CN107828756A - A kind of preparation method of the selectivity immobilized lipases of Sn 1,3 - Google Patents
A kind of preparation method of the selectivity immobilized lipases of Sn 1,3 Download PDFInfo
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- CN107828756A CN107828756A CN201710948748.8A CN201710948748A CN107828756A CN 107828756 A CN107828756 A CN 107828756A CN 201710948748 A CN201710948748 A CN 201710948748A CN 107828756 A CN107828756 A CN 107828756A
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- lipase
- resin
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- enzyme
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 110
- 102000004882 Lipase Human genes 0.000 title claims abstract description 110
- 239000004367 Lipase Substances 0.000 title claims abstract description 110
- 235000019421 lipase Nutrition 0.000 title claims abstract description 110
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 72
- 108090000790 Enzymes Proteins 0.000 claims abstract description 72
- 229920005989 resin Polymers 0.000 claims abstract description 60
- 239000011347 resin Substances 0.000 claims abstract description 60
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000001179 sorption measurement Methods 0.000 claims abstract description 15
- 239000007853 buffer solution Substances 0.000 claims abstract description 10
- 230000002745 absorbent Effects 0.000 claims abstract description 7
- 239000002250 absorbent Substances 0.000 claims abstract description 7
- 239000012535 impurity Substances 0.000 claims abstract description 5
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 27
- 229910021645 metal ion Inorganic materials 0.000 claims description 14
- 238000007654 immersion Methods 0.000 claims description 10
- 238000012545 processing Methods 0.000 claims description 10
- 230000000536 complexating effect Effects 0.000 claims description 9
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 claims description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 229960002319 barbital Drugs 0.000 claims description 3
- 229910021538 borax Inorganic materials 0.000 claims description 3
- 239000004328 sodium tetraborate Substances 0.000 claims description 3
- OTNVGWMVOULBFZ-UHFFFAOYSA-N sodium;hydrochloride Chemical compound [Na].Cl OTNVGWMVOULBFZ-UHFFFAOYSA-N 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 229910001626 barium chloride Inorganic materials 0.000 claims description 2
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 claims description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Inorganic materials [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 claims description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- 238000011010 flushing procedure Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 26
- 230000032050 esterification Effects 0.000 abstract description 9
- 238000005886 esterification reaction Methods 0.000 abstract description 9
- 238000011084 recovery Methods 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 230000003197 catalytic effect Effects 0.000 abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 20
- 239000008363 phosphate buffer Substances 0.000 description 15
- 230000008859 change Effects 0.000 description 14
- 235000019197 fats Nutrition 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000000843 powder Substances 0.000 description 10
- 238000002791 soaking Methods 0.000 description 9
- 238000000967 suction filtration Methods 0.000 description 9
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].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=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 8
- 150000002500 ions Chemical class 0.000 description 7
- 238000005070 sampling Methods 0.000 description 7
- 108010048733 Lipozyme Proteins 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 239000004519 grease Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 240000006439 Aspergillus oryzae Species 0.000 description 2
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 2
- 101710098556 Lipase A Proteins 0.000 description 2
- 101710099648 Lysosomal acid lipase/cholesteryl ester hydrolase Proteins 0.000 description 2
- 102100026001 Lysosomal acid lipase/cholesteryl ester hydrolase Human genes 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- -1 each gellike Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- BUCIWTBCUUHRHZ-UHFFFAOYSA-K potassium;disodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O BUCIWTBCUUHRHZ-UHFFFAOYSA-K 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- MJTSOZOWJWOKAY-UHFFFAOYSA-L P(=O)([O-])([O-])O.[Na+].[Na+].P Chemical compound P(=O)([O-])([O-])O.[Na+].[Na+].P MJTSOZOWJWOKAY-UHFFFAOYSA-L 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000006136 alcoholysis reaction Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000001455 metallic ions Chemical class 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a kind of preparation method of the selectivity immobilized lipases of Sn 1,3, comprise the following steps：S1. pretreatment is carried out to macroreticular resin and goes the removal of impurity；S2. lipase is dissolved in buffer solution, centrifuging and taking supernatant liquor, as enzyme liquid；S3. the enzyme liquid for taking step S1 to obtain, the macroreticular resin , Zhen Oscillating that step S1 has been pre-processed are added, filters, wash, dry, being fixed lipase；Wherein, the macroreticular resin includes nonpolar macroporous adsorption resin A and low pole macroporous absorbent resin B.Immobilised enzymes obtained by this method fixes rate up to 93% ~ 97%, and the enzyme activity rate of recovery is up to 98% ~ 118.9%.Found by zymologic property research, being fixed lipase of the invention has preferable esterification activity and the high selectivitys of Sn 1,3, and catalytic esterification rate has preferable operational stability up to more than 96%, reusable 50 times.
Description
Technical field
The present invention relates to lipase immobilization technical field, more particularly, to a kind of Sn-1 of denomination of invention, 3 selectivitys
The preparation method of immobilized lipase.
