CN102839166A - TL immobilization enzyme and application thereof - Google Patents
TL immobilization enzyme and application thereof Download PDFInfo
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- CN102839166A CN102839166A CN2011101707016A CN201110170701A CN102839166A CN 102839166 A CN102839166 A CN 102839166A CN 2011101707016 A CN2011101707016 A CN 2011101707016A CN 201110170701 A CN201110170701 A CN 201110170701A CN 102839166 A CN102839166 A CN 102839166A
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- enzyme
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- lypase
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- 229920005989 resin Polymers 0.000 claims abstract description 119
- 238000000034 method Methods 0.000 claims abstract description 73
- 238000001035 drying Methods 0.000 claims abstract description 69
- 108090001060 Lipase Proteins 0.000 claims abstract description 23
- 239000004367 Lipase Substances 0.000 claims abstract description 23
- 238000002203 pretreatment Methods 0.000 claims abstract description 23
- 102000004882 Lipase Human genes 0.000 claims abstract description 22
- 235000019421 lipase Nutrition 0.000 claims abstract description 22
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 20
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- 238000002360 preparation method Methods 0.000 claims abstract description 20
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 25
- 239000003463 adsorbent Substances 0.000 claims description 23
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- 102000004169 proteins and genes Human genes 0.000 claims description 14
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- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 3
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 3
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
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- 101000984201 Thermomyces lanuginosus Lipase Proteins 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 2
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- DCXXMTOCNZCJGO-UHFFFAOYSA-N Glycerol trioctadecanoate Natural products CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The present invention relates to a TL immobilization enzyme and an application thereof. Specifically the present invention provides a preparation method for an immobilization lipase. The method comprises the following steps: a) carrying out a pre-treatment on a carrier, wherein the carrier is selected from an ion exchange resin or a macroporous adsorption resin having a polystyrene skeleton; b) providing a lipase; c) carrying out contact of the pre-treated carrier from the step a) and the lipase provided by the step b), such that the lipase is immobilized on the pre-treated carrier; and d) drying. The present invention further provides the immobilization lipase prepared by using the method of the present invention and an application thereof. According to the present invention, the immobilization enzyme of the present invention has high activity and stability, and does not generate soap during a process of catalysis of an ester exchange reaction in food industry, operation is simple, and oil processing cost is substantially reduced.
Description
Technical field
The invention belongs to food and chemical field.More specifically, the present invention relates to TL lipase immobilization enzyme method and application thereof.
Background technology
Enzyme has substrate selective as a kind of biological catalyst, and reaction conditions is gentle, pollutes advantages such as little, has overcome the severe reaction conditions that the traditional industry catalyzer brings, and shortcomings such as byproduct of reaction contaminate environment have good application potential.The searching catalysis activity is high, the enzyme that reaction conditions is gentle, or make it more adapt to production application to existing enzyme transformation, be the main developing direction that following enzyme engineering is used in industry.
Enzyme immobilization technology be with the free enzyme through physics or chemical means with its constraint or be limited in certain zone, make it still can carry out distinctive catalyzed reaction and can reclaim reusable a kind of technology.Because above-mentioned advantage makes a lot of researchers launch the careful research to immobilized enzyme.In 1967, immobilized enzyme was used in first and splits in the amino acid industry.Along with the development of organic chemistry, protein chemistry, Materials science etc., enzyme immobilization technology has obtained significant progress in recent years.
Lypase is the class of enzymes of catalysis grease generation hydrolysis, transesterify, esterification, and it can bring into play catalytic activity specifically on water-oil interface, in industry such as food, papermaking, leather, washing composition, pharmacy, huge application is arranged.Yet the natural enzyme poor stability is difficult to purifying after the reaction, can not reuse, and has not only reduced the service efficiency of enzyme, has improved production cost, and is difficult to realize continuous operation, has limited its application in industry.Under this background, enzyme immobilization technology becomes a kind of effective way that solves above-mentioned production bottleneck.
TL lypase is a quasi-lipase of in thermophilic hyphomycete (Thermomyces Lanuginosus), finding, it is mainly used in the modification of greasy hydrolysis, Witepsol W-S 55 and the production of Tegin 55G and sweet diester.The TL lypase of liquid is mainly used in hydrolysis reaction.Immobilized TL enzyme is used for catalyzed transesterification, and its advantage is to reuse, and reduces production costs.
WO8906278A1 has described a kind of method of immobilization TL enzyme; Fungi through cultivating the mould genus of humic or obtain to contain the fermented liquid of lypase through the mould genus of the humic of genetic modification fungi is a fixation support with phenolic or acrylic type resin, and immobilization process is for adding a certain amount of certain pH value enzyme liquid in the weakly alkaline macroreticular ion exchange resin of handling well; At room temperature shake 8h; Washing then, room temperature vacuum-drying gets product.
EP1239045A1 has described a kind of method of immobilization TL enzyme, is specially lipase powder is dissolved in the phosphoric acid buffer, adds ethanol and Vestolen PP 7052 type Resin A CCUREL MP1001, and concussion is spent the night under the room temperature.Collect immobilized enzyme through filtering, use buffer solution for cleaning, carry out drying under the room temperature.
Yet up to the present, existing TL immobilized enzyme can not satisfy production requirement on the market.Find in its catalytic transesterification reaction to have a large amount of soaps in the product; Waste raw material in a large number; Increase cost, analyze its reason and possibly be and exist some technical problems that can't overcome to cause in the perhaps carrier and enzyme fixation procedure and the conflicting of reaction medium in dissimilar carriers and the enzyme bonded process.Therefore present immobilized TL needs a large amount of grease wash-outs, causes a large amount of wastage of material.Purify for the separation in downstream simultaneously and brought huge difficulty, limited its application on oil prodution industry.
