CN1408855A - method for improving lipase activity stability - Google Patents
method for improving lipase activity stability Download PDFInfo
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- CN1408855A CN1408855A CN 01126807 CN01126807A CN1408855A CN 1408855 A CN1408855 A CN 1408855A CN 01126807 CN01126807 CN 01126807 CN 01126807 A CN01126807 A CN 01126807A CN 1408855 A CN1408855 A CN 1408855A
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- lipase
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- damping fluid
- linking agent
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Abstract
The method of improving lipase activity stability includes dissolving lipase in buffering phosphate solution, centrifuging to eliminating impurity, ultrafiltering of the supernatant liquid in a ultrafilter to obtain ultrafiltered concentrated liquid, chromatographic separation to further concentrate enzyme, liquid crystallizing lipase via adding crystallization agent into the concentrated enzyme liquid, centrifuging to obtain lipase crystal, crosslinking the lipase crystal with crosslinking agent to obtain lipase with improved activity stability. The method has increased two-step purification before crystallization and the control on crosslinking step to raise the activity stability of lipase, avoid the re-dissolving of crystal and increase product yield.
Description
Technical field
The present invention relates to a kind of method that improves lipase activity stability.
Background technology
In prior art, United States Patent (USP) (patent No. US5618710, April 8 1997 applying date) discloses a kind of cross-linked enzyme crystallization, and lipase crystallization wherein is to derive from the lipase that cylindrical candiyeast fermentation produces.Its method that improves enzymic activity stability is: freeze dried cylindrical lipase from candida sp is dissolved in the 0.5mL deionized water, and concentration is 6mg/mL, and this enzyme solution is dialysed in low concentration of salt buffered soln.Dialysis can be carried out under the condition that multiple damping fluid exists.As being damping fluid, be respectively dialysis in 6,7,8 o'clock at pH with the 5mM sodium phosphate; With 20mM Tutofusin tris and HCl is cocktail buffer, is dialysis in 6.8 o'clock at pH; With 20mM Tutofusin tris, HCl, 1mMCaCl
2, 1mMMgCl
2Being cocktail buffer, is dialysis in 6.8 o'clock at pH.Obtain the thin square plate crystal of 0.05～0.1mm through crystallization in 6 hours after the dialysis, isolating after the crystallization with whizzer is 6.8 times washings 10 times with the 20mM damping fluid at pH again, the centrifugal crystallization that obtains is a cocktail buffer at 7.5% glutaraldehyde and 20mM Tutofusin tris and HCl again, be 6.8 at pH, under the room temperature crosslinked 30 minutes, this crystallization is a cocktail buffer with 20mM Tutofusin tris and HCl, at pH is 6.8 times washings, obtains the finished product.But aspects such as how this method prevents that crystallization from dissolving again in being further purified protoenzyme and technology are still waiting research.
U.S. Vertex pharmaceuticals has set up ALTUS Biologics company specially with the commercialization of cross-linked enzyme crystallization biological catalyst.
Summary of the invention
The purpose of this invention is to provide a kind of method that improves the biological catalyst lipase activity stability, adopt method of the present invention can obtain the biological catalyst of enzymic activity good stability---the enzyme crystallite.
The present invention realizes like this.A kind of method that improves lipase activity stability of the present invention comprises following operation steps:
(1) lipase is dissolved in the phosphate buffered saline buffer, and the centrifugal impurity of removing obtains supernatant liquor, feeds the ultra-filtration equipment ultrafiltration then, obtains ultrafiltration and concentration liquid,
(2) the ultrafiltration and concentration liquid that step (1) is obtained changes dialysis tubing over to, immerses in the damping fluid to dialyse, and enzyme liquid is further concentrated,
(3) add the crystalline deposit agent in the enzyme concentrated liquor that in step (2), obtains and make the lipase crystallization, obtain the lipase crystal after centrifugal,
(4) lipase crystal and linking agent mix carry out crosslinked, the crosslinked lipase that finishes the centrifugal enzymic activity stability that is improved in back.
Method of the present invention, dialysis tubing submerged damping fluid is that ammonium sulfate damping fluid, phosphate buffered saline buffer, molecular weight are 6000～8000 polyvinyl alcohol damping fluid, 2 (N-morpholino) propane sulfonic acid damping fluid during dialysis.
Method of the present invention, described crystalline deposit agent are that molecular weight is 4000～8000 polyvinyl alcohol, 2-acetone, 2-methyl-2,4-pentanediol, CaCl
2
Method of the present invention can add fine lipase crystal seed induced crystallization during crystallization, during induced crystallization, the add-on of fine lipase crystal seed is that fine lipase crystal seed accounts for 0.01～0.25% in total amount of liquid (percent by volume).
Method of the present invention, when lipase crystal and linking agent were crosslinked, the linking agent add-on was that linking agent accounts for 1～10% in total amount of liquid (percent by volume), and crosslinking time is 30～200 minutes, and crosslinking temperature is 10～50 ℃.
