CN102093994A - Method for synchronously purifying and immobilizing linoleate isomerase - Google Patents
Method for synchronously purifying and immobilizing linoleate isomerase Download PDFInfo
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- CN102093994A CN102093994A CN 201010576488 CN201010576488A CN102093994A CN 102093994 A CN102093994 A CN 102093994A CN 201010576488 CN201010576488 CN 201010576488 CN 201010576488 A CN201010576488 A CN 201010576488A CN 102093994 A CN102093994 A CN 102093994A
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- linoleate isomerase
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Abstract
The invention discloses a method for synchronously purifying and immobilizing linoleate isomerase. The method comprises the steps of: treating attapulgite by using hydrochloric acid, adjusting the pH value by using solution of sodium hydroxide, stirring uniformly, filtering, washing, and drying to obtain acid modified attapulgite; mixing the acid modified attapulgite and solution of magnesium sulfate, stirring, filtering, and drying to obtain attapulgite treated by the magnesium sulfate; mixing the attapulgite treated by the magnesium sulfate, gamma-(methacryloyloxy)propyltrimethoxysilane, water and dimethylbenzene and stirring, filtering, washing, and drying to obtain modified attapulgite; mixing enzyme fluid of linoleate isomerase produced from Lactobacillus delbrueckii subsp bulgaricus and the modified attapulgite, stirring, filtering, and drying to obtain the purified and immobilized linoleate isomerase. The method for synchronously purifying and immobilizing the linoleate isomerase is simple and convenient, simple in steps, low in cost, good in purification effect, and high in immobilization efficiency; the activity recovery rate of enzyme reaches over 60 percent during the purification; and the immobilized enzyme has better pH adaptability, thermal stability and operation stability.
Description
Technical field
The invention belongs to enzyme purification and immobilization field, be specifically related to the method for a kind of while purifying and immobilization linoleate isomerase.
Background technology
Linoleate isomerase is generally used for linolic acid is converted into conjugated linolic acid, and conjugated linolic acid has many important physiological actions: anticancer; Anti-oxidant; Reducing cholesterol; Suppress the fat accumulation; Prevent and treat diabetes; Anti-congee shape arteriosclerosis; Improve effects such as metabolism of bone tissue; At present, the essential fatty acid conjugated linolic acid receives increasing concern at the application potential of aspects such as medicines and health protection, food, feed.
Chemical method is mainly adopted in conjugated linolic acid production at present, as linoleic alkali isomerization, but the product that chemical method obtains is a series of have all positions of conjugated double bond and CLA mixtures of geometrical isomer, also has by product to exist simultaneously, obtains very difficulty of individual isomer and will separate; So biological process especially enzyme process synthesis of conjugated linoleic acid has received increasing concern, Production by Enzymes adopts the free linoleic acid isomerase at present mostly, but resolvase character instability, reaction back resolvase is difficult to recycling, be unfavorable for the later separation purifying of product and obtain the purification step of resolvase many, yield is lower.The immobilization technology research of the linoleate isomerase of lactobacillus reported in research one literary composition of the described Northeast Forestry University of Sun Qi Master's thesis Bacterium lacticum linoleate isomerase conversion sea-buckthorn conjugated linolic acid technology, this technology can prepare the immobilization linoleate isomerase, but it at first will carry out centrifugal to fermented liquid, collect thalline, twice ultrasonic is handled behind the combination of ultrasound N,O-Diacetylmuramidase broken wall, Tritron-100 extracts, centrifugal, grade ammonium sulfate salting-out, the dialysis of enzyme liquid, the multistep separation means such as concentrate and obtain crude enzyme liquid, adopt sodium alginate-calcium chloride entrapping method to fix again, the characteristics of this method are first preliminary purification enzyme liquid, application vector carries out immobilization again, has the cost height and implement step by step, energy consumption is big, complex operation, many shortcomings such as step is many.Number of patent application a kind of preparation method of immobilized lipase attapulgite clay that has been 200610039833.4 patent disclosure, the pure enzyme of commodity fat that this patent is crossed with purifying is made enzyme liquid, what adopt then is the attapulgite modified method that is fit to lipase immobilization, the shortcoming of this method is at first to obtain pure enzyme, then could immobilization.