Therefore; Press for the immobilization TL enzyme method that exploitation makes new advances in this area; Make the TL enzyme keep higher vigor and in reaction, do not produce soap, thereby enlarge the range of application of TL lypase, and overcome the problem that existing immobilization TL enzyme produces soap aborning in foodstuffs industry.
Summary of the invention
One of the object of the invention is just providing a kind of new TL enzyme immobilization method.Another main purpose of the present invention provides a kind of immobilized TL enzyme.The present invention also aims to provide immobilization TL enzyme of the present invention in Industrial Application.
In first aspect of the present invention, a kind of method for preparing immobilized lipase is provided, said method comprises step:
A) carrier is carried out pre-treatment, said carrier is selected from: ion exchange resin or skeleton are the macroporous adsorbent resin of PS;
B) lypase is provided, and the optional zymoprotein linking agent that adds therein;
C) lypase that in the carrier of pre-treatment and step b), is provided with gained in the step a) contacts, and makes said lypase be fixed in said carrier through pre-treatment;
D) carrier that is fixed with lypase of gained drying step c) is to obtain said immobilized lipase.
In a preference, the macroporous adsorbent resin of said PS can be the macroporous adsorbent resin of polystyrene-divinylbenzene skeleton.
In an embodiment of the invention, said carrier is selected from: polar macroporous adsorption resin, nonpolar macroporous adsorption resin, Zeo-karb or anionite-exchange resin.
In yet another embodiment of the present invention, said nonpolar macroporous adsorption resin is selected from: D101, HPD100A, AmberliteXAD-4, HPD100, HPD300, HPD700, X-5, XAD-4 or HP20;
Said polar macroporous adsorption resin is selected from: D201, LSA-10 or Amberlite XAD7HP;
Said Zeo-karb is selected from: D113, D001 or Ambetlite IRC50;
Said anionite-exchange resin is selected from: D301 or Styrene-DVB 201 * 2.
In yet another embodiment of the present invention; The pre-treatment of said macroporous adsorbent resin is carried out as follows: place water to soak impurity elimination in carrier; Soaked in solvent 1～24h is to remove organic impurity and insolubles then; The said solvent of water flush away again, wherein said solvent is: ethanol, normal hexane, acetone or sherwood oil;
The pre-treatment of said ion exchange resin is carried out as follows: alternately soak or wash said resin 1-5 time with the alkali lye of 0.05mol/L～1mol/L and acid solution; Only soak once more with the alkali lye of 0.05mol/L～1mol/L then or wash resin 1 time, water is washed till water and is neutral again.
In a preference, said water is deionized water, zero(ppm) water or distilled water.
In another preference, used ethanol is that concentration range is the ethanol of 100%-50% (V/V) in the pre-treatment of macroporous adsorbent resin.
In another preference, the addition scope of solvent described in the pre-treatment of macroporous adsorbent resin is 0.5-10 a times of resin volume, and preferred 1-5 doubly.
In another preference, the said acid in the pre-treatment of ion exchange resin is selected from: HCl, H
2SO
4Or H
3PO
4, said alkali is selected from: NaOH, ammoniacal liquor or KOH.
In another preference, the time of each alkali lye and acid soak or flushing is 1-24 hour.
In another preference; The pre-treating process of said ion exchange resin is: with resin and alkali lye or acid solution 1: 1～1: 5, preferred 1: 1.5～1: 3, more preferably 1: 2 weightmeasurement ratio (W/V) soaks or washes said resin; Order is acid behind the first alkali; Repeat for several times the back last all over handling with alkali lye, water is washed till water and is neutral then.
In another preference, with treated carrier be immersed in preserve in the deionized water (or zero(ppm) water, distilled water) subsequent use.
In yet another embodiment of the present invention, the lypase that is provided in the step b) is selected from: the tunning of thermophilic hyphomycete (Thermomyces Lanuginosus); The lypase that gets through genetically engineered or protein engineering transformation; Or commercially available lypase.
In a preference, said genetically engineered or protein engineering transformation and lypase be: the enzyme that the enzyme of chemically modified, the two mutants of protein engineering or genetic engineering bacterium produce.
In another preference, said commercially available lypase is preferably the Lipozyme TL100L of Denmark Novozymes Company.
In yet another embodiment of the present invention, said lypase is used for step c) without any pre-treatment.
In another preference, can dilute said lypase.
In yet another embodiment of the present invention, said zymoprotein linking agent is selected from: glutaraldehyde solution, polymine, Vestolen PP 7052 imines, polyvinylamine, gelatin or spermine.
In a preference, the consumption of said zymoprotein linking agent is the long-pending 0.001-0.1 (V/V) of enzyme liquid, and the consumption of preferred LUTARALDEHYDE is 1% glutaraldehyde solution or other concentration glutaraldehyde solution of equal proportion that adds 0.01～0.1ml in every ml enzyme liquid.
In yet another embodiment of the present invention, in the step c), said treated carrier and lypase are 25: 1～1: 25 in the by weight/volume of g/ml.
In a preference, said ratio is preferably 10: 1～and 1: 10, more preferably 1: 1.
In another preference, use shaking bath, gas bath shaking table, stirrer or magnetic stirring apparatus make said treated carrier contact with lypase and fix.
In another preference, survey the fixation degree that residual protein content is judged zymoprotein through getting supernatant, said residual protein Determination on content adopts the method that is selected from down group to carry out: Xylene Brilliant Cyanine G method, lorry method, biuret method, BCA method.
In another preference, when residual protein content is lower than 0.1mg/ml～0.5ml/ml, stop fixing step.
In another preference, said drying is to carry out through the method that is selected from down group: lyophilize, vacuum-drying, fluidised bed drying and drying at room temperature.
In second aspect of the present invention, a kind of immobilized lipase or its wrapped product are provided, described immobilized lipase prepares through method of the present invention.