Background technology
Lipase is a kind of special Acyl- hydrolase, and during as biocatalyst, ester can be both catalyzed on oil-water interfaces
Hydrolysis（ROOR*+H20→→RCOOH+R*OH）, again can catalytic synthesis, including esterification（RCOOH+R*OH→→
ROOR*+H20）, ester exchange（RCOOR*+R#COOR→→ROOR+R#COOR*）, alcoholysis（RCOOR*+ R#OH→→ROOR#+ R*
OH）And acidolysis（RCOOR*+ R#COOH→→R#COOR*+ RCOOH）, based on this, it is a kind of important industrial enzyme preparation.
Following defect in use easily be present in free-fat enzyme：1. enzyme is expensive, it is used only once, makes
With cost height；2. not dissolved in non-aqueous system, easily assemble in course of reaction, influence enzyme activity；3. easily by extraneous factor（Such as
Temperature, pH value etc.）Change and inactivate.If by lipase immobilization, the stability of enzyme can be not only improved, and is easy to point
From, can be used continuous use, reduce production cost.
Substantial amounts of research shows both at home and abroad, the 1 of triacylglycerol in grease, the reason of the aliphatic acid of 3 and 2 to grease
Change, have larger difference in terms of nutrition and physiological property, wherein optimal with 1,3 bit function.Due to Sn-1,3 positions selectivity
Lipase optionally hydrolyzes 1 and 3 of triacylglycerol, is widely used in production structured lipid, human milk fat replaces
Dai Pin, cacaolike butter and diglyceride etc., therefore obtain cheap and active high enzyme, efficient enzyme immobilization technology and grind
It is the emphasis studied from now on to send out enzyme reactor.
Domestic and international common Sn-1,3 selectivitys fat mainly has three kinds of commercialization fat of Novozyme companies of Denmark at present
Fat enzyme Lipozyme RM IM（Strain sourceRhizomucor.miehei）、Lipozyme TL IM（Strain sourceThermomyces.lanuginosus）、Lipozyme RM IM（Strain sourceCandida.antarctica）, its exist make
With cost it is high the characteristics of；Also there is being fixed of lipase of research and utilization Aspergillusoryzae fermentations the country and applied
In OPO synthesis（Li Yang, the immobilization of 2015, Sn-1,3 specific lipases and its answering in Structure grease OPO synthesis
With）.Retrieve Patents to find, the country is not detected dedicated for Sn-1, the patent of 3 selectivitys fat, presently disclosed
CN201310495368.5, CN201410614900.5, CN201410263491.9, CN201410534346.X etc. are to introduce
The fixation of lipase is carried out with the single carrier such as magnetic microsphere, material modified, natural porous material, each gellike, synthetic resin
Change, but there is also carrier preparation technology is more complicated, the typically expendable problem of carrier.
And the technique study for improving immobilized lipase enzymatic activity rare at present, ZL101736000B- improve immobilization fat
One kind is described in the method for fat enzymatic activity and stability from alcohol, ketone, ester inorganic agent to improve enzymatic activity and stability, still
In immobilized lipase due to using mostly organic solvent, certain safety in utilization be present.How easily in immobilization
The activity that immobilized lipase is improved in journey is also the breach for reducing immobilized lipase cost and improving its practicality.
The content of the invention
In view of this, the present invention is insufficient to overcome at least one described in above-mentioned prior art, there is provided and one kind has Sn-1,
The preparation method of 3 professional immobilized lipases, from Sn-1, the high free-fat enzyme powder A03 of 3 bit esterified activity is carried out
Immobilization, the free enzyme powder access times of solution are few, cost is high, the problems such as being not easy to separate from reaction system, to which its is extensive
Using in the production application with the structured lipid such as diglyceride, OPO.
In order to solve above-mentioned technical problem, the present invention uses following technical proposals：
A kind of Sn-1, the preparation method of 3 selectivity immobilized lipases, comprises the following steps：
S1. pretreatment is carried out to macroreticular resin and goes the removal of impurity；
S2. lipase is dissolved in buffer solution, centrifuging and taking supernatant liquor, as enzyme liquid；
S3. the enzyme liquid for taking step S1 to obtain, the macroreticular resin , Zhen Oscillating that step S1 has been pre-processed are added, filters, wash, dry, obtain
To immobilized lipase.
Wherein, the macroreticular resin includes nonpolar macroporous adsorption resin A and low pole macroporous absorbent resin B.
Further, the nonpolar macroporous adsorption resin include MA-NP6, MI-BN4, MI-300IDA, LX-1000EP,
GDX-104、LX-1000HA、LX-EP120、LX-EP130、LX-201A、SD300、DM11、D354、D730、SIP1100、
In SIP1200 at least with one kind；The low pole macroporous absorbent resin include MI-WP8, GDX-501, D201, D301,
At least one of HPD400, HPD500, Dowex A-1, D401, Diaion CR-10, LSC-100, LSC-200.
Further, the mass ratio of nonpolar macroporous adsorption resin and low pole macroporous absorbent resin in the macroreticular resin
For 1:1~1:2.
