/L ~ 0.3mol/L in step 4 in consoluet chitosan-acetic acid solution
2solution, to acyltransferase (Lysomax oil) being fixed.Other step is identical with embodiment one.
Embodiment five: the difference of present embodiment and embodiment one is to add the glutaraldehyde that concentration is 0.2% ~ 0.4% in step 4 in the mixing solutions of the smooth microcapsule ball formed, to acyltransferase (Lysomax oil) being fixed.Other step is identical with embodiment one.
Embodiment six: the difference of present embodiment and embodiment one is crosslinking time 6h ~ 8h in step 4, to acyltransferase (Lysomax oil) being fixed.Other step is identical with embodiment one.
Embodiment seven: the difference of present embodiment and embodiment one is to adopt Responds Surface Methodology to analyze best fixing condition further in step 5.Other step is identical with embodiment one.
Claims (7)
1. fix a method for Phospholipase A2 with Ah-ACMS, it is characterized in that fixing acyltransferase with Ah-ACMS complex carrier is realized by following steps:
Step one: be incubated the alginate calcium that concentration of ordinary dissolution is 0.1% ~ 5.0% in 40 DEG C of water-baths respectively, take simultaneously a certain amount of chitosan 5% acetum in 40 DEG C of water-baths, be incubated dissolving, then add a certain amount of CaCl in consoluet chitosan-acetic acid solution
2solution, fully mixes, and accurately pipettes 1ml and dilutes the enzyme liquid of 10 times in certain density 10mL alginate calcium solution, stir with liquid-transfering gun; After static for some time, suck above-mentioned mixed solution with the 10mL syringe after sterilizing, inject certain density 10mL chitosan-acetic acid solution, CaCl with about 5/s
2in mixing solutions, stir with rotating speed 180r/min, form smooth microcapsule ball, then add finite concentration glutaraldehyde, rapid stirring, is placed on 4 DEG C of environment immobilization certain hours simultaneously; By microcapsule with pH8.0 NaHCO
3, 0.1mo1/L NaCl, 0.lmol/L pH5.5 NaAc, after pH8.0 Tris-HCl sequential purge, then carry out lyophilize, obtained immobilized enzyme carries out enzyme activity determination analysis, determines alginate calcium concentration;
Step 2: select chitosan concentration to be respectively 0.1% ~ 5.0%, course synchronization rapid, determines chitosan concentration;
Step 3: selective chlorination calcium concn is respectively 0.1mol/L ~ 1.0 mol/L, course synchronization rapid, determines calcium chloride concentration;
Step 4: select glutaraldehyde concentration to be respectively 0.1% ~ 1.0%, course synchronization rapid, determines glutaraldehyde concentration;
Step 5: select crosslinking time to be respectively 2h ~ 10h, course synchronization rapid, determines crosslinking time.
2. the method for a kind of Ah-ACMS immobilized liposome acyltransferase according to claim 1, it is characterized in that in step one by concentration be 1.5% ~ 2.5% alginate calcium in 40 DEG C of water-baths, be incubated dissolving.
3. the method for a kind of Ah-ACMS immobilized liposome acyltransferase according to claim 1, it is characterized in that in step 2 by concentration be 1.5% ~ 2.5% chitosan 5% acetum in 40 DEG C of water-baths, be incubated dissolving.
4. the method for a kind of Ah-ACMS immobilized liposome acyltransferase according to claim 1, is characterized in that in consoluet chitosan-acetic acid solution, adding the CaCl that concentration is 0.1mol/L ~ 0.3mol/L in step 3
2solution.
5. the method for a kind of Ah-ACMS immobilized liposome acyltransferase according to claim 1, is characterized in that in the mixing solutions of the smooth microcapsule ball formed, adding the glutaraldehyde that concentration is 0.2% ~ 0.4% in step 4.
6. the method for a kind of Ah-ACMS immobilized liposome acyltransferase according to claim 1, is characterized in that crosslinking time 6h ~ 8h in step 5.
7. the method for a kind of Ah-ACMS immobilized liposome acyltransferase according to claim 1; it is characterized in that with the immobilized enzyme rate of recovery for response value; adopt response phase method to carry out analysis discussion and finally determine best fixing condition, and enzyme is lived, the rate of recovery can reach 80%.
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| CN201410497999.5A CN104263715A (en) | 2014-09-26 | 2014-09-26 | Method for immobilizing phospholipase A2 by calcium alginate-chitosan |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561215A (en) * | 2015-01-17 | 2015-04-29 | 东北农业大学 | Method for preparing plant sterol ester by using immobilized lipid acyltransferase |
| CN106591275A (en) * | 2016-11-30 | 2017-04-26 | 温县兴发生物科技有限公司 | Method for preparing calcium gluconate through co-immobilization of glucose oxidase and catalase |
| CN106754856A (en) * | 2016-11-30 | 2017-05-31 | 温县兴发生物科技有限公司 | A kind of method that co-immobilization glucose oxidase and catalase prepare ferrous gluconate |
| CN106754858A (en) * | 2016-11-30 | 2017-05-31 | 温县兴发生物科技有限公司 | A kind of method that co-immobilization glucose oxidase and catalase prepare sodium gluconate |
| CN106754857A (en) * | 2016-11-30 | 2017-05-31 | 温县兴发生物科技有限公司 | A kind of method that co-immobilization glucose oxidase and catalase prepare zinc gluconate |
-
2014
- 2014-09-26 CN CN201410497999.5A patent/CN104263715A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561215A (en) * | 2015-01-17 | 2015-04-29 | 东北农业大学 | Method for preparing plant sterol ester by using immobilized lipid acyltransferase |
| CN106591275A (en) * | 2016-11-30 | 2017-04-26 | 温县兴发生物科技有限公司 | Method for preparing calcium gluconate through co-immobilization of glucose oxidase and catalase |
| CN106754856A (en) * | 2016-11-30 | 2017-05-31 | 温县兴发生物科技有限公司 | A kind of method that co-immobilization glucose oxidase and catalase prepare ferrous gluconate |
| CN106754858A (en) * | 2016-11-30 | 2017-05-31 | 温县兴发生物科技有限公司 | A kind of method that co-immobilization glucose oxidase and catalase prepare sodium gluconate |
| CN106754857A (en) * | 2016-11-30 | 2017-05-31 | 温县兴发生物科技有限公司 | A kind of method that co-immobilization glucose oxidase and catalase prepare zinc gluconate |
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Application publication date: 20150107 |