In a preference, said wrapped product also comprises packing material, preferred container or packing bag.
In the third aspect of the invention, a kind of method of carrying out transesterify, esterification, acidolysis or alcoholysis reaction is provided, said method comprises uses immobilized lipase of the present invention as catalyzer.
In another aspect of this invention, the purposes of immobilized lipase of the present invention in transesterify, esterification, acidolysis or alcoholysis reaction is provided also.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Embodiment
The inventor has found a kind of TL immobilized enzyme method through long-term and deep research, is a kind of immobilized enzyme method that in reaction, does not produce or seldom produce soap furtherly.Said method mainly comprises following three steps: the selection of carrier and pre-treatment step, enzyme immobilization step and drying step.The immobilized lipase vigor in catalyzed reaction that adopts this method to make is higher, and can not produce a large amount of soaps, has very big application prospect.On this basis, the inventor has accomplished the present invention.
1, the selection of carrier and pre-treatment
Adopted among the present invention that not used polystyrene type macroporous adsorbent resin is a carrier as carrier or treated ion exchange resin in the prior art; Make unexpectedly and have high reactivity and stability with its immobilized lypase; And in the catalyzed transesterification process, do not produce soap; Simple to operate, greatly reduce the grease cost of processing.
As used herein; Term " macroporous adsorbent resin " is meant one type of polymer polymeric adsorbent that does not contain cation exchange groups and have macroporous structure; It has good macroporous netlike structure and bigger specific surface area, can be through physical adsorption adsorb organic compound selectively from solution.Term " polystyrene type macroporous adsorbent resin " is meant with the PS to be the macroporous adsorbent resin of skeleton.Should understand the various hypotypes that this term comprises the polystyrene type macroporous adsorbent resin, for example the resin of polystyrene-divinylbenzene skeleton.
The polystyrene type macroporous adsorbent resin that can be used among the present invention can include, but is not limited to: nonpolar D101, HPD100A, AmberliteXAD-4, HPD100, HPD300, HPD700, X-5, XAD-4 or HP20; Polar D201, LSA-10, Amberlite XAD7HP etc.Preferred in the present invention nonpolar polystyrene type macroporous adsorbent resin, more preferably D101, HPD100A, the X-5 of adopting.
As used herein, term " ion exchange resin " is meant and has functional group's (reactive group that exchange ion is arranged), has reticulated structure, insoluble macromolecular compound, the ion-exchange chromatography media of doing commonly used.
Can be used for ion exchange resin of the present invention includes, but is not limited to: anionite-exchange resin, like D301, Styrene-DVB201 * 2 etc.; Zeo-karb is like D113, D001, Ambetlite IRC50 etc.Preferably adopt anionite-exchange resin in the present invention.
Carrier of the present invention also can be based on the modification of above or other resin or modified resin, for example through diazotization or amination modified resin or adopt the resin of other chemical modification means through functionalization.But reference example such as Xu Jingliang etc., the carrier immobilized enzyme progress of amino functional chemical industry progress 201029 (3): 494-496.
It will be understood by those skilled in the art that; Can be used for polystyrene type macroporous adsorbent resin and ion exchange resin among the present invention except above with the specific product that trade(brand)name limited; Also comprise any commercial resins product with other trade(brand)name or the resin that obtains through preparation or processing voluntarily, as long as they have and the same or analogous structure of said specific product and absorption or switching performance.
Used these carriers (especially polystyrene macroporous resin) are not used carrier in the existing commercialization immobilized enzyme among the present invention, and the treatment process of carrier of the present invention also there are differences with conventional treatment method.
The treatment process of macroporous adsorbent resin: carrier is placed deionized water (also available zero(ppm) water; Distilled water) soaks impurity elimination in; Soak 1～24h with appropriate solvent then; Remove organic impurity and other insolubles, use deionized water (or zero(ppm) water, distilled water) flush away solvent again, place deionized water (or zero(ppm) water, distilled water) subsequent use.Wherein, solvent for use can be: ethanol, preferred concentration range for are the ethanol of 100%-50% (v/v); Or organic solvent such as normal hexane, acetone, sherwood oil.The addition scope of said solvent is 0.5-10 a times of resin volume, and preferred 1-5 doubly.
Ion exchange resin: with the alkali lye (for example NaOH, KOH or ammoniacal liquor) of lower concentration (0.05mol/L-1mol/L) and acid solution (for example HCl, H
2SO
4Or H
3PO
4) (treatment process for example can be (concentration of acid solution and alkali lye is 0.05mol/L-1mol/L) alternate treatment: earlier with dipping by lye resin 1～3h (preferred 2h); Be washed till neutrality then; With soak or after washing 1～3h (preferred 2h), be washed till neutrality again, repeat this process 1-5 time) resin 1-5 time; Alkali lye is only used in last processing; Treatment time is 1-24 hour at every turn, and water after disposing (preferred deionized water, zero(ppm) water or distilled water) is washed till water and is neutral, and it is subsequent use to be immersed in the middle preservation of water (preferred deionized water, zero(ppm) water or distilled water) again.The pH inner through the ion exchange resin of above-mentioned processing remains alkalescence, more helps its activity of enzyme performance.
2, enzyme immobilization
The carrier that takes by weighing a certain amount of processing is in container; Put into the TL enzyme liquid of certain volume, (can adopt the gas bath shaking table in 15～35 ℃ of (preferred 15～25 ℃, more preferably room temperature) shaking baths; Mechanical stirrer and magnetic stirring apparatus, the form of vortex stirrer) 150rpm (10-200rpm) is fixing in.After fixing the completion, take out the resin suction that is adsorbed with enzyme and remove residual liquid.