Summary of the invention
The objective of the invention is: the method for a kind of while purifying and immobilization linoleate isomerase is provided, and applying modified attapulgite is as raw material and carrier, and is easy and simple to handle, with low cost, simultaneously purifying and immobilization.
Technical solution of the present invention is that this purifying and process for fixation may further comprise the steps:
(1) record by described article of 2010 15 phases of Anhui agricultural sciences " lactobacillus delbruockii subspecies bulgaricus produces linoleate isomerase research preparation " prepares the linoleate isomerase fermented liquid, the centrifugal 10min of 8000rpm, collect thalline, by fermented liquid: damping fluid be the volume ratio of 5:2 thalline is suspended in the citric acid-sodium citrate buffer of 0.1mol/L pH=6.0 damping fluid, add the helicase that N,O-Diacetylmuramidase that quality accounts for damping fluid volume 0.5% and quality account for damping fluid volume 0.2% simultaneously, stir 60min, centrifugal 10min, get the supernatant crude enzyme liquid, in crude enzyme liquid, add the cationic polyacrylamide flocculant agent that quality accounts for damping fluid volume 0.02%, stir 10-15min, filter, collect filtrate;
(2) in filtrate and attapulgite modified attapulgite modified with the adding in the filtrate that foundation step 1 obtains of mass ratio 4:1 ratio, stirring at room 10-15h, vacuum filtration gets purifying and immobilization linoleate isomerase.
Wherein, described attapulgite modified preparation may further comprise the steps: a certain amount of attapulgite is joined in the three-necked bottle that has stirring and condensing works, the mass/volume of pressing 1:12 is than the hydrochloric acid that adds 2mol/L, stirring and refluxing 1-3h, sodium hydroxide solution with 3mol/L is regulated pH3.5, stirs, and filters, drying gets sour attapulgite modified; With the attapulgite modified Adlerika of pressing the mass volume ratio adding 2mol/L of 1:1 of acid, stirring at room 3h filters, and drying gets sal epsom and handles attapulgite; Sal epsom is handled the mass ratio of pressing 1:1:1:2-3 in the attapulgite and is added γ-(methacryloxypropyl) propyl trimethoxy silicon, water, dimethylbenzene, fully mixes 24-30h, filter, and washing, drying gets attapulgite modified.
Wherein, the molecular weight 6,000,000-8,000,000 of described cationic-type polyacrylamide flocculation agent.
The invention has the beneficial effects as follows:
1. the raw material of purifying and immobilization linoleate isomerase is attapulgite modified, and China's attapulgite aboundresources utilizes attapulgite as raw material, and is not only with low cost, and enlarged the range of application of attapulgite.
2. the purifying of linoleate isomerase provided by the invention and immobilization only need a step, have saved multistep, have not only reduced production cost, have more effectively shortened step and time, and this method is reliable and stable, is easy to scale operation.
3. adopting molecular weight is that 6,000,000-8,000,000 cationic-type polyacrylamide flocculation agents are handled fermented liquid, not only can remove thalline, also can remove most foreign protein and ion, saves multistep operations such as centrifugal, chromatography simultaneously, greatly reduces production cost and energy consumption.
4. the inventive method is easy and simple to handle reliable, mild condition, and the immobilization efficiency height, and save the purge process of resolvase, greatly reduce production cost.
5. the present invention combines the purge process and the immobilization process of enzyme, at the immobilized efficiently purifying of realizing enzyme simultaneously, in purifying, realize the immobilization of enzyme, polystep reaction is integrated in the single step reaction, realize the Cheap highly effective production of enzyme, belonged to original work.
6. immobilized enzyme has pH adaptability, temperature stability and operational stability preferably, has adaptability preferably between pH5.5-8.5, reuses 13 times, and enzyme is lived and still can be kept more than 60%.
7. attapulgite is as carrier, reliable in quality, and biologically stable and biocompatibility are better.
8. the present invention utilizes attapulgite modified purge process and immobilization with linoleate isomerase to combine, in the process of purifying, enzyme is realized immobilization simultaneously, with purge process and the highly coupling of one step of immobilization process, realize the high efficiency, low cost production of enzyme, this method has low cost, less energy-consumption, advantage such as easy and simple to handle.