Method of the present invention, the linking agent that adds when the lipase crystal is crosslinked is cyanogen bromide, polymine, glutaraldehyde, carbodiimide.
The present invention compared with prior art has the following advantages:
1. the method for raising lipase activity stability of the present invention adopts the purification process of the ultrafiltration removal of impurity, dialysis enrichment can improve the active princlple content of enzyme.
2. method of the present invention adopts and adds fine lipase crystal induced crystallization, is convenient to obtain the lipase crystallite.
3. the crosslinking process that method of the present invention adopts can prevent that lipase crystallization crystal self from dissolving again, increased the stability of product.
4. the method for the present invention's employing can obtain better fine crystalline of enzymic activity stability, can improve the rate of recovery of total enzyme simultaneously.
Embodiment
Further specify the present invention below by detailed description, but embodiment not a limitation of the present invention to specific embodiments of the invention.
Embodiment 1
(1) 0.8g lipase is dissolved in the 250mL phosphate buffered saline buffer, the centrifugal impurity of removing obtains supernatant liquor, and supernatant liquor obtains ultrafiltration and concentration liquid by the ultra-filtration equipment ultrafiltration, and this moment, the rejection of enzyme was 100%.
(2) ultrafiltration and concentration liquid is changed in the dialysis tubing, as damping fluid, dialysis made solution equilibria in 12 hours under pH=7.5, obtained further spissated enzyme liquid with phosphoric acid salt.
(3) get spissated lipase enzyme liquid 40mL, add phosphoric acid buffer 40mL, phosphoric acid buffer pH=7.5 adds precipitation agent 2-methyl-2 simultaneously, 4-pentanediol 40mL, CaCl
20.03g, add the lipase fine crystalline that accounts for 0.05% (percent by volume) in total amount of liquid again, under agitation carry out induced crystallization, crystallization finishes the centrifugal lipase crystal that obtains in back.
(4) resulting lipase crystal is mixed with the 50mL phosphate buffered saline buffer, add CaCl
20.03g with 2-methyl-2, the 4-pentanediol makes 2-methyl-2, the 4-pentanediol accounts for 30% (percent by volume) of total amount of liquid, adds the linking agent glutaraldehyde again, and its add-on is 3.5% (percent by volume) that glutaraldehyde accounts for total amount of liquid.Lipase crystal and linking agent under agitation carry out crosslinked, and crosslinking temperature is 25 ℃, and crosslinking time is 3 hours.Crosslinked end back centrifugation goes out the lipase crystallite, gets the finished product twice with phosphate buffered saline buffer washing, separating out fat enzyme crystallite.
Embodiment 2
Removing dialysis tubing submerged damping fluid is the ammonium sulfate damping fluid, and dialysis time is 16 hours; The precipitation agent that adds is 2-acetone 40mL, CaCl
20.03g, lipase fine crystalline not; Add CaCl
20.005g with 2-methyl-2, the 4-pentanediol makes 2-methyl-2, the 4-pentanediol accounts for 25% (percent by volume) of total amount of liquid, linking agent is a cyanogen bromide, accounts for 1% (percent by volume) of total amount of liquid, and crosslinking time is 200 minutes, crosslinking temperature is 45 ℃, and all the other are operated with embodiment 1.
Embodiment 3
Removing dialysis tubing submerged damping fluid is that molecular weight is 6000 polyvinyl alcohol damping fluid, and dialysis time is 24 hours; The precipitation agent that adds is 6000 polyvinyl alcohol 40mL, CaCl
20.03g, add the lipase fine crystalline that accounts for 0.1% (percent by volume) in total amount of liquid; Add CaCl
20.009g with 2-methyl-2, the 4-pentanediol makes 2-methyl-2, the 4-pentanediol accounts for 40% (percent by volume) of total amount of liquid, linking agent is a polymine, accounts for 10% (percent by volume) of total amount of liquid, and crosslinking time is 30 minutes, crosslinking temperature is 30 ℃, and all the other are operated with embodiment 1.
Embodiment 4
Removing dialysis tubing submerged damping fluid is that molecular weight is 8000 polyvinyl alcohol damping fluid, and dialysis time is 18 hours; The precipitation agent that adds is that molecular weight is 8000 polyvinyl alcohol 40mL, CaCl
20.03g, add the lipase fine crystalline that accounts for 0.2% (percent by volume) in total amount of liquid; Add CaCl
20.05g with 2-methyl-2, the 4-pentanediol makes 2-methyl-2, the 4-pentanediol accounts for 60% (percent by volume) of total amount of liquid, linking agent is a carbodiimide, accounts for 5% (percent by volume) of total amount of liquid, and crosslinking time is 120 minutes, crosslinking temperature is 10 ℃, and all the other are operated with embodiment 1.
