CN102839166B - Tl immobilized enzyme and application thereof - Google Patents
Tl immobilized enzyme and application thereof Download PDFInfo
- Publication number
- CN102839166B CN102839166B CN201110170701.6A CN201110170701A CN102839166B CN 102839166 B CN102839166 B CN 102839166B CN 201110170701 A CN201110170701 A CN 201110170701A CN 102839166 B CN102839166 B CN 102839166B
- Authority
- CN
- China
- Prior art keywords
- lipase
- enzyme
- resin
- carrier
- drying
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The present invention relates to TL immobilized enzyme and application thereof.Specifically, provide a kind of method preparing immobilized lipase in the present invention, it comprises step: a) carry out pre-treatment to carrier, and described carrier is selected from: ion exchange resin or skeleton are the macroporous adsorbent resin of polystyrene; B) lipase is provided; C) by step a) in the carrier through pre-treatment of gained and step b) in the lipase that provides contact, make described lipase be fixed on the described carrier through pre-treatment; D) drying step.The immobilized lipase and application thereof prepared by the inventive method is additionally provided in the present invention.Immobilized enzyme of the present invention has high activity and stability, and does not produce soap in catalyzed transesterification process in the food industry, simple to operate, greatly reduces the cost of fats and oils processing.
Description
Technical field
The invention belongs to food and chemical field.More specifically, the present invention relates to the immobilized enzyme method for TL lipase and application thereof.
Background technology
Enzyme, as a kind of biological catalyst, has substrate selective, and reaction conditions is gentle, and pollute the advantages such as little, overcome the severe reaction conditions that traditional industry catalyzer brings, the shortcomings such as byproduct of reaction contaminate environment, have good application potential.Searching catalysis activity is high, the enzyme of reaction conditions gentleness, or carrying out transformation to existing enzyme makes it more adapt to production application, is the main development direction that following enzyme engineering is applied in the industry.
Free enzyme is fettered or is limited to by physics or chemical means in certain area by enzyme immobilization technology, makes it still can carry out distinctive catalyzed reaction and can reclaim reusable a kind of technology.Due to above-mentioned advantage, a lot of researcher is made to expand careful research to immobilized enzyme.In 1967, immobilized enzyme was used in first and splits in amino acids industry.In recent years along with the development of organic chemistry, protein chemistry, Materials science etc., enzyme immobilization technology achieves significant progress.
Lipase is that catalysis grease is hydrolyzed, transesterify, esterification class of enzymes, it can play catalytic activity specifically on water-oil interface, has huge application food, papermaking, leather, washing composition, pharmacy etc. are industrial.But natural enzyme poor stability, is difficult to purifying after reaction, can not reuse, not only reduce the service efficiency of enzyme, improve production cost, and be difficult to realize continuous operation, limit its application industrially.In this context, enzyme immobilization technology becomes a kind of effective way solving above-mentioned production bottleneck.
TL lipase is the quasi-lipase found in the thermophilic hyphomycete (ThermomycesLanuginosus), and it is mainly used in the production of the hydrolysis of grease, the modification of Witepsol W-S 55 and Tegin 55G and DAG.The TL lipase of liquid is mainly used in hydrolysis reaction.Immobilized TL enzyme is used for catalyzed transesterification, and its advantage to reuse, and reduces production cost.
WO8906278A1 describes a kind of method of immobilization TL enzyme, by cultivating the fungi of the mould genus of humic or obtaining the fermented liquid containing lipase through the humic mould genus fungi of genetic modification, with phenol aldehyde type or acrylic type resin for fixation support, immobilization process is add in the weakly alkaline macroreticular ion exchange resin handled well by a certain amount of certain pH value enzyme liquid, at room temperature shake 8h, then wash, room temperature in vacuo is dry, obtains product.
EP1239045A1 describes a kind of method of immobilization TL enzyme, is specially and is dissolved in phosphoric acid buffer by lipase powder, adds ethanol and polypropylene based resin ACCURELMP1001, and under room temperature, concussion is spent the night.By collecting by filtration immobilized enzyme, by buffer solution for cleaning, under room temperature, carry out drying.
But up to the present, on market, existing TL immobilized enzyme can not meet production requirement.Find in product, to there is a large amount of soaps in the transesterification reaction of its catalysis, waste raw material in a large number, increase cost, analyze its reason may be exist in the process that is combined with enzyme of dissimilar carrier some technical problems that cannot overcome cause or carrier with in enzyme fixation procedure with the conflicting of reaction medium.Therefore current immobilized TL needs a large amount of grease wash-outs, causes a large amount of wastage of material.Simultaneously for the separating-purifying in downstream brings huge difficulty, limit its application on oil prodution industry.
Therefore, in the urgent need to developing the immobilization TL enzyme method made new advances in this area, make TL enzyme keep higher vigor and not produce soap in the reaction, thus expand the range of application of TL lipase in foodstuffs industry, and overcome the problem that existing immobilization TL enzyme produces soap aborning.
Summary of the invention
An object of the present invention is just being to provide a kind of new TL enzyme immobilization method.Another main purpose of the present invention is to provide a kind of immobilized TL enzyme.The present invention also aims to provide immobilization TL enzyme of the present invention application in the industry.
In a first aspect of the present invention, provide a kind of method preparing immobilized lipase, described method comprises step:
A) carry out pre-treatment to carrier, described carrier is selected from: ion exchange resin or skeleton are the macroporous adsorbent resin of polystyrene;
B) provide lipase, and optionally add zymoprotein linking agent wherein;
C) by step a) in the carrier through pre-treatment of gained and step b) in the lipase that provides contact, make described lipase be fixed on the described carrier through pre-treatment;
D) drying step c) in the carrier being fixed with lipase of gained, to obtain described immobilized lipase.
In a preference, the macroporous adsorbent resin of described polystyrene can be the macroporous adsorbent resin of polystyrene-divinylbenzene skeleton.