The source of lypase can get at suitable culture condition bottom fermentation for thermophilic hyphomycete; Also can be through genetically engineered; Modern biology means such as protein engineering are transformed and are got, and comprise the enzyme of chemically modified, the enzyme that the two mutants of protein engineering or genetic engineering bacterium produce.The source of lypase can also be for commercial obtainable, like the Lipozyme TL100L of Denmark Novozymes Company.
Enzyme liquid in the being used for fixing process can need not to change through any separation, purifying etc. the pre-treatment of its original composition or environment.Also can dilute or processing such as pH regulator, to be suitable for reaction enzyme liquid.
Being used for TL enzyme liquid of the present invention can include, but is not limited to:
1. the fermented liquid that passes through to cultivate thermophilic hyphomycete (Thermomyces Lanuginosus) acquisition is (for example referring to document Charlotte Pinholt; Mathias Fano; Charlotte Wiberg; Influence of glycosylation on the adsorption of Thermomyces lanuginosus lipase to hydrophobic and hydrophilicsurfaces, European Journal of Pharmaceutical Sciences 40 (2010): 273-281);
2. obtain lipase gene through cultivating from thermophilic hyphomycete; The lypase enzyme liquid of producing acquisition by fermentation with gene modification aspergillus oryzae is (for example referring to the same document Charlotte Pinholt; Mathias Fano; Charlotte Wiberg, European Journal of Pharmaceutical Sciences 40 (2010): 273-281).
3. commercially available commodity TL liquid aliphatic enzyme, for example the Lipozyme TL100L of Novozymes Company.
4. through in the solution of aforesaid lypase liquid or enzyme powder, adding acid-alkali accommodation TL enzyme liquid to suitable pH value and/or add enzyme liquid that entry (like zero(ppm) water, deionized water or distilled water) is adjusted to suitable protein concentration (for example referring to document Daniel Otzen; Differential adsorption of variants of the Thermomyces lanuginosus lipase on a hydrophobic surface suggests a role for local flexibility, biointerfaces 2008 64:223-228).
Also can in enzyme liquid, add a certain amount of zymoprotein linking agent (can play the material of crosslinked role of apoenzyme) in the present invention; Preferred said zymoprotein linking agent has hydroxyl or amino, for example glutaraldehyde solution, polymine, Vestolen PP 7052 imines, polyvinylamine, gelatin or spermine etc.The consumption of said zymoprotein linking agent is to add 0.0～10% (v/v) in every milliliter of enzyme liquid.For example the consumption of LUTARALDEHYDE is 1% glutaraldehyde solution that adds 0.01～0.1ml in every milliliter of enzyme liquid, also can adopt other concentration glutaraldehyde solution of equal proportion.
The weight (g) of resin carrier of handling and TL enzyme liquid/volume (ml) is than can be: 25: 1～1: 25, and preferred 10: 1～1: 10, more preferably 1: 1 (can according to circumstances suitably resize ratio).
The fixed mode can adopt shaking bath or gas bath shaking table, usual manners such as stirrer or magnetic stirring apparatus.
During fixing, can be at regular intervals (5min～1h) get supernatant to survey absorption/combination degree that residual protein content is judged zymoprotein for example.The residual protein Determination on content can adopt conventional quantification of protein detection methods such as Xylene Brilliant Cyanine G method, lorry method, biuret method, BCA method to carry out.Usually, when residual protein content is lower than 0.1mg/ml～0.5ml/ml, stop fixing step.Certainly, those of ordinary skills can adjust and control fixation degree according to concrete needs (like quality control requirement etc.).
Immobilized enzyme method of the present invention need not enzyme is carried out complicated pre-treatment (like separations, purifying etc.), and has obtained the significantly generation of reduction soap in the immobilized enzyme use unexpectedly, even do not produce the technique effect of soap.
3, drying
After the immobilization step was advanced to accomplish, through redundant moisture in the drying step removal system, the exsiccant mode can be selected from: conventional drying methods such as lyophilize, vacuum-drying, fluidised bed drying and drying at room temperature.
For example, lyophilize can the freeze drier of LABCNCO (manufacturer: U.S. Labcnco company, model: carry out freezone), parameter can be set to (below be each step parameter, this several steps constitutes a temperature-fall period):
1.5 ℃/min of-20 ℃ of rate of temperature fall, 2h (0.5-5h),
1.5 ℃/min of-10 ℃ of rate of temperature fall, 6h (3-10h)
1.01 ℃/min of 5 ℃ of rate of temperature fall, 15h, (10-24h)
1.16 ℃/min of 20 ℃ of rate of temperature fall, 20h (10-24h)
Vacuum-drying can (manufacturer: guest's moral Asia-Pacific (Hong Kong) ltd model: carry out VD23), it be under the 6mbar that parameter is set in vacuum tightness, 40 ℃ of dry 48h (24-60h) at for example Binder vacuum drying oven.
Fluidised bed drying can in fluidized-bed, carry out (the special company of Gera, Germany for example, model: midi glatt), parameter is set to 45～55 ℃ of EATs (preferred 50 ℃), be 0.5～5h (preferred 1.5h) time of drying.
Drying at room temperature can be the place placement seasoning in 3～5 days that sample is placed on air seasoning.
Those of ordinary skills can select the conventional drying method with condition as required.
4, other optional step
According to concrete needs, also can extraly have following one or more optional steps, for example (but being not limited to) in the method for the present invention:
1 removes by filter small molecules with enzyme liquid, the influence of enzyme being lived with the small molecules of eliminating in the enzyme liquid.Filter method for example can be: on the ultra-filtration membrane device, carry out ultrafiltration (manufacturer: U.S. Millipore Corp.; Model: small-sized); Add phosphoric acid buffer (for example pH is the phosphoric acid buffer of 5 0.1mol/L) then; Make gained enzyme liquid and original enzyme liquid system pH be consistent, and adjust to suitable zymoprotein concentration.
