CN105671017A - Phospholipase mark activation method for nonaqueous phase environmental catalysis - Google Patents
Phospholipase mark activation method for nonaqueous phase environmental catalysis Download PDFInfo
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- 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)
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- 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)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01004—Phospholipase A2 (3.1.1.4)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01005—Lysophospholipase (3.1.1.5)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01032—Phospholipase A1 (3.1.1.32)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/04—Phosphoric diester hydrolases (3.1.4)
- C12Y301/04003—Phospholipase C (3.1.4.3)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/04—Phosphoric diester hydrolases (3.1.4)
- C12Y301/04004—Phospholipase D (3.1.4.4)
Abstract
The invention provides a phospholipase mark activation method for nonaqueous phase environmental catalysis. The method includes the following steps that 1, a phospholipase mark is activated; 2, mark phospholipase is cured; kieselguhr is added, the mixture is fully shocked and mixed to be uniform and stands still, and immobilized adsorption is completed; 3, freeze-drying is conducted; 4, washing is conducted; 5, desolventizing is conducted; washed enzyme powder is subjected to vacuum drying, residual organic solvent is removed, and finished product enzyme powder is obtained. The phospholipase mark activation method is simple in process, obvious in activation effect, low in production cost and capable of improving enzyme catalysis activity and substrate specificity, the content of refined oil phospholipids is lowered when the method is applied to oil and fat refining, the yield of a refined oil product is raised, and quite high economic value is achieved.
Description
Technical field
The invention belongs to biochemical industry activation of catalyst field, particularly to a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis.
Background technology
Phospholipase (Phospholipase, EC3.1.1.4; EC3.1.1.3; EC3.1.4.4; EC3.1.1.5) it is the big class hydrolytic enzyme with important value, there is the ability of the multiple reactions such as the synthesis of catalysis phospholipids compounds, hydrolysis, transesterification, ester exchange, be Chemical Manufacture, one of most important enzyme in medicine synthesis and food processing. Common phospholipase mainly includes several classes such as E.C. 3.1.1.32, phospholipase A2, phospholipase C, Choline phosphatase. Such as E.C. 3.1.1.32 and phospholipase A2, can react on oil-water interfaces in catalysis phospholipid and triglyceride α position and the Ester hydrolysis of β position, alcoholysis, Lipase absobed, ester exchange etc., and at food, chemical industry, particularly multiple field such as oil and fat refining is widely used. Between phospholipase C catalyzing glycerol and phosphate group, the synthesis such as phosphoric acid ester bond and fracture, be mainly used in oil and fat refining industry. In Choline phosphatase catalysis phospholipid, hydrolysis, synthesis, the transesterification etc. such as between phosphate group and base, have critical role in medication chemistry industry.
For the conventional enzyme of the overwhelming majority, catalytic reaction carries out in aqueous environment, organic solvent can substantially reduce the catalytic capability of enzyme, even make enzyme deactivation, but synthesis and some important reaction needed much with the product of important value especially carry out in organic solvent in nonaqueous phase environment, such as the enzyme process refine of crude oil and miscella. Therefore, improve enzyme performance in nonaqueous phase catalytic process and just become the focus of research. Nonaqueous phase enzyme catalysis Objective Concept Zaks and Klibanov proposed in 1984, and they find that some lipase still has certain catalysis activity under organic solvent environment, and have synthesized a series of organic compound based on this. Through the development of decades, Non-aqueous Enzymatic Catalysis Technology obtains certain development, and solvent system is no longer confined to single organic solvent, and the kind of used enzyme have also been obtained further expansion. Compared to abiotic catalysis chemical industry and aqueous biochemical catalysis, nonaqueous phase enzyme catalysis has some superiority, is mainly manifested in increase substrate valid density; Molecular balance moves to compound direction; Microorganism is polluted few; Enzyme heat stability improves; Enzyme reusability improves; Downstream process energy consumption reduces; Reduce side reaction; Improve the aspect such as enzyme spcificity and selectivity. But, in nonaqueous phase, enzymatic activity is not high is restrict this technical development to enter the biggest factor of commercial Application all the time. Continuous propelling along with correlational study, the further investigated enzyme reason that vigor reduces in organic facies, find that these reasons are soluble, by strategy design rational, system, and enzyme activition modifiies, can many orders of magnitude improve enzyme vigor in organic facies, and be finally reached enzyme activity level in aqueous phase.