In an embodiment of the invention, described 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, described nonpolar macroporous adsorption resin is selected from: D101, HPD100A, AmberliteXAD-4, HPD100, HPD300, HPD700, X-5, XAD-4 or HP20;
Described polar macroporous adsorption resin is selected from: D201, LSA-10 or AmberliteXAD7HP;
Described Zeo-karb is selected from: D113, D001 or AmbetliteIRC50;
Described anionite-exchange resin is selected from: D301 or Styrene-DVB201 × 2.
In yet another embodiment of the present invention, the pre-treatment of described macroporous adsorbent resin is carried out as follows: carrier is placed in water and soaks impurity elimination, then soaked in solvent 1 ~ 24h is to remove organic impurity and insolubles, wash described solvent again with water, wherein said solvent is: ethanol, normal hexane, acetone or sherwood oil;
The pre-treatment of described ion exchange resin is carried out as follows: with alkali lye and the acid solution alternate immersion of 0.05mol/L ~ 1mol/L or rinse described resin 1-5 time, then only again soak with the alkali lye of 0.05mol/L ~ 1mol/L or rinse resin 1 time, then washing with water to water in neutral.
In a preference, described water is deionized water, distilled water or distilled water.
In another preference, the ethanol of ethanol used in the pre-treatment of macroporous adsorbent resin to be concentration range be 100%-50% (V/V).
In another preference, the addition scope of solvent described in the pre-treatment of macroporous adsorbent resin is 0.5-10 times of resin volume, and preferred 1-5 doubly.
In another preference, the described acid in the pre-treatment of ion exchange resin is selected from: HCl, H
2sO
4or H
3pO
4, described 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 described ion exchange resin is: soak with the weightmeasurement ratio (W/V) of resin and alkali lye or acid solution 1: 1 ~ 1: 5, preferably 1: 1.5 ~ 1: 3, more preferably 1: 2 or rinse described resin, order is acid after first alkali, repeat last time base extraction for several times, then wash with water to water in neutral.
In another preference, treated carrier is immersed in deionized water (or distilled water, distilled water) and saves backup.
In yet another embodiment of the present invention, step b) in the lipase that provides be selected from: the tunning of thermophilic hyphomycete (ThermomycesLanuginosus); The lipase obtained by genetically engineered or protein engineering transformation; Or commercially available lipase.
In a preference, the lipase that described genetically engineered or protein engineering are transformed and obtained is: the enzyme that the enzyme of chemically modified, the mutant of protein engineering or genetic engineering bacterium produce.
In another preference, described commercially available lipase is preferably the LipozymeTL100L of Novozymes Company of Denmark.
In yet another embodiment of the present invention, described lipase without any pre-treatment for step c) in.
In another preference, can dilute described lipase.
In yet another embodiment of the present invention, described zymoprotein linking agent is selected from: glutaraldehyde solution, polymine, PPI, polyvinylamine, gelatin or spermine.
In a preference, the consumption of described zymoprotein linking agent is the 0.001-0.1 (V/V) that enzyme liquid amasss, and the consumption of preferred glutaraldehyde is 1% glutaraldehyde solution or other concentration glutaraldehyde solution of equal proportion of adding 0.01 ~ 0.1ml in every ml enzyme liquid.
In yet another embodiment of the present invention, step c) in, described treated carrier and lipase in the weight/volume of g/ml for 25: 1 ~ 1: 25.
In a preference, described ratio is preferably 10: 1 ~ 1: 10, and more preferably 1: 1.
In another preference, shaking bath, gas bath shaking table, stirrer or magnetic stirring apparatus is used to make described treated carrier contact with lipase and fix.
In another preference, survey by getting supernatant the fixation degree that residual protein content judges zymoprotein, the mensuration of described residual protein content adopts the method being selected from lower group to carry out: Coomassie Brilliant Blue, 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, described drying is that the method by being selected from lower group is carried out: lyophilize, vacuum-drying, fluidised bed drying and drying at room temperature.
In a second aspect of the present invention, provide a kind of immobilized lipase or its wrapped product, described immobilized lipase is prepared by method of the present invention.
In a preference, described wrapped product also comprises packing material, preferred container or packing bag.
In a third aspect of the present invention, provide a kind of method of carrying out transesterify, esterification, acidolysis or alcoholysis reaction, described method comprises and uses immobilized lipase of the present invention as catalyzer.
In another aspect of this invention, additionally provide the purposes of immobilized lipase of the present invention in transesterify, esterification, acidolysis or alcoholysis reaction.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Embodiment
The present inventor, through long-term and deep research, has found a kind of TL immobilized enzyme method, is a kind of immobilized enzyme method not producing in the reaction or seldom produce soap furtherly.Described 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 adopting the method obtained is higher, and can not produce a large amount of soaps, has very big application prospect.On this basis, the present inventor completes the present invention.
1, the selection of carrier and pre-treatment
Have employed not used polystyrene type macroporous adsorbent resin in prior art in the present invention is carrier as carrier or treated ion exchange resin, make, with its immobilized lipase, there is high activity and stability unexpectedly, and soap is not produced in catalyzed transesterification process, simple to operate, greatly reduce the cost of fats and oils processing.
As used herein, term " macroporous adsorbent resin " refers to that a class does not contain cation exchange groups and has the preparation of macroporous structure, it has good macroreticular structure and larger specific surface area, can by physical adsorption adsorb organic compound selectively from solution.Term " polystyrene type macroporous adsorbent resin " refers to polystyrene to be the macroporous adsorbent resin of skeleton.The various hypotypes that this term comprises polystyrene type macroporous adsorbent resin should be understood, the such as resin of polystyrene-divinylbenzene skeleton.
The polystyrene type macroporous adsorbent resin that can be used in the present invention can include, but is not limited to: nonpolar D101, HPD100A, AmberliteXAD-4, HPD100, HPD300, HPD700, X-5, XAD-4 or HP20; D201, LSA-10, AmberliteXAD7HP etc. of polarity.Preferably adopt nonpolar polystyrene type macroporous adsorbent resin in the present invention, more preferably D101, HPD100A, X-5.