2 can add the stability that materials such as dextrin, BSA and/or trehalose strengthen enzyme in the immobilization step, their preferred additions separately are: dextrin 1～20% (W/V); BSA 0.05～5% (W/V); Trehalose 0.1-10% (W/V).Can add one or more above-mentioned substances, to obtain required stabilising effect.
3 pairs of carriers carry out modification to be handled, and connects reactive group through chemical means.For example, can be with reference to Xu Jingliang etc., the carrier immobilized enzyme progress of amino functional chemical industry progress 201029 (3): 494-496.
5, preferred implementation of the present invention
An exemplary preferred embodiment of the present invention is following:
The processing of step 1, carrier
Place deionized water to soak impurity elimination in the macroporous adsorbent resin carrier, spend the night with alcohol immersion then and remove organic impurity and other insolubless, with deionized water rinsing to do not have the alcohol flavor, be immersed in again preserve in the deionized water subsequent use.
Perhaps, ion exchange resin is used washed with de-ionized water,, and then handle, handle about two hours at every turn, handle to water with deionized water then and be neutral, in deionized water, soak subsequent use with 5%NaOH with 5%NaOH and 5%HCl alternate treatment 2～5 times.
Step 2, enzyme immobilization
Take by weighing resin carrier that 40g handled in the 250ml triangular flask; Put into 40ml TL enzyme liquid; 150rpm fixes in 25 ℃ of shaking baths, during get supernatant at regular intervals and survey the degree of absorption that residual protein content is judged zymoprotein, take out then and put into petridish and inhale and remove residual liquid.
Step 3, drying
The redundant moisture of after the immobilization step is finished, will going out in the system is promptly dry, and drying can adopt mode conventional in this area, for example lyophilize, vacuum-drying and drying at room temperature.
Should be understood that above-mentioned preferred implementation is exemplary, those of ordinary skills can change and optimize each Step By Condition under the prerequisite that does not break away from spirit and scope of the invention based on the general knowledge of the record among the application and this area.
6, the application of immobilized lipase of the present invention
Can immobilized lipase of the present invention directly be used for suitability for industrialized production, also can be made into wrapped product so that store, transport, sell and further use.
During packing, preferably in the container of can in cleaning under the cryogenic condition of aseptic drying.Storage condition is preferably cryodrying condition storage, and storing optimum temperuture is 4～20 ℃.Preferably under 4 ℃～25 ℃ temperature, transport.
Immobilized lipase of the present invention or its wrapped product can be used for catalysis grease generation hydrolysis, transesterify, esterification or catalysis acidolysis, alcoholysis etc., in industry such as food, papermaking, leather, washing composition, pharmacy, huge application are arranged.For example, immobilization TL lypase of the present invention can be used for particularly being preferred for catalyzed transesterification in the production of modification and Tegin 55G and sweet diester of hydrolysis, Witepsol W-S 55.
Advantage of the present invention:
1. adopt among the present invention a kind of novel method for preparing immobilization TL enzyme is provided; Adopted in the immobilization field uncommon macroporous adsorbent resin or treated ion exchange resin as fixation support in this method; And need not lypase is carried out complicated pre-treatment, thereby simplified production stage greatly, reduced production cost.
2. solved the problem of commodity TL enzyme generation soap in the grease reaction.
3. immobilization TL enzyme of the present invention can reuse, thereby has reduced production cost, has improved rate of utilization.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to the condition of normal condition described in " Biochemistry and Molecular Biology experiment study course " (Liang Songping edits Higher Education Publishing House), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
TL enzyme liquid is available from Denmark Novozymes Company, and commodity are called Lipozyme TL 100L (pH5.0), is used for embodiments of the invention without any processing or adjusting pH to 8.0 back (NaOH of 0.05mol/L～lmol/L).
Embodiment 1. adopts macroporous adsorbent resin to prepare immobilized enzyme
(D101 and HPD100A are available from the precious grace chemical industry in Cangzhou, Hebei ltd with macroporous adsorbent resin D101, HPD100A or X-5; X-5 is available from Shanghai China shake resin ltd) place deionized water to soak about 12h impurity elimination respectively; Spend the night with 95% alcohol immersion then and remove organic impurity and other insolubles; To there not being the alcohol flavor, place deionized water subsequent use with deionized water rinsing.
Prepare immobilized enzyme 1-19 according to following prescription and step:
1, takes by weighing the D101 resin 40g that handles well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5,150rpm vibration 2h in 25 ℃ of shaking tables takes out resin then and puts into the exsiccant petridish; Put into freeze drier and carry out drying, concrete freeze-drying parameter is set to:
1.5 ℃/min of-20 ℃ of rate of temperature fall, 2h,
1.5 ℃/min of-10 ℃ of rate of temperature fall, 6h
1.01 ℃/min of 5 ℃ of rate of temperature fall, 15h,
1.16 ℃/min of 20 ℃ of rate of temperature fall, 20h
Get immobilized enzyme 1 after the drying.
2, take by weighing the D101 resin 40g that handles well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5; 150rpm vibration 2h in 25 ℃ of shaking tables takes out resin then and puts into the exsiccant petridish, puts into Vacuumdrier and carries out drying (vacuum tightness is under the 6mbar; 40 ℃ of dry 48h), get immobilized enzyme 2 after the drying.
3, take by weighing the D101 resin 40g that handles well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5,150rpm vibration 2h in 25 ℃ of shaking tables takes out resin then and puts into the exsiccant petridish; Be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 3 after the drying.
4, take by weighing the D101 resin 40g that handles well in the 250ml triangular flask; The TL enzyme liquid and the 10ml zero(ppm) water that add 30ml pH5; 150rpm vibration 2h in 25 ℃ of shaking tables; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 4 after the drying.