Further, the mass ratio of the lipase and macroreticular resin is 1:0.5~1:2, most preferably lipase and macropore tree
The mass ratio of fat is 1:0.5~1:1.
Further, in step S1, macroreticular resin pretreatment includes：The removal of impurity is gone in priority ethanol, acid, alkali immersion, most
After be washed to neutrality, preserved under low temperature stand-by.
Further, in step S2, the buffer solution is Tris-HCl, barbital sodium-HCl, boric acid-borax, lemon
One or more in acid-sodium citrate, phosphate buffer, wherein with phosphate buffer best results.The buffer solution
PH value be 5.0 ~ 9.0.
Further, in step S3, fatty enzyme immobilizatio temperature is 25 ~ 40 DEG C, and the immobilization time is 4 ~ 10h.
Sn-1, the immobilization of 3 specific lipases, specifically it is made up of the method comprising the following steps：
（1）Sn-1,3 specific lipases screen：Using glycerine, oleic acid as substrate, a certain amount of lipase is added in reactor,
At each suitable temperature, stir and reacted when vacuumizing, with thin-layered chromatography and gas Chromatographic Determination wherein 1,3-
The content of diglyceride, it is target Sn-1 to filter out the high lipase of 1,3-DAG generation content, and 3 selectivitys are fatty
Enzyme.
（2）Sn-1,3 specific lipase immobilizations：
S1. resin pre-processes：Take a certain amount of macroreticular resin A, B to soak 4 ~ 12 h with 95wt% ethanol, during which stir for several times, very
Sky, which filters, removes cleaning solution, then soaks 2 with 2 ~ 5wt% hydrochloric acid solutions to without obvious ethanol smell with the repeated multiple times washing of distilled water
Neutrality is washed to after ~ 4 h, then with neutrality is washed to after the h of 1 ~ 2wt% soaking with sodium hydroxide 2 ~ 4, low temperature is protected after suction filtration removes moisture removal
Deposit standby.
S2. prepared by enzyme liquid：Lipase is taken, adds the buffer solutions of pH 5.0 ~ 9.0 in 25 ~ 40 DEG C, stirring 1h makes it fully molten
Solve, then 4000 r/min centrifuge 10 min, and it is enzyme liquid to take supernatant liquor, and determining its albumen by Coomassie Brilliant Blue contains
Amount.
S3. fatty enzyme immobilizatio：100 mL enzyme liquids are taken, add 10 g（Wet basis）The macroreticular resin pre-processed（A, B two
Kind resin adding proportion is 1:1~1:2）, 150 r/min Zhen Oscillating adsorb 4 ~ 10h, absorption in 25 ~ 40 DEG C of water bath with thermostatic control shaking tables
Filtered after end and remove remaining enzyme liquid, be drying to obtain immobilized lipase.
As optimization, in step S1, the preferred 2wt% of concentration of the hydrochloric acid solution, the concentration of sodium hydroxide solution it is preferred
2wt%。
As optimization, in step S2, buffer solution ionic strength is 0.025M ~ 0.075M, and pH scopes are 5.0 ~ 9.0.
As optimization, in step S3, drying mode can be freeze-drying（- 40 DEG C, 2 ~ 4 h）, vacuum drying（40~50℃、
2~5h）, heated-air drying（50 ~ 60 DEG C, 2 ~ 5h）.
The present invention also provides a kind of method that metal ion solution complexing processing prepares high vigor immobilized lipase, including
Sn-1, each step in the preparation method of 3 selectivity immobilized lipases, wherein, in step S1, the pretreatment bag of macroreticular resin
Include to use and contain Ca2+、K+、Ba2+Middle one or more metal ion solution complexing processing.Further, the metal ion solution
For CaCl2、CaCO3、KCl、KNO3、BaCl2、BaSO4At least one of.Further, the ion of the metal ion solution
Intensity is 0.02 ~ 0.05M.
The method that metal ion solution complexing processing prepares high vigor immobilized lipase, is comprised the following steps that：
（1）Resin pre-processes：Take a certain amount of macroreticular resin A, B to soak 4 ~ 12 h with 95wt% ethanol, during which stir for several times, very
Sky, which filters, removes cleaning solution, then soaks 2 with 2 ~ 5wt% hydrochloric acid solutions to without obvious ethanol smell with the repeated multiple times washing of distilled water
Be washed to neutrality after ~ 4 h, then with being washed to neutrality after the h of 1 ~ 2wt% soaking with sodium hydroxide 2 ~ 4, then with ionic strength be 0.02 ~
0.05M's contains Ca2+、K+、Ba2+One or more of which metal ion solution soaks 4 ~ 12 h, and suction filtration goes stand-by after moisture removal；
（2）The same foregoing description of other subsequent techniques.
The present invention has the advantages that compared with prior art：
（1）Selected fatty enzyme powder comes from aspergillus niger（Aspergillusnige）Lipase A 03 caused by fermentation, thus
Not only enzymatic production is easy for the immobilized lipase that being fixed makes, and cost of manufacture is relatively low, is easy to commercial application.