Description of drawings
The optimum temperuture of Fig. 1 resolvase and purifying and immobilized enzyme relatively.
The optimal pH of Fig. 2 resolvase and purifying and immobilized enzyme relatively.
The temperature stability of Fig. 3 resolvase and purifying and immobilized enzyme relatively.
The repeated use design sketch of Fig. 4 purifying and immobilized enzyme.
Embodiment
Below by specific embodiment technical scheme of the present invention is further described, but technical solution of the present invention is not limited to following embodiment.
Embodiment 1:
The 5g attapulgite is joined in the three-necked bottle that has stirring and condensing works, add the hydrochloric acid 60ml of 2mol/L, stirring and refluxing 1h regulates pH3.5 with the sodium hydroxide solution of 3mol/L, stirs, and filters, and drying gets sour attapulgite modified; With the attapulgite modified Adlerika that joins the 2mol/L of 5ml of acid, stirring at room 3h filters, and drying gets the attapulgite that sal epsom is handled; Add 5ml γ-(methacryloxypropyl) propyl trimethoxy silicon, 5ml water and 10ml dimethylbenzene respectively in the attapulgite that sal epsom is handled, fully mix 24h, filter, washing, drying gets attapulgite modified.
Record by described article of 2010 15 phases of Anhui agricultural sciences " lactobacillus delbruockii subspecies bulgaricus produces linoleate isomerase research preparation " prepares the linoleate isomerase fermented liquid, get 50ml linoleate isomerase fermented liquid, the centrifugal 10min of 8000rpm, collect thalline, thalline is suspended in the citric acid-sodium citrate buffer of 0.1mol/L pH6.0 of 20ml, add 0.1g N,O-Diacetylmuramidase and 0.04g helicase simultaneously, stir 40min, the centrifugal 10min of 6000rpm, get the supernatant crude enzyme liquid, in crude enzyme liquid, add 0.004g molecular weight 6,000,000 polyacrylamide flocculants, stir 10min, filter, collect filtrate; Join in the filtrate 5g is attapulgite modified, stirring at room 10h, vacuum filtration, the immobilization linoleate isomerase.
Embodiment 2:
The 20g attapulgite is joined in the three-necked bottle that has stirring and condensing works, add the hydrochloric acid 240ml of 2mol/L, stirring and refluxing 2h regulates pH3.5 with the sodium hydroxide solution of 3mol/L, stirs, and filters, and drying gets sour attapulgite modified; With the attapulgite modified Adlerika that joins the 2mol/L of 20ml of acid, stirring at room 3h filters, and drying gets the attapulgite that sal epsom is handled; Add 20ml γ-(methacryloxypropyl) propyl trimethoxy silicon, 20ml water and 50ml dimethylbenzene respectively in the attapulgite that sal epsom is handled, fully mix 27h, filter, washing, drying gets attapulgite modified.
Record by described article of 2010 15 phases of Anhui agricultural sciences " lactobacillus delbruockii subspecies bulgaricus produces linoleate isomerase research preparation " prepares the linoleate isomerase fermented liquid, get 200ml linoleate isomerase fermented liquid, the centrifugal 10min of 8000rpm, collect thalline, thalline is suspended in the citric acid-sodium citrate buffer of 0.1mol/L pH6.0 of 80ml, add 0.4g N,O-Diacetylmuramidase and 0.16g helicase simultaneously, stir 40min, the centrifugal 10min of 6000rpm, get the supernatant crude enzyme liquid, adding the 0.016g molecular weight in crude enzyme liquid is 7,000,000 polyacrylamide flocculants, stirs 12min, filter, collect filtrate; Join in the filtrate 20g is attapulgite modified, stirring at room 13h, vacuum filtration, the immobilization linoleate isomerase.
Embodiment 3:
The 50g attapulgite is joined in the three-necked bottle that has stirring and condensing works, add the hydrochloric acid 600ml of 2mol/L, stirring and refluxing 3h regulates pH3.5 with the sodium hydroxide solution of 3mol/L, stirs, and filters, and drying gets sour attapulgite modified; With the attapulgite modified Adlerika that joins the 2mol/L of 50ml of acid, stirring at room 3h filters, and drying gets the attapulgite that sal epsom is handled; Add 50ml γ-(methacryloxypropyl) propyl trimethoxy silicon, water and 150ml dimethylbenzene respectively in the attapulgite that sal epsom is handled, fully mix 30h, filter, washing, drying gets attapulgite modified.