5, take by weighing the D101 resin 40g that handles well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH8,150rpm vibration 2h in 25 ℃ of shaking tables takes out resin then and puts into the exsiccant petridish; Be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 5 after the drying.
6, take out the D101 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5; With the glutaraldehyde solution of 0.8ml 1%, 150rpm vibration 2h in 25 ℃ of shaking tables, resin takes out puts into the exsiccant petridish; Be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 6 after the drying.
7, take out the D101 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid and the 10ml zero(ppm) water that add 30ml pH5; With the glutaraldehyde solution of 0.8ml 1%, 150rpm vibration 2h in 25 ℃ of shaking tables, resin takes out puts into the exsiccant petridish; Be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 7 after the drying.
8, take out the D101 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 8 after the drying.
The D101 resin 40g that 9 taking-ups are handled well is in the 250ml triangular flask; The TL enzyme liquid and the 10ml zero(ppm) water that add 30ml pH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 9 after the drying.
10, take out the D101 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid and the 10ml zero(ppm) water that add 30ml pH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 10 after the drying.
11, take out the D101 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 11 after the drying.
12, take out the HPD100A resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 0.8ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 12 after the drying.
13, take out the HPD100A resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 13 after the drying.
14, take out the HPD100A resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 0.8ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 14 after the drying.
15, take out the HPD100A resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 15 after the drying.
16, take out the X-5 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 0.8ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 16 after the drying.
17, take out the X-5 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 17 after the drying.
18, take out the X-5 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 0.8ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 18 after the drying.
19, take out the X-5 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40ml pH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, be placed on the good place of room ventilated and carry out drying, get immobilized enzyme 19 after the drying.
More than handle the mensuration that finishes afterwards supernatant to be carried out protein content at immobilization process, measuring method is the Xylene Brilliant Cyanine G method, and its residual protein content is below 0.1mol/L (as shown in table 1) all.Enzyme immobilization efficiency calculation formula is following:
Table 1. enzyme immobilization efficient
| The original enzyme liquid protein concentration | Absorption back supernatant protein concentration | Enzyme immobilization efficient |
| 5.5mol/L | ＜0.1mol/L | ＞98％ |
Summed up the preparation parameter of immobilized enzyme 1～19 in the table 2.
Table 2. adopts the immobilized enzyme of macroporous resin preparation
Summed up the preparation of the embodiment of enzyme and carrier different ratios in the table 3
Table 3. adopts different enzymes and the prepared immobilized enzyme of carrier ratio (V/W)
Embodiment 2. adopts ion exchange resin to prepare immobilized enzyme
Styrene-DVB201 * 2 resins (available from Tianjin Nankai Hecheng S&T Co., Ltd.) are used earlier washed with de-ionized water; Alternately soak 2 times with 5%NaOH and 5%HCl then; And then with 5%NaOH immersion 1 time; The each processing about two hours is washed till water with deionized water then and is neutral, in deionized water, soaks subsequent use.
Prepare immobilized enzyme 20-24 according to following prescription and step:
1, takes out the Styrene-DVB201 * 2 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40mlpH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 0.8ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 20 after the drying.
2, take out the Styrene-DVB201 * 2 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40mlpH5; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 21 after the drying.
3, take out the Styrene-DVB201 * 2 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40mlpH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 0.8ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 22 after the drying.
4, take out the Styrene-DVB201 * 2 resin 40g handle well in the 250ml triangular flask; The TL enzyme liquid that adds 40mlpH8; Glutaraldehyde solution 150rpm vibration 2h in 25 ℃ of shaking tables with 1.6ml 1%; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 23 after the drying.
Styrene-DVB201 * 2 resin 40g that 5 taking-ups are handled well are in the 250ml triangular flask; The TL enzyme liquid that adds 40mlpH5; 150rpm vibration 2h in 25 ℃ of shaking tables; Then resin is taken out and put into the exsiccant petridish, put into draughty place and carry out natural air drying, get immobilized enzyme 24 after the drying.
More than handle fixing the end and all (seeing table 4) more than 96% through calculating enzyme immobilization efficient after detect the supernatant protein content all below 0.2mg/ml.
Table 4. enzyme immobilization efficient
| The original enzyme liquid protein concentration | Absorption back supernatant protein concentration | Enzyme immobilization efficient |
| 5.5mol/L | ＜0.2mol/L | ＞96％ |
Summed up the preparation parameter of immobilized enzyme 20-23 in the table 5.
Table 5. adopts the immobilized enzyme of ion exchange resin preparation
The transesterify vigor test of embodiment 3. immobilized enzyme
Test and method of calculation
With purified soyabean oil (available from (Shanghai) foodstuffs industry ltd in good; Lot number 2B011121) and the deep hydrogenation VT 18 (available from (Shanghai) special oil ltd in good; Lot number 3H011104) mixture is substrate (w/w=73: 27), be 1: 20 with immobilized enzyme and substrate mass ratio, at 70 ℃ of reaction 30min; Reaction is finished the back and is taken out product, surveys its solid fats content of 40 ℃ (being SFC content).
40 ℃ SFC content assaying method is: the solid fat pipe that sample will be housed is put into 100 ℃ of baking oven 15min respectively; 60 ℃ of water-bath 5min; 0 ℃ of water-bath 60min, 40 ℃ of water-bath 30min use NMR (manufacturer: German Brooker spectral instrument company model: mq20) survey its solid fat content then.The calculation formula of transesterify vigor is:
Transesterify vigor=(SFC
Blank-SFC
Sample)/30 * 1260
Wherein, SFC
BlankBe meant that the substrate grease that does not pass through the enzyme process transesterification reaction is at 40 ℃ of following SFC values, SFC
SampleBe meant the SFC value of 40 ℃ of following enzyme reaction product samples.
Experimental result is the MV of 3 parallel laboratory tests.