As used herein, term " ion exchange resin " refers to functional group's (having the active group of exchange ion), has reticulated structure, insoluble macromolecular compound, is commonly used for ion-exchange chromatography media.
Ion exchange resin used in the present invention includes, but is not limited to: anionite-exchange resin, as D301, Styrene-DVB201 × 2 etc.; Zeo-karb, as D113, D001, AmbetliteIRC50 etc.Preferably adopt anionite-exchange resin in the present invention.
Carrier of the present invention also can be modification based on above or other resin or modified resin, such as, by diazotization or amination modified resin or adopt other chemical modification means through the resin of functionalization.Can see, for example Xu Jingliang etc., amino functional carrier immobilized enzyme progress 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 in the present invention and ion exchange resin except the specific product limited by trade(brand)name above, also comprise any there is other trade(brand)name commercial resins product or by preparing voluntarily or processing the resin obtained, as long as they have and the same or analogous structure of described specific product and absorption or switching performance.
These carriers (especially polystyrene macroporous resin) used in the present invention are not used carrier in existing commercialization immobilized enzyme, 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 in deionized water and (also can uses distilled water, distilled water) middle immersion impurity elimination, then suitable solvent soaking 1 ~ 24h is used, remove organic impurity and other insolubles, use deionized water (or distilled water, distilled water) to wash away solvent again, be placed in deionized water (or distilled water, distilled water) for subsequent use.Wherein, solvent for use can be: ethanol, and preferred concentration range is the ethanol of 100%-50% (v/v); Or the organic solvent such as normal hexane, acetone, sherwood oil.The addition scope of described solvent is 0.5-10 times of resin volume, and preferred 1-5 doubly.
Ion exchange resin: with alkali lye (such as NaOH, KOH or ammoniacal liquor) and acid solution (such as HCl, H of lower concentration (0.05mol/L-1mol/L)
2sO
4or H
3pO
4) (treatment process can be such as (concentration of acid solution and alkali lye is 0.05mol/L-1mol/L) alternate treatment: first use dipping by lye resin 1 ~ 3h (preferred 2h), then neutrality is washed till, again by acid soak or after rinsing 1 ~ 3h (preferred 2h), be washed till neutrality, repeat this process 1-5 time) resin 1-5 time, last process only uses alkali lye, each treatment time is 1-24 hour, with water (preferred deionized water after being disposed, distilled water or distilled water) be washed till water in neutral, be immersed in water (preferred deionized water again, distilled water or distilled water) in save backup.Remain alkalescence through the pH of the ion exchange resin inside of above-mentioned process, be more conducive to enzyme and play its activity.
2, enzyme immobilization
Take a certain amount of carrier processed in container, put into the TL enzyme liquid of certain volume, in 15 ~ 35 DEG C (preferably 15 ~ 25 DEG C, more preferably room temperature) shaking bath (can adopt gas bath shaking table, mechanical stirrer and magnetic stirring apparatus, the form of vortex stirrer) in 150rpm (10-200rpm) fix.After fixedly completing, take out the resin being adsorbed with enzyme and suck residual liquid.
The source of lipase can for thermophilic hyphomycete is fermented under proper culture conditions and obtain, also can be pass through genetically engineered, the modern biology means such as protein engineering are transformed and obtain, and comprise the enzyme of chemically modified, the enzyme that the mutant of protein engineering or genetic engineering bacterium produce.The source of lipase can also be commercially available, as the LipozymeTL100L of Novozymes Company of Denmark.
Can without the need to changing the pre-treatment of its original composition or environment through any separation, purifying etc. for the enzyme liquid in immobilization process.Also can dilute enzyme liquid or the process such as pH regulator, to be suitable for reaction.
For including, but is not limited in TL enzyme liquid of the present invention:
1. by cultivating thermophilic hyphomycete (ThermomycesLanuginosus) fermented liquid that obtains (for example, see document CharlottePinholt, MathiasFano, CharlotteWiberg, InfluenceofglycosylationontheadsorptionofThermomyceslanu ginosuslipasetohydrophobicandhydrophilicsurfaces, EuropeanJournalofPharmaceuticalSciences40 (2010): 273-281);
2. obtain lipase gene by cultivating from thermophilic hyphomycete, the lipase enzyme liquid of acquisition is produced by fermentation (for example, see the same document CharlottePinholt with gene-modified aspergillus oryzae, MathiasFano, CharlotteWiberg, EuropeanJournalofPharmaceuticalSciences40 (2010): 273-281).
3. commercially available commodity TL liquid aliphatic enzyme, the LipozymeTL100L of such as Novozymes Company.
4. by adding acid-alkali accommodation TL enzyme liquid to suitable pH value and/or add water (as distilled water, deionized water or distilled water) and be adjusted to the enzyme liquid of suitable protein concentration (for example, see document DanielOtzen in the solution of lipase liquid as above or enzyme powder, DifferentialadsorptionofvariantsoftheThermomyceslanugino suslipaseonahydrophobicsurfacesuggestsaroleforlocalflexi bility, biointerfaces200864:223-228).
Also a certain amount of zymoprotein linking agent (material of crosslinked role of apoenzyme can be played) can be added in enzyme liquid in the present invention, preferred described zymoprotein linking agent has hydroxyl or amino, such as glutaraldehyde solution, polymine, PPI, polyvinylamine, gelatin or spermine etc.The consumption of described zymoprotein linking agent is add 0.0 ~ 10% (v/v) in every milliliter of enzyme liquid.The consumption of such as glutaraldehyde is 1% glutaraldehyde solution adding 0.01 ~ 0.1ml in every milliliter of enzyme liquid, also can adopt other concentration glutaraldehyde solution of equal proportion.
Weight (the g)/volume (ml) of the resin carrier processed and TL enzyme liquid is than can be: 25: 1 ~ 1: 25, and preferably 10: 1 ~ 1: 10, more preferably 1: 1 (can according to circumstances suitably resize ratio).