Record by described article of 2010 15 phases of Anhui agricultural sciences " lactobacillus delbruockii subspecies bulgaricus produces linoleate isomerase research preparation " prepares the linoleate isomerase fermented liquid, get 500ml linoleate isomerase fermented liquid, the centrifugal 10min of 8000rpm, collect thalline, thalline is suspended in the citric acid-sodium citrate buffer of 0.1mol/L pH6.0 of 200ml, add 1g N,O-Diacetylmuramidase and 0.4g helicase simultaneously, stir 40min, the centrifugal 10min of 6000rpm, get the supernatant crude enzyme liquid, adding the 0.04g molecular weight in crude enzyme liquid is 8,000,000 polyacrylamide flocculants, stirs 15min, filter, collect filtrate; Join in the filtrate 50g is attapulgite modified, stirring at room 15h, vacuum filtration, the immobilization linoleate isomerase.
The mensuration of enzyme adsorptive capacity: adopt the Bradford method, bovine serum albumin is as standard substance, measure the concentration of linoleate isomerase in the immobilized enzyme water lotion and through the concentration and the enzyme liquid concentration of residual linoleate isomerase after attapulgite modified purifying and the immobilization, calculate linoleate isomerase immobilization amount on attapulgite modified.
The enzyme activity definition: the resolvase enzyme is lived definition referring to " lactobacillus delbruockii subspecies bulgaricus produces linoleate isomerase research preparation ", described article of 2010 15 phases of Anhui agricultural sciences.
The immobilized enzyme enzyme activity determination: get siritch 1ml, 2ml tween 80 constant volume is to 100ml, abundant stirring and emulsifying 1h, and the 0.22um membrane filtration is standby; Get the attapulgite purifying and immobilized enzyme 3g puts into the 20ml test tube, the citric acid-sodium citrate damping fluid 5ml that adds 0.1mol/L pH=6.0, substrate 0.2ml, behind the 200rpm constant temperature 30 degree concussion reaction 4h, add 10% trichoroacetic acid(TCA) termination reaction, in test tube, add the 8ml normal hexane, at the extraction conjugated linolic acid, centrifugal breakdown of emulsion is got organic phase, use the wherein light absorption value of 233nm of spectrophotometry, not add the attapulgite immobilized enzyme is that blank is added the equivalent damping fluid, and the light absorption value of sample deducts the barren light absorption value and is self-defined immobilized enzyme work.
The optimum temperuture of purifying that embodiment 1 obtains and immobilized enzyme and resolvase more as shown in Figure 1; The optimal pH of purifying that embodiment 2 obtains and immobilized enzyme and resolvase more as shown in Figure 2; The temperature stability of purifying that embodiment 3 obtains and immobilized enzyme and resolvase more as shown in Figure 3; The repeated use effect of purifying that embodiment 3 obtains and immobilized enzyme as shown in Figure 4.
Claims (3)
1. method of purifying and immobilization linoleate isomerase simultaneously is characterized in that this purifying and process for fixation may further comprise the steps:
(1) record by described article of 2010 15 phases of Anhui agricultural sciences " lactobacillus delbruockii subspecies bulgaricus produces linoleate isomerase research preparation " prepares the linoleate isomerase fermented liquid, the centrifugal 10min of 8000rpm, collect thalline, by fermented liquid: damping fluid be the volume ratio of 5:2 thalline is suspended in the citric acid-sodium citrate buffer of 0.1mol/L pH=6.0 damping fluid, add the helicase that N,O-Diacetylmuramidase that quality accounts for damping fluid volume 0.5% and quality account for damping fluid volume 0.2% simultaneously, stir 60min, centrifugal 10min, get the supernatant crude enzyme liquid, in crude enzyme liquid, add the cationic polyacrylamide flocculant agent that quality accounts for damping fluid volume 0.02%, stir 10-15min, filter, collect filtrate;
(2) by filtrate and attapulgite modified attapulgite modified with mass ratio 4:1 adding in the filtrate that foundation step (1) obtains, stirring at room 10-15h, vacuum filtration gets purifying and immobilization linoleate isomerase.