Domestic and international common Sn-1,3 selectivitys fat mainly has three kinds of commercial lipases of Novozyme companies of Denmark at present
Lipozyme RM IM（Strain sourceRhizomucor.miehei）、Lipozyme TL IM（Strain sourceThermomyces. lanuginosus）、Lipozyme RM IM（Strain sourceCandida.antarctica）, the high spy of use cost be present in it
Point；Also there is being fixed of lipase of research and utilization Aspergillusoryzae fermentations the country and applied in OPO synthesis
（Li Yang, the immobilization of 2015, Sn-1,3 specific lipases and its application in Structure grease OPO synthesis）.
（2）Immobilized lipase is made in the macroporous resin adsorption immobilization that the present invention uses, and technique is simple, available for industry
Change production application, cost is cheap, reusable more than 30 times, and carrier can carry out regeneration use.
（3）The present invention uses low pole-polar macroporous resin-bonded immobilization, effectively avoids single resin adsorption
The characteristics of lipase is insufficient.
（4）Immobilized lipase produced by the present invention has preferable heat endurance, and optimum temperature is by 40 DEG C after immobilization
Lifted to 65 DEG C；
（5）Metallic ions Ca has been complexed in immobilized lipase preparation process produced by the present invention2+、K+、Ba2+, three can stablize
The conformation of catalytic activity of lipase, its reactive group is set to be in required 3 D tropism, and by coordinate bond, by enzyme-to-substrate group
It is combined, not only increases the binding ability of enzyme and carrier moreover, have activated the activity of immobilized lipase.
Brief description of the drawings
Fig. 1 is immobilized lipase 00S difference access times enzyme activity stability
Fig. 2 is influence of the different buffer types to immobilized lipase 00S
Fig. 3 is influence of the pH of buffer to immobilized lipase 00S
Fig. 4 is different lipase-influence of the macroreticular resin ratio to immobilized lipase 00S
Fig. 5 is influence of the different immobilization temperature to immobilized lipase 00S
Fig. 6 is influence of the immobilization time to immobilized lipase 00S
Fig. 7 is immobilized lipase 00S optimum temperature curve
Fig. 8 is the immobilized lipase that different resins type obtains
Fig. 9 is fixation support resin micro-structure diagram
Figure 10 is immobilized lipase 00S micro-structure diagrams.
Embodiment
The present invention is described in further details with reference to specific embodiment.
Embodiment 1
A kind of preparation method of Sn-1,3 selectivitys immobilized lipase：
Take 10 g lipase As 03（Enzyme powder）, 100 mL, six kinds of different types of buffer solns are dissolved in respectively（Tris-HCl,
Barbital sodium-HCl, boric acid-borax, citric acid-sodium citrate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-phosphorus
Acid dihydride sodium）In, fully 1 h of dissolving is stirred at 40 DEG C, is then centrifuged for taking upper strata enzyme liquid.
Add 10 g the macroreticular resin HA-WP8 and LX-EP120 to be handled well（Adding proportion 1:1, first with 95wt% ethanol
4 h are embathed, with 2wt% hydrochloric acid solutions immersion 2h after being washed with water to neutrality, are washed to neutrality, then with 2wt% soaking with sodium hydroxide 2h
After be washed to neutrality, then soak 4h with ionic strength 0.05M CaCl solution, suction filtration removes moisture removal）.
The macroreticular resin that has pre-processed is placed in the Oscillating that shake of 150 r/min in 30 DEG C of shaking tables and adsorbs 5 h, timing after being mixed with enzyme liquid
Sampling determines the change of protein adsorption quantity in fixation procedure with Coomassie Brilliant Blue；Filtered off after the completion of immobilization except residual enzyme
Liquid, and immobilized lipase is rinsed with corresponding phosphate buffer, it is true in 45 DEG C to filter the immobilized lipase cleaned up
Sky dries 3 h, produces final Sn-1,3 selectivity immobilized lipase 00S.Obtained fixation under different buffer types
Change effect as shown in Fig. 2 wherein with phosphate（Disodium hydrogen phosphate-potassium dihydrogen phosphate）Immobilization effect is most under buffer solution system
Sn-1 that is good, being obtained under this method, 3 selectivity immobilized lipase 00S enzyme immobilizations rates are up to 91.50%, the esterification of unit interval
Rate is up to 66.11%.
Embodiment 2
A kind of preparation method of Sn-1,3 selectivitys immobilized lipase：
Take 10 g lipase As 03（Enzyme powder）, it is dissolved in 100 mL difference pH value（5.0、5.5、6.0、6.5、7.0、7.5、8.0、
9.0）Phosphate buffer
（Ion concentration is 0.05M）In, fully 1 h of dissolving is stirred at 40 DEG C, is then centrifuged for taking upper strata enzyme liquid.
Add 10 g the macroreticular resin HA-WP8 and LX-EP120 to be handled well（Adding proportion 1:1, first with 95wt% ethanol
4 h are embathed, with 2wt% hydrochloric acid solutions immersion 2h after being washed with water to neutrality, are washed to neutrality, then with 2wt% soaking with sodium hydroxide 2h
After be washed to neutrality, then soak 4h with ionic strength 0.05M CaCl solution, suction filtration removes moisture removal）.