Fixing mode, can adopt shaking bath or gas bath shaking table, the usual manner such as stirrer or magnetic stirring apparatus.
Fixing period, (such as 5min ~ 1h) supernatant survey residual protein content can be got to judge the absorption/combination degree of zymoprotein at regular intervals.The mensuration of residual protein content can adopt the common protein quantitative detecting methods such as Coomassie Brilliant Blue, 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 skill in the art can adjust fixation degree according to concrete needs (as quality control requirement etc.) and control.
Immobilized enzyme method of the present invention without the need to carrying out complicated pre-treatment (as separation, purifying etc.) to enzyme, and unexpectedly achieves the generation of significantly reduction soap in immobilized enzyme use procedure, does not even produce the technique effect of soap.
3, dry
After immobilization step has been entered, by moisture unnecessary in drying step removal system, dry mode can be selected from: the conventional drying methods such as lyophilize, vacuum-drying, fluidised bed drying and drying at room temperature.
Such as, lyophilize can in the freeze drier of LABCNCO (manufacturer: Labcnco company of the U.S., model: freezone) on carry out, parameter can be set to (be each step parameter below, these steps form a temperature-fall period):
-20 DEG C of rate of temperature fall 1.5 DEG C/min, 2h (0.5-5h),
-10 DEG C of rate of temperature fall 1.5 DEG C/min, 6h (3-10h)
5 DEG C of rate of temperature fall 1.01 DEG C/min, 15h, (10-24h)
20 DEG C of rate of temperature fall 1.16 DEG C/min, 20h (10-24h)
Vacuum-drying can be carried out in such as Binder vacuum drying oven (manufacturer: guest's moral Asia-Pacific (Hong Kong) company limited model: VD23), and optimum configurations is under vacuum tightness is 6mbar, 40 DEG C of dry 48h (24-60h).
Fluidised bed drying can carry out in fluidized-bed (the special company of such as Gera, Germany, model: midiglatt), and optimum configurations is inlet temperature 45 ~ 55 DEG C (preferably 50 DEG C), and time of drying is 0.5 ~ 5h (preferred 1.5h).
Drying at room temperature can be place sample being placed on air seasoning and places seasoning in 3 ~ 5 days.
Those of ordinary skill in the art can select conventional drying methods with condition as required.
4, other optional step
According to concrete needs, also following one or more optional steps additionally can be had in method of the present invention, such as (but being not limited to):
Enzyme liquid to pass through and filtered small molecules, the impact of living on enzyme with the small molecules eliminated in enzyme liquid by 1.Filter method can be such as: on ultra-filtration membrane device, carry out ultrafiltration (manufacturer: Millipore Corp. of the U.S., model: small-sized), then phosphoric acid buffer (such as pH is the phosphoric acid buffer of the 0.1mol/L of 5) is added, gained enzyme liquid and original enzyme liquid system pH are consistent, and adjust to suitable zymoprotein concentration.
2 can add the stability that the material such as dextrin, albumin and/or trehalose strengthens enzymes in immobilization step, and their respective preferred additions are: dextrin 1 ~ 20% (W/V); Albumin 0.05 ~ 5% (W/V); Trehalose 0.1-10% (W/V).One or more above-mentioned substances can be added, to obtain required stabilising effect.
3 pairs of carriers carry out modification, connect active group by chemical means.Such as, can with reference to Xu Jingliang etc., amino functional carrier immobilized enzyme progress chemical industry progress 201029 (3): 494-496.
5, the preferred embodiment of the present invention
An exemplary preferred embodiment of the present invention is as follows:
The process of step one, carrier
Macroporous adsorbent resin carrier is placed in deionized water and soaks impurity elimination, then spend the night by alcohol immersion and remove organic impurity and other insolubless, with deionized water rinsing to without alcohol taste, then soak and save backup in deionized water.
Or, by ion exchange resin washed with de-ionized water, by 5%NaOH and 5%HCl alternate treatment 2 ~ 5 times, and then with 5%NaOH process, each process two hours, then with deionized water process to water in neutral, soak for subsequent use in deionized water.
Step 2, enzyme immobilization
Take the resin carrier of 40g process in 250ml triangular flask, put into 40mlTL enzyme liquid, in 25 DEG C of shaking baths, 150rpm fixes, and period gets supernatant at regular intervals and surveys residual protein content to judge the degree of absorption of zymoprotein, and then taking-up is put into culture dish and sucked residual liquid.
Step 3, drying
Moisture unnecessary in system of will going out after immobilization step has been carried out is namely dry, and drying can adopt mode conventional in this area, such as lyophilize, vacuum-drying and drying at room temperature.
Should be understood that above-mentioned preferred implementation is exemplary, those of ordinary skill in the art without departing from the spirit and scope of the present invention, can change each Step By Condition and optimize based on the general knowledge of the record in the application and this area.
6, the application of immobilized lipase of the present invention
Can immobilized lipase of the present invention be directly used in suitability for industrialized production, also can be made into wrapped product so that carry out storing, transport, sell and using further.
During packaging, preferably under the condition of aseptic dry low temperature in the filling container in cleaning.Storage condition is preferably cryodrying condition storage, and storing optimum temperuture is 4 ~ 20 DEG C.Preferably transport at the temperature of 4 DEG C ~ 25 DEG C.
Immobilized lipase of the present invention or its wrapped product can be used for that catalysis grease is hydrolyzed, transesterify, esterification or catalysis acidolysis, alcoholysis etc., have huge application food, papermaking, leather, washing composition, pharmacy etc. are industrial.Such as, immobilization TL lipase of the present invention can be used for, in hydrolysis, the modification of Witepsol W-S 55 and the production of Tegin 55G and DAG, being particularly preferred for catalyzed transesterification.
advantage of the present invention:
1. adopt in the present invention and provide a kind of novel method preparing immobilization TL enzyme, have employed in the method at the uncommon macroporous adsorbent resin in immobilization field or treated ion exchange resin as fixation support, and without the need to carrying out complicated pre-treatment to lipase, thus enormously simplify production stage, reduce production cost.