2. the method for while purifying according to claim 1 and immobilization linoleate isomerase, it is characterized in that described attapulgite modified preparation may further comprise the steps: a certain amount of attapulgite is joined in the three-necked bottle that has stirring and condensing works, the mass/volume of pressing 1:12 is than the hydrochloric acid that adds 2mol/L, stirring and refluxing 1-3h, sodium hydroxide solution with 3mol/L is regulated pH3.5, stirs, and filters, drying gets sour attapulgite modified; With the attapulgite modified Adlerika of pressing the mass volume ratio adding 2mol/L of 1:1 of acid, stirring at room 3h filters, and drying gets sal epsom and handles attapulgite; Sal epsom is handled the mass ratio of pressing 1:1:1:2-3 in the attapulgite and is added γ-(methacryloxypropyl) propyl trimethoxy silicon, water, dimethylbenzene, fully mixes 24-30h, filter, and washing, drying gets attapulgite modified.
3. the method for while purifying according to claim 1 and immobilization linoleate isomerase is characterized in that: the molecular weight 6,000,000-8,000,000 of cationic-type polyacrylamide flocculation agent.
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| CN 201010576488 CN102093994A (en) | 2010-12-07 | 2010-12-07 | Method for synchronously purifying and immobilizing linoleate isomerase |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104974996A (en) * | 2014-04-02 | 2015-10-14 | 内蒙古农业大学 | Method of preparing linoleate isomerase micro-capsules on the basis of polyelectrolyte layer-by-layer self-assembly technology |
| CN106591279A (en) * | 2017-02-08 | 2017-04-26 | 光合强化（北京）生物科技有限公司 | Modified attapulgite used for immobilizing polycyclic aromatic hydrocarbon degrading bacteria, and preparation method thereof |
| CN112194658A (en) * | 2020-10-23 | 2021-01-08 | 内蒙古拜克生物有限公司 | Separation and purification method of pyrroloquinoline quinone |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999032604A1 (en) * | 1997-12-23 | 1999-07-01 | Dcv, Inc. Doing Business As Bio-Technical Resources | Linoleate isomerase |
| WO2001000846A2 (en) * | 1999-06-30 | 2001-01-04 | Dcv, Inc. D.B.A Bio-Technical Resources | Linoleate isomerase |
| CN101092613A (en) * | 2007-05-25 | 2007-12-26 | 中国农业科学院农产品加工研究所 | Method for extracting pyrified isomerase of linoleic acid from strain of producing isomerase of linoleic acid |
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2010
- 2010-12-07 CN CN 201010576488 patent/CN102093994A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999032604A1 (en) * | 1997-12-23 | 1999-07-01 | Dcv, Inc. Doing Business As Bio-Technical Resources | Linoleate isomerase |
| WO2001000846A2 (en) * | 1999-06-30 | 2001-01-04 | Dcv, Inc. D.B.A Bio-Technical Resources | Linoleate isomerase |
| CN101092613A (en) * | 2007-05-25 | 2007-12-26 | 中国农业科学院农产品加工研究所 | Method for extracting pyrified isomerase of linoleic acid from strain of producing isomerase of linoleic acid |
Non-Patent Citations (1)
| Title |
|---|
| 《安徽农业科学》 20100520 游庆红，尹秀莲 德氏乳杆菌保加利亚亚种产亚油酸异构酶研究 8193，8271 1-3 第38卷, 第15期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104974996A (en) * | 2014-04-02 | 2015-10-14 | 内蒙古农业大学 | Method of preparing linoleate isomerase micro-capsules on the basis of polyelectrolyte layer-by-layer self-assembly technology |
| CN106591279A (en) * | 2017-02-08 | 2017-04-26 | 光合强化（北京）生物科技有限公司 | Modified attapulgite used for immobilizing polycyclic aromatic hydrocarbon degrading bacteria, and preparation method thereof |
| CN112194658A (en) * | 2020-10-23 | 2021-01-08 | 内蒙古拜克生物有限公司 | Separation and purification method of pyrroloquinoline quinone |
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Application publication date: 20110615 |