Embodiment 5
Removing dialysis tubing submerged damping fluid is 2 (N-morpholino) propane sulfonic acid damping fluid, and dialysis time is 8 hours; The precipitation agent that adds is that molecular weight is 6000 polyvinyl alcohol 40mL, CaCl
20.03g, add the lipase fine crystalline that accounts for 0.25% (percent by volume) in total amount of liquid; Add CaCl
20.015g with 2-methyl-2, the 4-pentanediol makes 2-methyl-2, the 4-pentanediol accounts for 50% (percent by volume) of total amount of liquid, linking agent is a glutaraldehyde, accounts for 4% (percent by volume) of total amount of liquid, and crosslinking time is 150 minutes, crosslinking temperature is 20 ℃, and all the other are operated with embodiment 1.
Embodiment 6
Removing dialysis tubing submerged damping fluid is phosphate buffered saline buffer, and dialysis time is 15 hours; The precipitation agent that adds is 2-acetone 40mL, CaCl
20.03g, add the lipase fine crystalline that accounts for 0.01% (percent by volume) in total amount of liquid; Add CaCl
20.01g with 2-methyl-2, the 4-pentanediol makes 2-methyl-2, the 4-pentanediol accounts for 45% (percent by volume) of total amount of liquid, linking agent is a cyanogen bromide, accounts for 1% (percent by volume) of total amount of liquid, and crosslinking time is 200 minutes, crosslinking temperature is 45 ℃, and all the other are operated with embodiment 1.
Embodiment 7
The mensuration of enzymic activity stability:
The lipase crystallite that obtains among the above embodiment still keeps more than 90% through recording enzymic activity after 5 days in 50% tetrahydrofuran solution.Compare with untreated lipase, the transformation period time lengthening of its enzymic activity nearly a hundred times.
Claims (7)
1, a kind of method that improves lipase activity stability is characterized in that this method comprises the steps:
(1) lipase is dissolved in the phosphate buffered saline buffer, and the centrifugal impurity of removing obtains supernatant liquor, feeds the ultra-filtration equipment ultrafiltration then, obtains ultrafiltration and concentration liquid,
(2) the ultrafiltration and concentration liquid that step (1) is obtained changes dialysis tubing over to, immerses in the damping fluid to dialyse, and enzyme liquid is further concentrated,
(3) add the crystalline deposit agent in the enzyme concentrated liquor that in step (2), obtains and make the lipase crystallization, obtain the lipase crystal after centrifugal,
(4) lipase crystal and linking agent mix carry out crosslinked, the crosslinked lipase that finishes the centrifugal enzymic activity stability that is improved in back.
2, the method for claim 1, dialysis tubing submerged damping fluid is that ammonium sulfate damping fluid, phosphate buffered saline buffer, molecular weight are 6000～8000 polyvinyl alcohol damping fluid, 2 (N-morpholino) propane sulfonic acid damping fluid when it is characterized in that dialysing.
3, the method for claim 1 is characterized in that described crystalline deposit agent molecule amount is 4000～8000 polyvinyl alcohol, 2-acetone, 2-methyl-2,4-pentanediol, CaCl
2
4, the method for claim 1 can add fine lipase crystal seed induced crystallization when it is characterized in that crystallization.
5, method as claimed in claim 4, when it is characterized in that induced crystallization, the add-on of fine lipase crystal seed is that fine lipase crystal seed accounts for 0.01～0.25% in total amount of liquid (percent by volume).
6, the method for claim 1, when it is characterized in that lipase crystal and linking agent are crosslinked, the linking agent add-on is that linking agent accounts for 1～10% in total amount of liquid (percent by volume), and crosslinking time is 30～200 minutes, and crosslinking temperature is 10～50 ℃.
7,, it is characterized in that the linking agent that adds when the lipase crystal is crosslinked is cyanogen bromide, polymine, glutaraldehyde, carbodiimide as claim 1 or 6 described methods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01126807 CN1257270C (en) | 2001-09-19 | 2001-09-19 | method for improving lipase activity stability |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01126807 CN1257270C (en) | 2001-09-19 | 2001-09-19 | method for improving lipase activity stability |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1408855A true CN1408855A (en) | 2003-04-09 |
| CN1257270C CN1257270C (en) | 2006-05-24 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01126807 Expired - Fee Related CN1257270C (en) | 2001-09-19 | 2001-09-19 | method for improving lipase activity stability |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139579B (en) * | 2007-08-02 | 2010-12-08 | 张家港澳洁生物科技有限公司 | Method for preparing cross-linking bacillus subtilis proteinase crystal |
| CN102015028A (en) * | 2008-04-22 | 2011-04-13 | 丹尼斯科美国公司 | Oral care composition containing oxidase enzyme |
-
2001
- 2001-09-19 CN CN 01126807 patent/CN1257270C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139579B (en) * | 2007-08-02 | 2010-12-08 | 张家港澳洁生物科技有限公司 | Method for preparing cross-linking bacillus subtilis proteinase crystal |
| CN102015028A (en) * | 2008-04-22 | 2011-04-13 | 丹尼斯科美国公司 | Oral care composition containing oxidase enzyme |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1257270C (en) | 2006-05-24 |
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