2. solve the problem that commodity TL enzyme produces soap in grease reaction.
3. immobilization TL enzyme of the present invention can reuse, thus reduces production cost, improves rate of utilization.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, the usual conveniently condition of condition as described in " Biochemistry and Molecular Biology experiment textbook " (Liang Songping edits Higher Education Publishing House), or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
TL enzyme liquid is purchased from Novozymes Company of Denmark, and commodity are called LipozymeTL100L (pH5.0), without (NaOH of 0.05mol/L ~ lmol/L) after any process or adjustment pH to 8.0 in embodiments of the invention.
Embodiment 1. adopts macroporous adsorbent resin to prepare immobilized enzyme
By macroporous adsorbent resin D101, HPD100A or X-5, (D101 and HPD100A is purchased from Cangzhou, Hebei Bao En Chemical Co., Ltd.; X-5 is purchased from Shanghai Hua Zhen resin company limited) be placed in deionized water respectively and soak about 12h impurity elimination; then spend the night by 95% alcohol immersion and remove organic impurity and other insolubles; with deionized water rinsing to without alcohol taste, be placed in deionized water for subsequent use.
Immobilized enzyme 1-19 is prepared according to following formula and step:
1, the D101 resin 40g handled well is taken in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5,150rpm vibration 2h in 25 DEG C of shaking tables, then takes out resin and puts into dry culture dish, put into freeze drier and carry out drying, concrete freeze-drying optimum configurations is:
-20 DEG C of rate of temperature fall 1.5 DEG C/min, 2h,
-10 DEG C of rate of temperature fall 1.5 DEG C/min, 6h
5 DEG C of rate of temperature fall 1.01 DEG C/min, 15h,
20 DEG C of rate of temperature fall 1.16 DEG C/min, 20h
Immobilized enzyme 1 is obtained after drying.
2, the D101 resin 40g handled well is taken in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5,150rpm vibration 2h in 25 DEG C of shaking tables, then resin is taken out and put into dry culture dish, put into Vacuumdrier to carry out drying (vacuum tightness is under 6mbar, 40 DEG C of dry 48h), obtain immobilized enzyme 2 after drying.
3, the D101 resin 40g handled well is taken in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5,150rpm vibration 2h in 25 DEG C of shaking tables, then takes out resin and puts into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 3.
4, the D101 resin 40g handled well is taken in 250ml triangular flask, add TL enzyme liquid and the 10ml distilled water of 30mlpH5,150rpm vibration 2h in 25 DEG C of shaking tables, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 4.
5, the D101 resin 40g handled well is taken in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8,150rpm vibration 2h in 25 DEG C of shaking tables, then takes out resin and puts into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 5.
6, the D101 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with the glutaraldehyde solution of 0.8ml1%, 150rpm vibration 2h in 25 DEG C of shaking tables, resin takes out puts into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 6.
7, the D101 resin 40g handled well is taken out in 250ml triangular flask, add TL enzyme liquid and the 10ml distilled water of 30mlpH5, with the glutaraldehyde solution of 0.8ml1%, 150rpm vibration 2h in 25 DEG C of shaking tables, resin takes out puts into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 7.
8, the D101 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 8.
9 take out the D101 resin 40g that handles well in 250ml triangular flask, add TL enzyme liquid and the 10ml distilled water of 30mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 9.
10, the D101 resin 40g handled well is taken out in 250ml triangular flask, add TL enzyme liquid and the 10ml distilled water of 30mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 10.
11, the D101 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 11.
12, the HPD100A resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 0.8ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 12.
13, the HPD100A resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 13.
14, the HPD100A resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 0.8ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 14.
15, the HPD100A resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 15.
16, the X-5 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 0.8ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 16.
17, the X-5 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 17.
18, the X-5 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 0.8ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 18.
19, the X-5 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, be placed on the good place of room ventilated and carry out drying, after drying, obtain immobilized enzyme 19.
More than process the mensuration of supernatant liquor being carried out to protein content after immobilization process terminates, measuring method is Coomassie Brilliant Blue, and its residual protein content is all at below 0.1mol/L (as shown in table 1).Enzyme immobilization efficiency calculation formula is as follows:
Table 1. enzyme immobilization efficiency
| Original enzyme liquid protein concentration | Supernatant protein concentration after absorption | Enzyme immobilization efficiency |
| 5.5mol/L | ＜0.1mol/L | ＞98％ |
The preparation parameter of immobilized enzyme 1 ~ 19 is summarized in table 2.
The immobilized enzyme that table 2. adopts macroporous resin to prepare
The preparation of the embodiment of enzyme and carrier different ratios is summarized in table 3
Table 3. adopts different enzyme and the immobilized enzyme prepared by carrier ratio (V/W)
Embodiment 2. adopts ion exchange resin to prepare immobilized enzyme
Styrene-DVB201 × 2 resin (purchased from Tianjin Nankai Hecheng S&T Co., Ltd.) is first used washed with de-ionized water, then 5%NaOH and 5%HCl alternate immersion is used 2 times, and then soak 1 time with 5%NaOH, each process two hours, then be washed till water in neutral with deionized water, soak for subsequent use in deionized water.
Immobilized enzyme 20-24 is prepared according to following formula and step:
1, the Styrene-DVB201 × 2 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 0.8ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 20.
2, the Styrene-DVB201 × 2 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 21.
3, the Styrene-DVB201 × 2 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 0.8ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 22.
4, the Styrene-DVB201 × 2 resin 40g handled well is taken out in 250ml triangular flask, add the TL enzyme liquid of 40mlpH8, with glutaraldehyde solution 150rpm vibration 2h in 25 DEG C of shaking tables of 1.6ml1%, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 23.
5 take out the Styrene-DVB201 × 2 resin 40g that handles well in 250ml triangular flask, add the TL enzyme liquid of 40mlpH5,150rpm vibration 2h in 25 DEG C of shaking tables, then resin is taken out and put into dry culture dish, put into draughty place and carry out natural air drying, after drying, obtain immobilized enzyme 24.