Experimental result and discussion
Table 6 has been summed up the transesterify vitality test result of immobilized enzyme 1-24 of the present invention.For clear relatively for the purpose of, table 7～12 have shown the LUTARALDEHYDE addition that adopts under different drying modes, different TL enzyme pH value, the condition of different pH respectively, have reached the influence of different resins to immobilized enzyme transesterify vigor.
The transesterify vigor of table 6. immobilized enzyme 1-23
Table 7. adopts the transesterify vigor of the D101 immobilization TL enzyme of different drying mode preparations
| Enzyme number | Resin | Drying mode | The transesterify vigor |
| 1 | D101 | Lyophilize | 406 |
| 2 | D101 | Vacuum-drying | 411 |
| 3 | D101 | Drying at room temperature | 557 |
The transesterify vigor data of the D101 immobilization TL enzyme of the different drying modes preparations of the employing enumerated in the table 7 show, adopt different drying modes to the transesterify vigor of immobilized enzyme and do not make significant difference.Therefore, can be as required in the preparation of immobilized enzyme and working condition, adopt various drying mode.
The transesterify vigor of the immobilization TL enzyme of the TL enzyme preparation of the different pH values of table 8. employing
It is under 5 the condition that table 8 result is presented at pH, adopts the fixedly TL enzyme activity of the macroporous adsorbent resin D101 TL enzyme activity that compares under the pH8 condition fixedly to want height.D101 adds LUTARALDEHYDE and HPD100A, and to add these two groups of LUTARALDEHYDEs also like this.Be better than acidic conditions (there was no significant difference) slightly and adopt the X-5 alkaline condition to fix down.Being fixed with under the acidic conditions possibly be the reason of avoiding in the transesterify process, producing soap, in actual fabrication process, can select suitable pH value according to the character of different carriers.
Under the table 9.pH5 condition, adopt the transesterify vigor of D101, HPD100A or the X-5 immobilization TL enzyme of different LUTARALDEHYDE consumption preparations
| Enzyme number | Resin (40g) | The consumption of 1% LUTARALDEHYDE (ml) | The transesterify vigor |
| 3 | D101 | 0 | 557 |
| 6 | D101 | 0.8 | 582 |
| 8 | D101 | 1.6 | 556 |
| 12 | HPD100A | 0.8 | 645 |
| 13 | HPD100A | 1.6 | 612 |
| 16 | X-5 | 0.8 | 554 |
| 17 | X-5 | 1.6 | 555 |
Table 9 result shows concerning D101 and two kinds of resins of HPD100A, and the enzyme when under pH is 5 condition, adding 1% LUTARALDEHYDE amount the enzyme work of immobilized enzyme being higher than 1% LUTARALDEHYDE addition and being 1.6ml when being 0.8ml is lived, but there was no significant difference.And concerning the X-5 resin, to be higher than 1% LUTARALDEHYDE addition be that the enzyme of 0.8ml is lived to the immobilized enzyme vigor when 1% LUTARALDEHYDE addition was 1.6ml, but also there was no significant difference.
This presentation of results is to reach high enzyme work can add suitable LUTARALDEHYDE amount according to the character of carrier to different resins, but also can select not add LUTARALDEHYDE.
Under table 10. condition of different pH, adopt the HPD100A of different LUTARALDEHYDE consumption preparations or the transesterify vigor of X-5 immobilization TL enzyme
Table 10 result is illustrated under the identical situation of other condition, and fixed TL enzyme is higher than fixed TL enzyme activity under the pH8 condition under the pH5.Be under 8 the condition at pH, different resins adds enzyme that the LUTARALDEHYDE amounts of different amounts obtain and lives also differently, waits until and further investigates (referring to embodiment 4) in the follow-up successive reaction.
The transesterify vigor of enzyme and carrier different ratios immobilization TL enzyme under the table 11.pH5 condition
Table 11 result shows that the different ratios of carrier and enzyme can obtain different enzymes and live; Generally speaking the addition of enzyme is directly proportional with enzyme work; Live in order to obtain higher enzyme, except that D101 fixedly the embodiment of TL enzyme, it is 1: 1 (V/W) that other embodiment all select the ratio of enzyme and carrier.
Table 12. adopts the transesterify vigor of the fixed on ion exchange resin TL enzyme of different pH and the preparation of LUTARALDEHYDE consumption
Table 12 is the result show: the immobilized enzyme enzyme work that ion exchange resin obtains under pH is 5 condition is higher than the immobilized enzyme enzyme that other condition obtains under the pH8 condition when identical and lives.The enzyme that enzyme work when the add-on of LUTARALDEHYDE is 0.8ml is higher than when not adding LUTARALDEHYDE or LUTARALDEHYDE add-on and being 1.6ml is lived, and explain that the LUTARALDEHYDE to Styrene-DVB201 * 2 resins interpolation 0.8ml 1% is suitable.
Comparison through above experimental result in the present embodiment can be known: can obtain higher enzyme at the LUTARALDEHYDEs that do not change the different amounts of interpolation on the pH basis and live, in practical application, can not change under its original acidic conditions through adding the vigor that an amount of LUTARALDEHYDE improves enzyme.
Embodiment 4. adopts the saponified matter content and solid fat content measuring of immobilized enzyme through the reacted product of many wheels
Testing method
Ratio preparation reaction substrate with ST (palm stearin, Southseas Specialty Fats Industrial (Shanghai) Co., Ltd.) 65% and PKO (palm-kernel oil, Southseas Specialty Fats Industrial (Shanghai) Co., Ltd.) 35% (W/W); In the 250ml triangular flask, add the 40g substrate; The 8g immobilized enzyme, 200rpm reaction 30min takes out product then in 70 ℃ of water-baths; Immobilized enzyme is stayed in the reactor drum; Continue to add the 40g substrate and react next time, carry out altogether 5 times, the saponified matter content of detection reaction product and 40 ℃ of solid fat content (SFC).
Experimental result and discussion
Following table 13～14 shown respectively the LUTARALDEHYDE addition that adopts under different drying modes, different TL enzyme pH value, the condition of different pH, and different resins to the influence of immobilized enzyme transesterify vigor.
Immobilized enzyme with prior art: Novi's letter TL IM immobilized enzyme is contrast.This immobilized enzyme is that NOVOINDUSTRI company produces, and with the lypase of thermophilic hyphomycete, prepares according to method described in the WO89/06278.According to comparing, the preparation of method of the present invention and the letter TL IM of Novi immobilized enzyme has following difference:
1) carrier is different: the carrier that WO89/06278 adopted is the carrier of weakly alkaline phenolic aldehyde skeleton or acrylic type skeleton, the polystyrene type carrier that adopts among the present invention.
2) the vehicle treated method is different: WO89/06278 does not mention the pre-treatment of carrier in patent, carrier of the present invention is through pre-treatment.
3) process for fixation is different: WO89/06278 immobilization time in instance is 8h, and water-washing step is arranged after the immobilization, and adopts vacuum-drying, and the set time of the present invention is for being merely about 2h, and can adopt more easy, energy-conservation drying at room temperature method.
4) preparation of enzyme liquid is different, and the enzyme liquid that WO89/06278 adopted is the solution of being dissolved into the enzyme powder, and directly adopts the enzyme liquid of buying among the present invention.
Table 13. different fixing enzyme saponified matter content of product separately after each takes turns reaction
By the digital proof in the table 13, the product saponified matter content that the immobilized enzyme that adopts the inventive method to prepare is produced aborning significantly is lower than the immobilized enzyme that adopts the art methods preparation, does not produce soap (being that the product saponified matter content is 0) even.
Table 14. different fixing enzyme is taken turns the solid fat content (SFC) of reaction after product at each
The greasy SFC of substrate is 19.61
Data in the table 14 have also further proved, adopt the immobilized enzyme of the inventive method preparation to have higher activity aborning, and vigor and existing commercial enzyme are comparable, are superior to existing commercial enzyme even.5 take turns various immobilized enzyme catalysis products in the reaction SFC all far below substrate SFC, the effective catalysis of immobilized enzyme has been described transesterification reaction.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. method for preparing immobilized lipase, said method comprises step:
A) carrier is carried out pre-treatment, said carrier is selected from: ion exchange resin or skeleton are the macroporous adsorbent resin of PS;
B) lypase is provided, and the optional zymoprotein linking agent that adds therein;
C) lypase that in the carrier of pre-treatment and step b), is provided with gained in the step a) contacts, and makes said lypase be fixed in said carrier through pre-treatment;
D) carrier that is fixed with lypase of gained drying step c) is to obtain said immobilized lipase.
2. the method for claim 1 is characterized in that, said carrier is selected from: polar macroporous adsorption resin, nonpolar macroporous adsorption resin, Zeo-karb or anionite-exchange resin.
3. method as claimed in claim 2 is characterized in that,
Said nonpolar macroporous adsorption resin is selected from: D101, HPD100A, AmberliteXAD-4, HPD100, HPD300, HPD700, X-5, XAD-4 or HP20;
Said polar macroporous adsorption resin is selected from: D201, LSA-10 or Amberlite XAD7HP;
Said Zeo-karb is selected from: D113, D001 or Ambetlite IRC50;
Said anionite-exchange resin is selected from: D301 or Styrene-DVB201 * 2.
4. method as claimed in claim 2 is characterized in that,
The pre-treatment of said macroporous adsorbent resin is carried out as follows: place water to soak impurity elimination in carrier; Soaked in solvent 1～24h is to remove organic impurity and insolubles then; The said solvent of water flush away again, wherein said solvent is: ethanol, normal hexane, acetone or sherwood oil;
The pre-treatment of said ion exchange resin is carried out as follows: alternately soak or wash said resin 1-5 time with the alkali lye of 0.05mol/L～lmol/L and acid solution; Only soak once more with the alkali lye of 0.05mol/L～lmol/L then or wash resin 1 time, water is washed till water and is neutral again.
5. the method for claim 1 is characterized in that, the lypase that is provided in the step b) is selected from: the tunning of thermophilic hyphomycete (Thermomyces Lanuginosus); The lypase that gets through genetically engineered or protein engineering transformation; Or commercially available lypase.
6. method as claimed in claim 5 is characterized in that said lypase is used for step c) without any pre-treatment.
7. the method for claim 1 is characterized in that, said zymoprotein linking agent is selected from: glutaraldehyde solution, polymine, Vestolen PP 7052 imines, polyvinylamine, gelatin or spermine.
8. the method for claim 1 is characterized in that, in the step c), said treated carrier and lypase are 25: 1～1: 25 in the by weight/volume of g/ml.
9. an immobilized lipase or its wrapped product is characterized in that, described immobilized lipase is through each described method preparation among the claim 1-8.
10. method of carrying out transesterify, esterification, acidolysis or alcoholysis reaction, said method comprise uses the described immobilized lipase of claim 9 as catalyzer.
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| CN103468668A (en) * | 2012-06-06 | 2013-12-25 | 益海(佳木斯)生物质能发电有限公司 | Method for immobilization of lipase and its application |
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| CN104694526A (en) * | 2013-12-06 | 2015-06-10 | 丰益(上海)生物技术研发中心有限公司 | Sn-1,3 selective immobilized lipase catalyzing esterification and transesterification and preparing method thereof |
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| CN108486094A (en) * | 2018-04-20 | 2018-09-04 | 中国科学院南海海洋研究所 | A kind of immobilized lipase and preparation method thereof |
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