The macroreticular resin that has pre-processed is placed in the Oscillating that shake of 150 r/min in 30 DEG C of shaking tables and adsorbs 5 h, timing after being mixed with enzyme liquid
Sampling determines the change of protein adsorption quantity in fixation procedure with Coomassie Brilliant Blue；Filtered off after the completion of immobilization except residual enzyme
Liquid, and immobilized lipase is rinsed with corresponding phosphate buffer, it is true in 45 DEG C to filter the immobilized lipase cleaned up
Sky dries 3 h, produces final Sn-1,3 selectivity immobilized lipase 00S.Obtained fixation under different pH of cushioning fluid
Change effect as shown in figure 3, wherein the pH of cushioning fluid of best results is 7.5, the Sn-1 obtained under this method, 3 selectivitys are fixed
Change lipase 00S enzyme immobilizations rate up to 91.50%, the enzyme activity rate of recovery is up to 104.45%, and esterification activity is up to 800.23 U/g.
Embodiment 3
A kind of preparation method of Sn-1,3 selectivitys immobilized lipase：
5g, 7.5g, 10 g, 15g, 20g lipase A 03 are taken respectively（Enzyme powder）, it is dissolved in the phosphate buffers of 100 mL pH 7.5
（Ion concentration is 0.05M）In, fully 1 h of dissolving is stirred at 40 DEG C, is then centrifuged for taking upper strata enzyme liquid.
Add 10 g the macroreticular resin HA-WP8 and LX-EP120 to be handled well（Adding proportion 1:1, first with 95wt% ethanol
4 h are embathed, with 2wt% hydrochloric acid solutions immersion 2h after being washed with water to neutrality, are washed to neutrality, then with 2wt% soaking with sodium hydroxide 2h
After be washed to neutrality, then soak 4h with ionic strength 0.05M CaCl solution, suction filtration removes moisture removal）.
The macroreticular resin that has pre-processed is placed in the Oscillating that shake of 150 r/min in 30 DEG C of shaking tables and adsorbs 5 h, timing after being mixed with enzyme liquid
Sampling determines the change of protein adsorption quantity in fixation procedure with Coomassie Brilliant Blue；Filtered off after the completion of immobilization except residual enzyme
Liquid, and immobilized lipase is rinsed with corresponding phosphate buffer, it is true in 45 DEG C to filter the immobilized lipase cleaned up
Sky dries 3 h, produces final Sn-1,3 selectivity immobilized lipase 00S.It is made under different lipase-macroreticular resin ratio
The immobilization effect obtained is as shown in figure 4, wherein the enzyme of best results and resin ratio are 1：1.3, the Sn-1 obtained under this method,
3 selectivity immobilized lipase 00S enzyme immobilizations rates are up to 94.50%, and the enzyme activity rate of recovery is up to 103.28%, and esterification activity is up to 865.12
U/g。
Embodiment 4
A kind of preparation method of Sn-1,3 selectivitys immobilized lipase：
Take 7.5g lipase As 03（Enzyme powder）, it is dissolved in 100 mL pH 7.5 phosphate buffer（Ion concentration is 0.05M）
In, fully 1 h of dissolving is stirred at 40 DEG C, is then centrifuged for taking upper strata enzyme liquid.
Add 10 g the macroreticular resin HA-WP8 and LX-EP120 to be handled well（Adding proportion 1:1, first with 95wt% ethanol
4 h are embathed, with 2wt% hydrochloric acid solutions immersion 2h after being washed with water to neutrality, are washed to neutrality, then with 2wt% soaking with sodium hydroxide 2h
After be washed to neutrality, then soak 4h with ionic strength 0.05M CaCl solution, suction filtration removes moisture removal）.
The macroreticular resin that has pre-processed is respectively placed in 20 DEG C, 30 DEG C, 40 DEG C, 150 r/ in 50 DEG C of shaking tables after being mixed with enzyme liquid
Min Zhen Oscillating adsorb 5 h, and timing sampling determines the change of protein adsorption quantity in fixation procedure with Coomassie Brilliant Blue；Immobilization is complete
Filtered off after except remaining enzyme liquid, and immobilized lipase is rinsed with corresponding phosphate buffer, filter consolidating of cleaning up
Immobilized lipase is dried in vacuo 3 h in 45 DEG C, produces final Sn-1,3 selectivity immobilized lipase 00S.Different immobilizations
At a temperature of obtained immobilization effect as shown in figure 5, wherein the immobilization temperature of best results is 30 DEG C, obtained under this method
Sn-1,3 selectivity immobilized lipase 00S enzyme immobilizations rates are up to 94.32%, and the enzyme activity rate of recovery is up to 103.68%, esterification activity
Up to 845.42 U/g.