More than process through detecting Supernatant protein content all at below 0.2mg/ml after fixing end, enzyme immobilization efficiency all more than 96% (see table 4) as calculated.
Table 4. enzyme immobilization efficiency
| Original enzyme liquid protein concentration | Supernatant protein concentration after absorption | Enzyme immobilization efficiency |
| 5.5mol/L | ＜0.2mol/L | ＞96％ |
The preparation parameter of immobilized enzyme 20-23 is summarized in table 5.
The immobilized enzyme that table 5. adopts ion exchange resin to prepare
The ester exchange activity test of embodiment 3. immobilized enzyme
measurement and Computation method
With purified soyabean oil (purchased from (Shanghai) foodstuffs industry company limited in good, lot number 2B011121) and deep hydrogenation soybean oil (purchased from (Shanghai) special oil company limited in good, lot number 3H011104) mixture be substrate (w/w=73: 27), with immobilized enzyme and substrate mass ratio for 1: 20, at 70 DEG C of reaction 30min, reaction takes out product after finishing, and surveys its solid fats content of 40 DEG C (i.e. SFC content).
The SFC content assaying method of 40 DEG C is: the solid fat pipe that sample is housed is put into 100 DEG C of baking oven 15min respectively, 60 DEG C of water-bath 5min, 0 DEG C of water-bath 60min, 40 DEG C of water-bath 30min, then use nuclear magnetic resonance analyser (manufacturer: German Brooker spectral instrument company model: mq20) to survey its solid fat content.The calculation formula of ester exchange activity is:
Ester exchange activity=(SFC
blank-SFC
sample)/30 × 1260
Wherein, SFC
blankrefer to not through substrate grease SFC value at 40 DEG C of enzyme process transesterification reaction, SFC
samplerefer to the SFC value of enzyme reaction product sample at 40 DEG C.
Experimental result is the mean value of 3 parallel laboratory tests.
experimental result and discussion
Table 6 summarizes the ester exchange activity measurement result of immobilized enzyme 1-24 of the present invention.For the purpose of clear comparison, table 7 ~ 12 respectively illustrate and adopt glutaraldehyde addition under different drying mode, different TL enzyme pH value, condition of different pH and different resins on the impact of immobilized enzyme ester exchange activity.
The ester exchange activity of table 6. immobilized enzyme 1-23
The ester exchange activity of the D101 immobilization TL enzyme that table 7. adopts different drying mode to prepare
| Enzyme number | Resin | Drying mode | Ester exchange activity |
| 1 | D101 | Lyophilize | 406 |
| 2 | D101 | Vacuum-drying | 411 |
| 3 | D101 | Drying at room temperature | 557 |
The ester exchange activity data of D101 immobilization TL enzyme prepared by the different drying mode of the employing enumerated in table 7 show, adopt different drying modes to the ester exchange activity of immobilized enzyme and do not make significant difference.Therefore, as required and working condition, various different drying mode can be adopted in the preparation of immobilized enzyme.
The ester exchange activity of the immobilization TL enzyme that table 8. adopts the TL enzyme of different pH value to prepare
It is under the condition of 5 that table 8 result is presented at pH, adopts the fixing TL enzyme activity of macroporous adsorbent resin D101 to compare to fix TL enzyme activity under pH8 condition to want high.D101 adds glutaraldehyde and HPD100A, and to add glutaraldehyde these two groups also like this.And adopt and fixingly under X-5 alkaline condition be slightly better than acidic conditions (there was no significant difference).Being fixed with under acidic conditions may be the reason avoiding producing soap in Exchange Ester Process, can select suitable pH value in actual fabrication process according to the character of different carriers.
Under table 9.pH5 condition, the ester exchange activity of D101, HPD100A or X-5 immobilization TL enzyme adopting different glutaraldehyde consumption to prepare
| Enzyme number | Resin (40g) | The consumption (ml) of 1% glutaraldehyde | Ester exchange activity |
| 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 D101 and HPD100A two kinds of resins, is that enzyme when the enzyme work of immobilized enzyme is 1.6ml higher than 1% glutaraldehyde addition when to add 1% glutaraldehyde amount under the condition of 5 be 0.8ml is lived at pH, but there was no significant difference.And concerning X-5 resin, when 1% glutaraldehyde addition is 1.6ml, immobilized enzyme is lived higher than the enzyme that 1% glutaraldehyde addition is 0.8ml, but also there was no significant difference.
It is reach high enzyme work to add suitable glutaraldehyde amount according to the character of carrier that this result illustrates for different resins, but also can select not add glutaraldehyde.
Under table 10. condition of different pH, the ester exchange activity of HPD100A or the X-5 immobilization TL enzyme adopting different glutaraldehyde consumption to prepare
Table 10 result shows when other condition is identical, and TL enzyme fixing under pH5 is higher than TL enzyme activity fixing under pH8 condition.Be under the condition of 8 at pH, the enzyme that the glutaraldehyde that different resins adds different amount measures is lived also different, waits until in follow-up successive reaction and investigates (see embodiment 4) further.
The ester exchange activity of enzyme and carrier different ratios immobilization TL enzyme under 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 lived to enzyme and is directly proportional, live to obtain higher enzyme, fixing except the embodiment of TL enzyme except D101, other embodiments all select the ratio of enzyme and carrier to be 1: 1 (V/W).
The ester exchange activity of the fixed on ion exchange resin TL enzyme that table 12. adopts different pH and glutaraldehyde consumption to prepare
Table 12 result shows: ion exchange resin pH be the immobilized enzyme enzyme work that obtains under the condition of 5 identical higher than other condition time pH8 condition under the immobilized enzyme enzyme that obtains live.Enzyme work when the add-on of glutaraldehyde is 0.8ml is lived higher than the enzyme do not added when glutaraldehyde or glutaraldehyde add-on are 1.6ml, illustrates that the glutaraldehyde for adding 0.8ml1% the resin of Styrene-DVB201 × 2 is suitable.