Phospholipid is one of major impurity in crude oil, is dispersed in solute state in crude oil or miscella.Phospholipid is under the high temperature conditions, it is easy to occurs chemical reaction to generate dark pigment and odorous compound, dehydration coking under higher temperature, has a strong impact on edible oil quality. The higher edible oil of content of phospholipid in later stage storage, very easily oxidative rancidity, quality deterioration. Therefore, the phospholipid in removal crude oil is a ring important in oil and fat refining process, and this process is also degumming raw oil. The phospholipid imbibition that the hydrophilic such as industrial conventional aquation degumming, utilizes the amphipathic characteristic of phospholipid molecule, adds a certain amount of water in crude oil, lecithin are stronger, agglomerate forms gelatinous precipitate and separates from crude oil. But non-hydratable phospholipid, particularly phosphatidic acid calcium and magnesium salt, it is impossible to separate from crude oil in this form. Enzymatic degumming can utilize the catalysis activity of phospholipase, non-hydratable phospholipid is converted into the higher lysophosphatide of hydrophilic and then aquation separates, or excision phosphate group, is translated into diacylglycerol, to reach the purpose of elimination phospholipid. Owing to degumming raw oil system is bigger with conventional aqueous phase reactions architectural difference, particularly in miscella system, enzyme activity is had strong inhibition effect by normal hexane or light solvent oil, a lot of phospholipase can not show higher vigor as in aqueous, improves the phosphide enzyme vigor when nonaqueous phase significant.
Summary of the invention
It is an object of the invention to propose a kind of phosphide enzyme ecphoria method suitable in nonaqueous phase environmental catalysis.
The present invention is achieved by the following technical solutions: a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis, comprises the following steps:
(1) the phospholipase ecphoria: phospholipase is dissolved in the buffer containing imprinting factor and activates, imprinting factor is phospholipid or derivatives thereof;
(2) trace phospholipase immobilization: add kieselguhr, fully shakes mixing, stands, and completes immobilization absorption;
(3) lyophilizing: the trace phospholipase completing immobilization and freeze proof protection is carried out pre-freeze, then to carry out vacuum lyophilization be powder, is immobilization trace phospholipase powder;
(4) washing: adding cleaning mixture in immobilization trace phospholipase powder, abundant vortex oscillation is disperseed, elimination solvent after standing, and repeated washing washes away imprinting factor;
(5) precipitation: the enzyme powder washed carries out vacuum drying, removes the organic solvent of residual, obtains finished product enzyme powder.
Preferably, in step (1), buffer concentration is 10～500mMol/L, and activation condition is 4～40 DEG C, 2～30min.
Preferably, the imprinting factor described in step (1) is 1:10～250 with the volume ratio of buffer.
Preferably, the buffer described in step (1) is citric acid or phosphoric acid or Tris-HCl; Described phospholipase is the one in E.C. 3.1.1.32, phospholipase A2, phospholipase C, Choline phosphatase; Described phospholipid or derivatives thereof is the one of phosphatidic acid, phospholipid calcium salt, phospholipid magnesium salt. Wherein citric acid and phosphoric acid are acidulant the most frequently used in existing oil and fat refining industry, and Tris-HCl is the conventional pH buffer system of biochemical reaction, and acid-salt buffer scope is consistent with the present invention relates to enzyme optimum pH scope, and without interference with the use of finished product enzyme agent.
Preferably, the temperature stood in step (2) is 4～40 DEG C, the time is 2～30min.
Preferably, the kieselguhr in step (2) is to add according to 10～100% (w/v) of previous step system volume.
Preferably, in step (3), the temperature of pre-freeze is-20～-80 DEG C, and the condition of vacuum lyophilization is temperature is-60～-80 DEG C, vacuum 1～500Pa.When temperature is higher than-20 DEG C, the adverse consequencess such as the trace system containing imprinting factor, pheron and buffer salt is difficult to fully freeze, and a large amount of bubble can be caused in process of vacuum drying to bloat, finished product yield reduction.
Preferably, in step (4), adding cleaning mixture according to 2～10 times of volumes of immobilization trace phospholipase powder, the scattered temperature of vortex oscillation is 4～40 DEG C, and time of repose is 2～30min, and repeated washing number of times is 3～4 times. Ecphoria agent difficult imurity-removal in oil and fat refining process that the present invention uses, this activator is easily soluble in non-polar organic solvent, consider that the degeneration of pheron is affected by follow-up precipitation condition and organic solvent, it is preferable that cleaning mixture is normal hexane, petroleum ether, the one in ether. Wash conditions is lower than this proposition scope, and the harmful effect of follow-up use can not be left in the basket by imprinting factor residual.
Preferably, between step (1), (2), also carry out freeze proof protection, the trace phospholipase activated adds freezeproof protectant, after mix homogeneously, standing, completes freeze proof protection.
Preferably, described freezeproof protectant is glycerol or mannose; Freezeproof protectant concentration is 0～500mMol/L, and the temperature of standing is 4～40 DEG C, the time is 2～30min. Glycerol and mannose are biochemical conventional freezeproof protectants, and have nontoxic, water-soluble, and subsequent step and finished product are used the feature having no adverse effects. Within the scope of this; freezeproof protectant can effectively not reduce by defence enzyme activity in pre-freeze and lyophilisation link, and when the freezeproof protectant of higher concentration, washing imprinting factor link difficulty strengthens; and finished product enzyme agent not easily keeps characters powder, affect follow-up preservation and use.
The invention have the advantage that
The present invention utilizes phospholipase protein molecular conformation flexibility in aqueous phase, adopts cryodesiccated method to be undertaken induced curing by phospholipase protein conformation, so as to keep being suitable for the state with Binding Capacity, is beneficial to catalytic reaction and carries out. Due to phospholipase protein conformation relative stiffness in non-aqueous media, the phospholipase high catalytic activity conformation that the method provides is easily available maintenance. On the basis of Bio-imprinting process, by being combined with fixation support by trace phospholipase molecule, it is possible to improve phospholipase dispersibility in organic solvent, improving phospholipase catalytic capability further, basis is reclaimed in the offer of reusing for phospholipase simultaneously.
It addition, present invention process is simple, activation effect is obvious, and production cost is low, has wide prospects for commercial application and economic worth. After ecphoria immobilization, phospholipase vigor in non-aqueous phase medium can improve 1～2 order of magnitude, and substrate specificity is also remarkably reinforced, and in commercial production, particularly has good application prospect in oil and fat refining processing.
Detailed description of the invention
Embodiment 1
Measuring 50g E.C. 3.1.1.32 (LecitaseUltra, 12000IU/g), be dissolved in the 300mL pH4.5 containing 5% phosphatidic acid calcium salt, concentration is in the citrate buffer solution emulsion of 20mMol/L, under 20 DEG C of conditions, stands 10min. Add 8g (148mMol/L) mannose, after fully dissolving mixing, under 20 DEG C of conditions, stand 10min. Adding 150g (50%, w/v) kieselguhr, abundant vortex oscillation, mix homogeneously, 20 DEG C stand 10min. Pre-freeze is to fully charge at-20 DEG C for mixture, and through-80 DEG C, 1Pa low-temperature freeze drying becomes immobilization trace E.C. 3.1.1.32. Add 900mL normal hexane, mixing of fully vibrating, stand 10min at 20 DEG C, be filtered to remove solvent, in triplicate.The immobilization trace phospholipase vacuum drying washing away imprinting factor is removed organic solvent, and 4 DEG C of lower seals preserve. After measured, under the normal hexane environment of moisture 4%, with phosphatidic acid calcium for substrate catalytic hydrolysis reaction, ecphoria E.C. 3.1.1.32 vigor improves 79 times than un-activation phospholipase, using oleic three ester to measure as competitive substrate, zymolyte specificity improves 213 times.
Embodiment 2
Measuring: 5g E.C. 3.1.1.32 powder (Serratiaprofundus originates, 14000IU/g), be dissolved in the 100mL pH7.0 containing 0.4% phosphatidic acid calcium salt, concentration is in the citrate buffer solution emulsion of 10mMol/L, under 4 DEG C of conditions, stands 30min. Adding 10g (10%, w/v) kieselguhr, abundant vortex oscillation, mix homogeneously, 4 DEG C stand 30min. Pre-freeze is to fully charge at-80 DEG C for mixture, and through-70 DEG C, 200Pa low-temperature freeze drying becomes immobilization trace E.C. 3.1.1.32. Add 1000mL ether, mixing of fully vibrating, stand 30min at 4 DEG C, be filtered to remove solvent, in triplicate. The immobilization trace phospholipase vacuum drying washing away imprinting factor is removed organic solvent, and-20 DEG C of lower seals preserve. After measured, under the normal hexane environment of moisture 4%, with phosphatidic acid calcium for substrate catalytic hydrolysis reaction, ecphoria E.C. 3.1.1.32 vigor improves 53 times than un-activation E.C. 3.1.1.32, using oleic three ester to measure as competitive substrate, zymolyte specificity improves 158 times.
Embodiment 3
Measure 10g phospholipase A2 (10L, 8500IU/g), it is dissolved in the 100mL pH5.5 containing 5% phosphatidic acid magnesium salt, concentration is in the phosphoric acid buffer liquid emulsion of 500mMol/L, under 40 DEG C of conditions, stands 2min. Add 3g (167mMol/L) mannose, fully dissolve mixing, under 40 DEG C of conditions, stand 2min. Adding 100g (100%, w/v) kieselguhr, abundant vortex oscillation, mix homogeneously, 40 DEG C stand 2min. Pre-freeze is to fully charge at-80 DEG C for mixture, and through-80 DEG C, 500Pa low-temperature freeze drying becomes immobilization trace phospholipase. Add 200mL petroleum ether (boiling range 60～90 DEG C), mixing of fully vibrating, stand 2min at 40 DEG C, be filtered to remove solvent, be repeated four times. The phospholipase vacuum drying washing away imprinting factor is removed organic solvent, and 25 DEG C of lower seals preserve. After measured, under the normal hexane environment of moisture 4%, with phosphatidic acid calcium for substrate catalytic hydrolysis reaction, ecphoria phospholipase activity improves 82 times than un-activation phospholipase A2, uses oleic three ester to measure zymolyte specificity as competitive substrate and improves 216 times.
Embodiment 4
Measure 105The phospholipase C (Purifine, 200000IU/g) of IU, is dissolved in the 100mL pH5.5 containing 10% phosphatidic acid, and concentration is in the Tris-HCl buffer emulsion of 50mMol/L, under 30 DEG C of conditions, stands 10min. Add 4.6g (500mMol/L) glycerol, fully dissolve mixing, under 20 DEG C of conditions, stand 10min. Adding 80g (80%, w/v) kieselguhr, abundant vortex oscillation, mix homogeneously, 20 DEG C stand 10min. Pre-freeze is to fully charge at-80 DEG C for mixture, through-60 DEG C, and 100Pa low-temperature freeze drying, become immobilization trace phospholipase. Add 250mL normal hexane, mixing of fully vibrating, at 20 DEG C, stand 10min, be filtered to remove solvent, in triplicate. The phospholipase vacuum drying washing away imprinting factor is removed organic solvent, and 4 DEG C of lower seals preserve. After measured, under the normal hexane environment of moisture 4%, with phosphatidic acid calcium for substrate catalytic hydrolysis reaction, ecphoria phospholipase activity improves 58 times than un-activation enzyme, uses oleic three ester to measure as competitive substrate, and zymolyte specificity improves 330 times.
Embodiment 5
Take Semen sojae atricolor lixiviate miscella (normal hexane content 50%, v/v, phosphorus content 203mg/kg) 3L, being placed in 5L triangular flask, heating, to 50 DEG C, adds the citric acid solution of 4.5mL50%, after 30min is hatched in 1200rpm stirring, add NaOH solution that concentration is 4% to grain slag phase pH4.5. Measuring immobilization trace E.C. 3.1.1.32 in 150IU embodiment 1 and add the miscella after neutralizing, after stirring dispersion, add 45mL soft water, be incubated 50 DEG C, 6h is hatched in 1200rpm stirring. After enzyme digestion reaction completes, after adding NaOH tune pH to 7.0, the extra 30mL concentration that adds is the NaOH solution of 4%, 50 DEG C, and 30min is hatched in 1200rpm stirring. Being centrifuged off oil foot precipitation through 4000g, supernatant is degumming miscella. Measuring through molybdenum blue method, phosphorus content is 8.3mg/kg. Degumming miscella yield is 98.4%. Using original phospholipase C to process, phosphorus content is 80.6mg/kg.
Embodiment 6
Taking Semen sojae atricolor squeezing crude oil (phosphorus content 323mg/kg) 1L, be placed in 2.5L triangular flask, heating, to 70 DEG C, adds 1.5mL50% citric acid solution, after 30min is hatched in 1200rpm stirring, adds NaOH solution that concentration is 4% to grain slag phase pH5.5. After being cooled to 45 DEG C, measuring immobilization trace phospholipase C in 100IU embodiment 4 and add the crude oil after neutralizing, after stirring dispersion, be incubated 45 DEG C, 8h is hatched in 1200rpm stirring. After enzyme digestion reaction completes, adding 50mL soft water, 30min is hatched in 1200rpm stirring. Being centrifuged off bottom through 4000g to precipitate on a small quantity and aqueous phase, supernatant oil phase is degumming miscella. Measuring through molybdenum blue method, phosphorus content is 3.3mg/kg. Degumming crude oil yield is 97.3%. Using original E.C. 3.1.1.32 to process, phosphorus content is 12.2mg/kg, and degumming crude oil yield is 96.7%.
The foregoing is only the preferred embodiment of the invention; not in order to limit the invention; the any amendment made within all spirit in the invention and principle, equivalent replacement and improvement etc., should be included within the protection domain of the invention.
Claims (10)
1. the phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis, it is characterised in that comprise the following steps:
(1) the phospholipase ecphoria: phospholipase is dissolved in the buffer containing imprinting factor and activates, imprinting factor is phospholipid or derivatives thereof;
(2) trace phospholipase immobilization: add kieselguhr, fully shakes mixing, stands, and completes immobilization absorption;
(3) lyophilizing: the trace phospholipase completing immobilization and freeze proof protection is carried out pre-freeze, then to carry out vacuum lyophilization be powder, is immobilization trace phospholipase powder;
(4) washing: adding cleaning mixture in immobilization trace phospholipase powder, abundant vortex oscillation is disperseed, elimination solvent after standing, and repeated washing washes away imprinting factor;
(5) precipitation: the enzyme powder washed carries out vacuum drying, removes the organic solvent of residual, obtains finished product enzyme powder.
2. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterised in that in step (1), buffer concentration is 10～500mMol/L, and activation condition is 4～40 DEG C, 2～30min.
3. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterised in that the volume ratio of the imprinting factor described in step (1) and buffer is 1:10～250.
4. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterised in that the buffer described in step (1) is citric acid or phosphoric acid or Tris-HCl;Described phospholipase is the one in E.C. 3.1.1.32, phospholipase A2, phospholipase C, Choline phosphatase; Described phospholipid or derivatives thereof is the one of phosphatidic acid, phospholipid calcium salt, phospholipid magnesium salt.
5. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterised in that the temperature stood in step (2) is 4～40 DEG C, the time is 2～30min.
6. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterised in that the kieselguhr in step (2) is to add according to 10～100% (w/v) of previous step system volume.
7. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterized in that, in step (3), the temperature of pre-freeze is-20～-80 DEG C, and the condition of vacuum lyophilization is temperature is-60～-80 DEG C, vacuum 1～500Pa.
8. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterized in that, in step (4), cleaning mixture is added according to 2～10 times of volumes of immobilization trace phospholipase powder, the scattered temperature of vortex oscillation is 4～40 DEG C, and time of repose is 2～30min, and repeated washing number of times is 3～4 times, cleaning mixture is normal hexane, petroleum ether, the one in ether.
9. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 1, it is characterised in that also carry out freeze proof protection between step (1), (2); The trace phospholipase activated adds freezeproof protectant, after mix homogeneously, standing, completes freeze proof protection.
10. a kind of phosphide enzyme ecphoria method for nonaqueous phase environmental catalysis according to claim 9, it is characterised in that described freezeproof protectant is glycerol or mannose; Freezeproof protectant concentration is 0～500mMol/L, and the temperature of standing is 4～40 DEG C, the time is 2～30min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947753A (en) * | 2017-02-21 | 2017-07-14 | 西北大学 | A kind of phospholipase D process for fixation for improving phosphatidyl glycerol synthetic reaction vigor |
| CN112725174A (en) * | 2020-12-28 | 2021-04-30 | 黑龙江省农业科学院生物技术研究所 | Preparation device and method for biological imprinted enzyme |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337256A (en) * | 2011-09-20 | 2012-02-01 | 东北农业大学 | Method for entrapping and cross-linking phosphatidase A1 aggregates |
-
2016
- 2016-02-22 CN CN201610097117.5A patent/CN105671017A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337256A (en) * | 2011-09-20 | 2012-02-01 | 东北农业大学 | Method for entrapping and cross-linking phosphatidase A1 aggregates |
Non-Patent Citations (3)
| Title |
|---|
| AYELET FISHMAN ET AL.: "Bio-imprinting of lipases with fatty acids", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
| ZHEMIN LI ET AL.: "Enhancing the performance of a phospholipase A1 for oil degumming by bio-imprinting and immobilization", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
| 蔡奕璇等: ""构象记忆"的辣根过氧化物酶的微水相共价固定化", 《生物工程学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947753A (en) * | 2017-02-21 | 2017-07-14 | 西北大学 | A kind of phospholipase D process for fixation for improving phosphatidyl glycerol synthetic reaction vigor |
| CN106947753B (en) * | 2017-02-21 | 2020-04-21 | 西北大学 | Phospholipase D immobilization method for improving phosphatidylglycerol synthesis reaction activity |
| CN112725174A (en) * | 2020-12-28 | 2021-04-30 | 黑龙江省农业科学院生物技术研究所 | Preparation device and method for biological imprinted enzyme |
| CN112725174B (en) * | 2020-12-28 | 2022-09-06 | 黑龙江省农业科学院生物技术研究所 | Preparation device and method for biological imprinting enzyme |
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