Embodiment 5
A kind of preparation method of Sn-1,3 selectivitys immobilized lipase：
Take 7.5g lipase As 03（Enzyme powder）, it is dissolved in 100 mL pH 7.5 phosphate buffer（Ion concentration is 0.05M）
In, fully 1 h of dissolving is stirred at 40 DEG C, is then centrifuged for taking upper strata enzyme liquid.
Add 10 g the macroreticular resin HA-WP8 and LX-EP120 to be handled well（Adding proportion 1:1, first with 95wt% ethanol
4 h are embathed, with 2wt% hydrochloric acid solutions immersion 2h after being washed with water to neutrality, are washed to neutrality, then with 2wt% soaking with sodium hydroxide 2h
After be washed to neutrality, then contain Ca with ionic strength 0.05M respectively2+、K+、Ba2+、Zn2+、Mn2+、Fe2+、Mg2+Salting liquid enters
Row immersion complexing processing 4h, suction filtration removes moisture removal）.
The macroreticular resin that has pre-processed is placed in the Oscillating that shake of 150 r/min in 30 DEG C of shaking tables and adsorbs 5 h, timing after being mixed with enzyme liquid
Sampling determines the change of protein adsorption quantity in fixation procedure with Coomassie Brilliant Blue；Filtered off after the completion of immobilization except residual enzyme
Liquid, and immobilized lipase is rinsed with corresponding phosphate buffer, it is true in 45 DEG C to filter the immobilized lipase cleaned up
Sky dries 3 h, produces final Sn-1,3 selectivity immobilized lipase 00S.Different metal ions complexing processing is lower obtained
Immobilization effect it is as shown in table 1：
Influence of the different metal ions of table 1 processing to immobilized lipase
| Different disposal | Untreated fish group | Ca2+ | K+ | Ba2+ | Zn2+ | Mn2+ | Fe2+ | Mg2+ |
| Fixed rate | 92% | 94.32% | 95.02% | 93.98% | 91.74% | 89.66% | 89.85% | 91.02% |
| The enzyme activity rate of recovery | 85% | 114.7% | 119% | 112.0% | 70.1% | 76.5% | 68.3% | 84.3% |
| Esterification activity changes | —— | Increase by 35% | Increase by 40% | Increase by 32% | Reduce by 17.5% | Reduce by 10% | Reduce by 20% | It is almost unchanged |
Embodiment 6
A kind of preparation method of Sn-1,3 selectivitys immobilized lipase：
Take 7.5g lipase As 03（Enzyme powder）, it is dissolved in 100 mL pH 7.5 phosphate buffer（Ion concentration is 0.05M）
In, fully 1 h of dissolving is stirred at 40 DEG C, is then centrifuged for taking upper strata enzyme liquid.
Add 10 g the macroreticular resin HA-WP8 and LX-EP120 to be handled well（Adding proportion 1:1, first with 95wt% ethanol
4 h are embathed, with 2wt% hydrochloric acid solutions immersion 2h after being washed with water to neutrality, are washed to neutrality, then with 2wt% soaking with sodium hydroxide 2h
After be washed to neutrality, then soak 4h with ionic strength 0.05M CaCl solution, suction filtration removes moisture removal）.
The macroreticular resin that has pre-processed be placed in after being mixed with enzyme liquid the 150 r/min points of Oscillating absorption 1h that do not shake in 30 DEG C of shaking tables,
2h, 3h, 4h, 5h, 6h, 8h, 10h, timing sampling determine the change of protein adsorption quantity in fixation procedure with Coomassie Brilliant Blue；Gu
Filtered off after the completion of fixedization except remaining enzyme liquid, and immobilized lipase is rinsed with corresponding phosphate buffer, it is dry to filter cleaning
Net immobilized lipase is dried in vacuo 3 h in 45 DEG C, produces final Sn-1,3 selectivity immobilized lipase 00S.It is different
Obtained immobilization effect under the immobilization time is as shown in fig. 6, its fixation rate increases, the immobilization time with time lengthening
Tended towards stability for immobilization effect after 5h, the Sn-1 obtained under this method, 3 selectivity immobilized lipase 00S enzyme immobilization rates
Up to 90% ~ 97%.
Embodiment 7
A kind of preparation method of Sn-1,3 selectivitys immobilized lipase：
Take 7.5g lipase As 03（Enzyme powder）, it is dissolved in 100 mL pH 7.5 phosphate buffer（Ion concentration is 0.05M）In,
Fully 1 h of dissolving is stirred at 40 DEG C, is then centrifuged for taking upper strata enzyme liquid.
The five groups of macroreticular resins to be handled well are taken respectively：1. HA-WP8,2. LX-EP120,3. HA-WP8 and LX-EP120 are mixed
Close（2:1）, 4. HA-WP8 and LX-EP120 mixing（1:1）, 5. HA-WP8 and LX-EP120 mixing（1:2）（First use 95wt% ethanol
4 h are embathed, with 2wt% hydrochloric acid solutions immersion 2h after being washed with water to neutrality, are washed to neutrality, then with 2wt% soaking with sodium hydroxide 2h
After be washed to neutrality, then soak 4h with ionic strength 0.05M CaCl solution, suction filtration removes moisture removal）10g, mixed with enzyme liquid
After be placed in 150 r/min in 30 DEG C of shaking tables shake Oscillating adsorb 5 h, timing sampling with Coomassie Brilliant Blue determine fixation procedure in albumen
The change of adsorbance；Filtered off after the completion of immobilization except remaining enzyme liquid, and immobilization fat is rinsed with corresponding phosphate buffer
Fat enzyme, filter the immobilized lipase cleaned up and be dried in vacuo 3 h in 45 DEG C, produce final Sn-1,3 selectivity immobilizations
Lipase 00S.
Obtained immobilization effect is as shown in table 2 under different resins bond type：
Table 2 is nonpolar-the different additions of polar resin under influence to immobilized lipase
Contrast is it can be seen that immobilization enzyme immobilizatio rate, enzyme activity are returned made from nonpolar-polar macroporous resin-bonded immobilization
Yield is more nonpolar, the independent immobilization effect of polar resin is high；Wherein with HA-WP8 and LX-EP120 with 1:Mixed under 1 ratio
Close best results.
It is follow-up also to have carried out Sn-1,3 selectivity immobilized lipase 00S application test, this method is obtained obtained
Immobilised enzymes be used for be catalyzed free fatty and glycerine synthesis 1,3-DAG in, as shown in figure 1, continuous use 15 times after its
Catalytic esterification vigor remains at more than 97%, and the Sn-1 that this method obtains, 3 selectivity immobilized lipase 00S tools have been said in checking
There are good practicality and operational stability.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair
The restriction of embodiments of the present invention.For those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms.There is no necessity and possibility to exhaust all the enbodiments.It is all this
All any modification, equivalent and improvement made within the spirit and principle of invention etc., should be included in the claims in the present invention
Protection domain within.
Claims (10)
1. a kind of Sn-1, the preparation method of 3 selectivity immobilized lipases, comprise the following steps：
S1. pretreatment is carried out to macroreticular resin and goes the removal of impurity；
S2. lipase is dissolved in buffer solution, centrifuging and taking supernatant liquor, as enzyme liquid；
S3. the enzyme liquid for taking step S2 to obtain, the macroreticular resin , Zhen Oscillating that step S1 has been pre-processed are added, filters, wash, dry, obtain
To immobilized lipase；
Characterized in that, the macroreticular resin includes nonpolar macroporous adsorption resin and low pole macroporous absorbent resin.
2. Sn-1 according to claim 1, the preparation method of 3 selectivity immobilized lipases, it is characterised in that described non-
Polar macroporous adsorption resin includes MA-NP6, MI-BN4, MI-300IDA, LX-1000EP, GDX-104, LX-1000HA, LX-
In EP120, LX-EP130, LX-201A, SD300, DM11, D354, D730, SIP1100, SIP1200 at least with one kind；Institute
State low pole macroporous absorbent resin include MI-WP8, GDX-501, D201, D301, HPD400, HPD500, Dowex A-1,
At least one of D401, Diaion CR-10, LSC-100, LSC-200.
3. Sn-1 according to claim 1 or 2, the preparation method of 3 selectivity immobilized lipases, it is characterised in that institute
It is 1 to state the mass ratio of nonpolar macroporous adsorption resin and low pole macroporous absorbent resin in macroreticular resin:1~1:2.
4. Sn-1 according to claim 1 or 2, the preparation method of 3 selectivity immobilized lipases, it is characterised in that institute
The mass ratio for stating lipase and macroreticular resin is 1:0.5~1:2.
5. Sn-1 according to claim 1 or 2, the preparation method of 3 selectivity immobilized lipases, it is characterised in that step
In rapid S1, macroreticular resin pretreatment includes：The removal of impurity is gone in priority ethanol, acid, alkali immersion, neutrality is finally washed to, under low temperature
Preserve stand-by.
6. Sn-1 according to claim 1 or 2, the preparation method of 3 selectivity immobilized lipases, it is characterised in that step
In rapid S2, the buffer solution is Tris-HCl, barbital sodium-HCl, boric acid-borax, citric acid-sodium citrate, phosphate delay
One or more in fliud flushing, the pH value of the buffer solution is 5.0 ~ 9.0.
7. Sn-1 according to claim 1 or 2, the preparation method of 3 selectivity immobilized lipases, it is characterised in that step
In rapid S3, fatty enzyme immobilizatio temperature is 25 ~ 40 DEG C, and the immobilization time is 4 ~ 10h.
8. a kind of metal ion solution complexing processing prepares high vigor Sn-1, the method for 3 selectivity immobilized lipases, its feature
It is, including each step as described in any one of claim 1 ~ 7, in step S1, the pretreatment of macroreticular resin is included with containing Ca2 +、K+、Ba2+Middle one or more metal ion solution complexing processing.
9. metal ion solution complexing processing according to claim 8 prepares high vigor Sn-1,3 selectivity immobilized lipases
The method of enzyme immobilization lipase, it is characterised in that the metal ion solution is CaCl2、CaCO3、KCl、KNO3、BaCl2、
BaSO4At least one of.
10. metal ion solution complexing processing according to claim 8 or claim 9 prepares high vigor Sn-1,3 selectivity immobilizations
The method of lipase immobilization lipase, it is characterised in that the ionic strength of the metal ion solution is 0.02 ~ 0.05M.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109371006A (en) * | 2018-12-12 | 2019-02-22 | 江南大学（如皋）食品生物技术研究所 | A kind of process for fixation of sucrose phosphorylase |
| CN110241107A (en) * | 2019-06-11 | 2019-09-17 | 中国科学院南海海洋研究所 | A kind of method using amino resins immobilized lipase and immobilized lipase obtained by this method |
| CN113073090A (en) * | 2021-04-06 | 2021-07-06 | 江南大学 | Method for immobilizing lipase for enriching polyunsaturated fatty acids |
| CN114480360A (en) * | 2021-12-14 | 2022-05-13 | 山东省农业科学院 | Method for preparing diglyceride by immobilized Sn-2 lipase |
| CN117625586A (en) * | 2023-11-23 | 2024-03-01 | 广东惠尔泰生物科技有限公司 | Sn-1,3 specific immobilized lipase, and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110053229A1 (en) * | 2008-04-21 | 2011-03-03 | Kao Corporation | Method for production of immobilized enzyme |
| CN103805617A (en) * | 2012-11-09 | 2014-05-21 | 丰益(上海)生物技术研发中心有限公司 | 1,3-specific lipase and encoding gene sequence and application thereof |
| CN104140961A (en) * | 2014-07-04 | 2014-11-12 | 浙江大学 | Immobilized lipase having Sn-1,3 specificity as well as preparation method and application of immobilized lipase |
| 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 |
| CN107012136A (en) * | 2017-06-12 | 2017-08-04 | 浙江工业大学 | A kind of method of immobilization Thermomyces lanuginosus lipase |
-
2017
- 2017-10-12 CN CN201710948748.8A patent/CN107828756B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110053229A1 (en) * | 2008-04-21 | 2011-03-03 | Kao Corporation | Method for production of immobilized enzyme |
| CN103805617A (en) * | 2012-11-09 | 2014-05-21 | 丰益(上海)生物技术研发中心有限公司 | 1,3-specific lipase and encoding gene sequence and application thereof |
| 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 |
| CN104140961A (en) * | 2014-07-04 | 2014-11-12 | 浙江大学 | Immobilized lipase having Sn-1,3 specificity as well as preparation method and application of immobilized lipase |
| CN107012136A (en) * | 2017-06-12 | 2017-08-04 | 浙江工业大学 | A kind of method of immobilization Thermomyces lanuginosus lipase |
Non-Patent Citations (3)
| Title |
|---|
| 李阳: "Sn-1,3专一性脂肪酶的固定化及其在结构油脂OPO合成中的应用", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 段筱杉等: "混合大孔树脂纯化海芦笋总黄酮工艺的研究", 《海洋科学》 * |
| 赵丽芳: "脂肪酶的固定化及其催化性能的研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109371006A (en) * | 2018-12-12 | 2019-02-22 | 江南大学（如皋）食品生物技术研究所 | A kind of process for fixation of sucrose phosphorylase |
| CN110241107A (en) * | 2019-06-11 | 2019-09-17 | 中国科学院南海海洋研究所 | A kind of method using amino resins immobilized lipase and immobilized lipase obtained by this method |
| CN113073090A (en) * | 2021-04-06 | 2021-07-06 | 江南大学 | Method for immobilizing lipase for enriching polyunsaturated fatty acids |
| CN114480360A (en) * | 2021-12-14 | 2022-05-13 | 山东省农业科学院 | Method for preparing diglyceride by immobilized Sn-2 lipase |
| 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 |
| CN117625586A (en) * | 2023-11-23 | 2024-03-01 | 广东惠尔泰生物科技有限公司 | Sn-1,3 specific immobilized lipase, and preparation method and application thereof |
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Inventor after: Liu Fang Inventor after: Cao Yong Inventor after: Dai Weijie Inventor after: Huang Zaocheng Inventor after: Zheng Zhongmu Inventor after: Guo Baoyan Inventor after: Zhou Cong Inventor before: Liu Fang Inventor before: Cao Yong Inventor before: Dai Weijie Inventor before: Huang Cong |
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Denomination of invention: A Preparation Method of Sn-1,3 Specific Immobilized Lipase Effective date of registration: 20221205 Granted publication date: 20201127 Pledgee: Agricultural Bank of China Limited Guangzhou Development Zone Branch Pledgor: GUANGDONG HUIERTAI BIOTECHNOLOGY CO.,LTD. Registration number: Y2022980024848 |
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Denomination of invention: Preparation method of Sn-1,3 specific immobilized lipase Granted publication date: 20201127 Pledgee: Agricultural Bank of China Limited Guangzhou Development Zone Branch Pledgor: GUANGDONG HUIERTAI BIOTECHNOLOGY CO.,LTD. Registration number: Y2024980037188 |
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