By relatively can knowing of experimental result above in the present embodiment: higher enzyme can being obtained in the glutaraldehyde not changing the different amount of interpolation on pH basis and live, can the vigor improving enzyme under its original acidic conditions by adding appropriate glutaraldehyde not changed in actual applications.
Embodiment 4. adopts immobilized enzyme to take turns the saponified matter content of reacted product and solid fat content measuring through more
testing method
With ST (palm stearin, Southseas Specialty Fats Industrial (Shanghai) Co., Ltd.) 65% and PKO (palm-kernel oil, Southseas Specialty Fats Industrial (Shanghai) Co., Ltd.) the proportions reaction substrate of 35% (W/W), 40g substrate is added in 250ml triangular flask, 8g immobilized enzyme, in 70 DEG C of water-baths, 200rpm reacts 30min, then product is taken out, immobilized enzyme stays in the reactor, continue to add 40g substrate to react next time, carry out 5 times altogether, the saponified matter content of detection reaction product and 40 DEG C of solid fat content (SFC).
experimental result and discussion
Following table 13 ~ 14 respectively illustrates and adopts different drying mode, different TL enzyme pH value, glutaraldehyde addition under condition of different pH and different resins on the impact of immobilized enzyme ester exchange activity.
Immobilized enzyme with prior art: Novi's letter TLIM immobilized enzyme is contrast.This immobilized enzyme is NOVOINDUSTRI Company, with the lipase of thermophilic hyphomycete, prepares according to method described in WO89/06278.According to comparing, method of the present invention and Novi believe that the preparation of TLIM immobilized enzyme has following difference:
1) carrier is different: the carrier that WO89/06278 adopts is the carrier of weakly alkaline phenolic aldehyde skeleton or acrylic type skeleton, the polystyrene type carrier adopted in the present invention.
2) vehicle treated method is different: WO89/06278 does not mention the pre-treatment of carrier in the patent, and carrier of the present invention is through pre-treatment.
3) process for fixation is different: the immobilization time is 8h to WO89/06278 in instances, has water-washing step, and adopt vacuum-drying after immobilization, and the set time of the present invention for being only 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 adopts is the solution be dissolved into enzyme powder, and directly adopts the enzyme liquid buied in the present invention.
The different immobilized enzyme of table 13. is at each saponified matter content of taking turns the rear product separately of reaction
By the digital proof in table 13, adopt the immobilized enzyme prepared of the inventive method product saponified matter content produced aborning significantly lower than the immobilized enzyme adopting art methods to prepare, do not produce even soap (namely product saponified matter content is 0).
The different immobilized enzyme of table 14. is each solid fat content (SFC) of taking turns reaction after product
The SFC of substrate grease is 19.61
Data in table 14 also further demonstrate, and adopt the immobilized enzyme prepared of the inventive method to have higher activity aborning, and vigor and existing commercial enzyme comparable, be better than even existing commercial enzyme.Take turns the SFC of various immobilized enzyme catalysis product in reaction all far below substrate SFC 5, immobilized enzyme effective catalysis transesterification reaction is described.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. prepare a method for immobilization TL lipase, described method comprises step:
A) carry out pre-treatment to carrier, described carrier is selected from the nonpolar macroporous adsorption resin that skeleton is polystyrene;
B) provide TL lipase, condition is, except optional adds except zymoprotein linking agent, optional dilution and optional pH regulator process, this TL lipase is not carried out to the pre-treatment of its original composition of any change or environment;
C) by step a) in the carrier through pre-treatment of gained and step b) in the TL lipase that provides contact, make described TL lipase be fixed on the described carrier through pre-treatment;
D) drying step c) in the carrier being fixed with TL lipase of gained, to obtain described immobilization TL lipase.
2. the method for claim 1, is characterized in that,
Described nonpolar macroporous adsorption resin is selected from: D101, HPD100A, AmberliteXAD-4, HPD100, HPD300, HPD700, X-5, XAD-4 or HP20.
3. the method for claim 1, is characterized in that,
The pre-treatment of described macroporous adsorbent resin is carried out as follows: carrier is placed in water and soaks impurity elimination, then soaked in solvent 1 ~ 24h is to remove organic impurity and insolubles, wash described solvent again with water, wherein said solvent is: ethanol, normal hexane, acetone or sherwood oil.
4. the method for claim 1, is characterized in that, step b) in the TL lipase that provides be selected from: thermophilic hyphomycete (
thermomycesLanuginosus) tunning; The TL lipase obtained by genetically engineered or protein engineering transformation; Or commercially available TL lipase.
5. method as claimed in claim 4, is characterized in that, described TL lipase without any pre-treatment for step c) in.
6. the method for claim 1, is characterized in that, step b) in, in described TL lipase, add zymoprotein linking agent.
7. method as claimed in claim 6, it is characterized in that, described zymoprotein linking agent is selected from: glutaraldehyde solution, polymine, PPI, polyvinylamine, gelatin or spermine.
8. the method for claim 1, is characterized in that, step c) in, described treated carrier and TL lipase in the weight/volume of g/ml for 25:1 ~ 1:25.
9. immobilization TL lipase or its wrapped product, is characterized in that, described immobilization TL lipase is prepared by the method according to any one of claim 1-8.
10. carry out a method for transesterify, esterification, acidolysis or alcoholysis reaction, described method comprises and uses immobilization TL lipase described in claim 9 as catalyzer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110170701.6A CN102839166B (en) | 2011-06-23 | 2011-06-23 | Tl immobilized enzyme and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110170701.6A CN102839166B (en) | 2011-06-23 | 2011-06-23 | Tl immobilized enzyme and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102839166A CN102839166A (en) | 2012-12-26 |
| CN102839166B true CN102839166B (en) | 2015-11-25 |
Family
ID=47366885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110170701.6A Active CN102839166B (en) | 2011-06-23 | 2011-06-23 | Tl immobilized enzyme and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102839166B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468668B (en) * | 2012-06-06 | 2017-10-24 | 益海(佳木斯)生物质能发电有限公司 | A kind of method of fixed fat enzyme and application thereof |
| CN104130860B (en) * | 2013-05-03 | 2020-03-31 | 丰益(上海)生物技术研发中心有限公司 | Method for enriching long-chain polyunsaturated fatty acid by utilizing immobilized thermomyces lanuginosus lipase |
| CN104694526B (en) * | 2013-12-06 | 2019-01-08 | 丰益(上海)生物技术研发中心有限公司 | Catalytic esterification and the Sn-1,3 selectivity immobilized lipase of transesterification and preparation method thereof |
| CN105039298A (en) * | 2015-07-02 | 2015-11-11 | 曹庸 | Preparation method of immobilized tannase |
| CN105368886A (en) * | 2015-12-11 | 2016-03-02 | 江苏万年长药业有限公司 | Process of using resin-immobilized halohydrin dehalogenase to catalytically synthesize (R)-4-cyan-3-hydroxy ethyl butyrate |
| CN105385675A (en) * | 2015-12-31 | 2016-03-09 | 厦门大学 | Immobilization method of candida antarctica lipase B |
| CN106434616A (en) * | 2016-12-19 | 2017-02-22 | 山东思科新材料有限公司 | Preparation and application method of immobilized esterifying enzyme for baijiu |
| CN106701729B (en) * | 2017-02-15 | 2019-12-17 | 南京工业大学 | Immobilized enzyme taking polypeptide-modified amino resin as carrier and preparation method thereof |
| CN107012136A (en) * | 2017-06-12 | 2017-08-04 | 浙江工业大学 | A kind of method of immobilization Thermomyces lanuginosus lipase |
| 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 |
| CN110938619A (en) * | 2019-12-19 | 2020-03-31 | 北京农学院 | Immobilized enzyme transformed radix scutellariae and application thereof in repairing acetaminophen damage |
| CN114480360B (en) * | 2021-12-14 | 2024-03-12 | 山东省农业科学院 | Method for preparing diglyceride by immobilized Sn-2 lipase |
| CN114657218A (en) * | 2022-03-25 | 2022-06-24 | 陕西海斯夫生物工程有限公司 | Preparation method and application of composite catalyst for preparing biodiesel |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101086000A (en) * | 2007-04-13 | 2007-12-12 | 北京化工大学 | Method for synthesizing EGCG fatty acid ester catalyzed by immobilized enzyme |
| CN101712951A (en) * | 2009-10-15 | 2010-05-26 | 福建师范大学 | Immobilization method-based lipase fixing method and application of lipase in ferulic acid esterification |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009010561A1 (en) * | 2007-07-18 | 2009-01-22 | Novozymes A/S | Immobilization of enzymes |
-
2011
- 2011-06-23 CN CN201110170701.6A patent/CN102839166B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101086000A (en) * | 2007-04-13 | 2007-12-12 | 北京化工大学 | Method for synthesizing EGCG fatty acid ester catalyzed by immobilized enzyme |
| CN101712951A (en) * | 2009-10-15 | 2010-05-26 | 福建师范大学 | Immobilization method-based lipase fixing method and application of lipase in ferulic acid esterification |
Non-Patent Citations (1)
| Title |
|---|
| 一种选择性拆分布洛芬对映体的固定化脂肪酶;唐良华等;《过程工程学报》;20080630;第8卷(第3期);567-571 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102839166A (en) | 2012-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102839166B (en) | Tl immobilized enzyme and application thereof | |
| CN104694526B (en) | Catalytic esterification and the Sn-1,3 selectivity immobilized lipase of transesterification and preparation method thereof | |
| CN110331139B (en) | Immobilization method of candida antarctica lipase B | |
| CN107828756A (en) | A kind of preparation method of the selectivity immobilized lipases of Sn 1,3 | |
| CN110229807B (en) | Preparation method of organophosphorus immobilized enzyme catalyst, organophosphorus immobilized enzyme catalyst and application thereof | |
| JP2001037475A (en) | Production of immobilized lipase, enzyme catalytic or enantio-selective conversion of such lipase and substrate, production of optically active compound and use of immobilized lipase | |
| CN103468668B (en) | A kind of method of fixed fat enzyme and application thereof | |
| CN103224926A (en) | Method of preparing immobilized lipase | |
| CN108611380A (en) | A kind of method of selectivity synthesis 2,5- dihydroxymethyl furans | |
| Weng et al. | Immobilization of recombinant endoglucanase (CelA) from Clostridium thermocellum on modified regenerated cellulose membrane | |
| CN104293764A (en) | Phosphatidase A1 immobilization method | |
| CN101671663B (en) | Preparation method of corncob fixed lipase and product thereof | |
| CN109456959A (en) | A kind of method of modified magnetic graphene oxide immobilized lipase | |
| CN112301025A (en) | Modification method and application of immobilized enzyme carrier | |
| CN102154256A (en) | Carrier-free immobilized lipase and preparation method thereof | |
| CN101914515B (en) | Immobilized laccase, preparation method and application thereof | |
| CN105483111A (en) | Immobilization lipase and preparation method thereof | |
| KR20200102361A (en) | Immobilized lipase for diisononyl adipate synthesis, preparation method of thereof, and preparation method of diisononyl adipate using thereof | |
| CN104928279A (en) | Carrier-free immobilized rhizopus oryzae lipase as well as preparation method thereof and application thereof in producing biodiesel | |
| CN107815445B (en) | Dry powder type immobilized lipase and production process thereof | |
| CN110656102A (en) | Preparation method of beta-glucosidase immobilized enzyme | |
| CN106867989B (en) | Immobilized enzyme and preparation method thereof | |
| CN103756991B (en) | Immobilization cutinase, preparation method and the application of phthalate in removing water body thereof | |
| CN105462952B (en) | A kind of method of fixed fat enzyme B in fermented supernatant fluid from antarctic candida | |
| KR102468425B1 (en) | Synthesis method of magnetic rice straw for lipase immobilization and esterification reaction using lipase immobilized on magnetic rice straw |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |