CN108753755A - A kind of crosslinked bio enzyme catalyst and its application - Google Patents
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Abstract
The present invention relates to a kind of crosslinked bio enzyme catalyst and its applications, are prepared by the following method：Bovine serum albumin(BSA) is dissolved in phosphate buffer, obtain bovine serum albumin solution, add the stirring of candida antarctica lipase B solution, obtain enzyme-mixed liquid of protein, precipitating reagent and crosslinking agent is added, it is stirred under the conditions of ice-water bath, collect solid shaped bodies, solid shaped bodies are washed with phosphate buffer, obtain cross-linking enzyme aggressiveness, phosphate buffer is added to be disperseed, add crosslinking agent, it is stirred under the conditions of ice-water bath, obtain aldehyde group modified cross-linking enzyme aggressiveness, candida antarctica lipase B solution is added, it is stirred under the conditions of ice-water bath, product is collected by centrifugation, washing, after freeze-drying, obtain product.The crosslinked bio enzyme catalyst of preparation shows excellent catalytic activity in Kinetic Resolution and Dynamic Kinetic Resolution α-phenylethylamine, shows the series of advantages such as stability height, separable recycling, repeatable utilization, simple for process, cheap.
Description
Technical field
The invention belongs to catalyst technical fields, and in particular to a kind of crosslinked bio enzyme catalyst and its application.
Background technology
α-phenylethylamine is a kind of highly important chemical intermediate feed, its optics single enantiomer (R)-α-benzene
Ethamine and (S)-α-phenylethylamine serve not only as the chiral resolution examination of many racemic organic acids, alcohol, ester or phenolic compound
Agent, is also used as the chiral raw material of chiral auxiliary and asymmetric syntheses, and purposes is very extensive.The derivative of optical voidness α-phenylethylamine
Object is also widely used in the fields such as medicine, fragrance, dyestuff and emulsifier, there is very good application prospect.
It is mainly the method by asymmetric syntheses and fractionation to prepare optically pure α-phenylethylamine at present.Asymmetric syntheses master
It is specific to divide again if introducing one or more molecule with chiral centre into reactant molecule by chemical reaction
For application chiral catalyst method, addition chiral auxiliary method and use chiral reagent method.But asymmetric syntheses complex process is cumbersome,
Expensive disadvantage limits its commercial Application.Split includes Kinetic Resolution and Dynamic Kinetic Resolution, dynamics again
Split Method is a kind of classical separation method, and production cost is relatively low, is that industrial production prepares most having for single optical activity body
One of efficacious prescriptions method.α-phenylethylamine is split at present and mainly uses enzyme Split Method, and it is candida antarctica lipase B to commonly use enzyme.Dynamically
Kinetic Resolution is by the racemization of substrate and Kinetic Resolution resolution reaction associated with one pot simultaneously, in Kinetic Resolution
On the basis of more steps remaining reaction substrate is synchronized to the reaction of racemization, overcome Kinetic Resolution highest theoretical yield 50%
Defect, can theoretically chiral substrates be made to be completely converted into target product, realize 100% conversion.
Immobilised enzymes is continuous controllable, repeatable because specific ionization enzyme shows higher thermal stability, chemical stability, operation
Using and the series of advantages such as easily separated, be widely used in biocatalysis field.But immobilised enzymes is because shared by its carrier
Large percentage causes enzyme activity to be diluted, and declines so as to cause ratio defective product, therefore immobilised enzymes starts to turn to carrier-free immobilization
Method prepares cross-linking enzyme aggressiveness.Traditional cross-linking enzyme aggressiveness is simple for process, is made first by precipitating reagent close to each other between enzyme molecule
Aggregation, then carries out the enzyme molecule aggregation being settled out using crosslinking agent to be crosslinked obtained enzyme aggressiveness.Penta 2 are often used in industry
Aldehyde makees crosslinking agent, and mechanism of action is that schiff base reaction occurs for the amino in the aldehyde radical and enzyme molecule of glutaraldehyde.For itself ammonia
For the less enzyme of base, because its crosslinked action is too weak so the protein protective agent being commonly incorporated into more than amino content carries out auxiliary crosslinking.
But the enzyme dimeric active position prepared causes to be limited by serious mass transfer when its catalyzing reaction of macromolecule substrate mostly in inside
System, influences enzyme activity.
Therefore, a kind of crosslinked bio enzyme catalyst not limiting substrate transmission is prepared, for Kinetic Resolution and moves
State kinetic resolution of racemic α-phenylethylamine is of great significance.
Invention content
The purpose of the present invention is exactly to solve the above-mentioned problems and provides a kind of crosslinked bio enzyme catalyst and its application.
The purpose of the present invention is achieved through the following technical solutions：
A kind of crosslinked bio enzyme catalyst, the crosslinked bio enzyme catalyst are prepared by the following method to obtain：
(1) bovine serum albumin(BSA) is dissolved in phosphate buffer, obtains bovine serum albumin solution, add South Pole vacation
Silk Yeast-lipase B solution stirring, obtains enzyme-mixed liquid of protein；
(2) precipitating reagent and crosslinking agent are added into enzyme-mixed liquid of protein, is stirred under the conditions of ice-water bath, collects solid shaped bodies,
Solid shaped bodies are washed with phosphate buffer, after removing extra precipitating reagent and crosslinking agent, obtain cross-linking enzyme aggressiveness；
(3) phosphate buffer is added into cross-linking enzyme aggressiveness to be disperseed, crosslinking agent is added, under the conditions of ice-water bath
Stirring, obtains aldehyde group modified cross-linking enzyme aggressiveness；
(4) candida antarctica lipase B solution is added into aldehyde group modified cross-linking enzyme aggressiveness, under the conditions of ice-water bath
Stirring, is collected by centrifugation product, to get to product after washing, freeze-drying.
Preferably, a concentration of 25-75mg/mL of step (1) described bovine serum albumin(BSA).
Preferably, step (1) described phosphate buffering liquid concentration is 20mM, pH=7.5.
Preferably, a concentration of 8mg/mL of step (1) and step (4) described candida antarctica lipase B, South Pole vacation silk
The ratio of the volume of Yeast-lipase B and the quality of bovine serum albumin(BSA) is 1L/50-150g.
Preferably, the quality of step (1) described bovine serum albumin(BSA) and the volume ratio of phosphate buffer are 50-150g/
2L, the quality of step (3) described bovine serum albumin(BSA) and the volume ratio of phosphate buffer are 50-150g/3L.
Preferably, step (2) described precipitating reagent is ammonium sulfate, and the quality of ammonium sulfate and the mass ratio of bovine serum albumin(BSA) are
7-22。
Preferably, step (2) and step (3) described crosslinking agent are glutaraldehyde, the addition volume of step (2) crosslinking agent and ox
Sero-abluminous mass ratio is 3-7 μ L/5-15mg, the quality of the addition volume and bovine serum albumin(BSA) of step (3) crosslinking agent
Than for 1 μ L/1-3mg.
Preferably, step (2) stirs under the conditions of ice-water bath carries out the time of cross-linking reaction as 2h.
Preferably, step (4) stirs under the conditions of ice-water bath carries out the time of cross-linking reaction as 1h.
The crosslinked bio enzyme catalyst is applied to Kinetic Resolution α-phenylethylamine or Dynamic Kinetic Resolution α-phenylethylamine,
Metallic catalyst used is industrialized pd/C catalyst in the reaction of Dynamic Kinetic Resolution α-phenylethylamine.
The present invention uses secondary cross-linking solid carrier technology, and by precipitating reagent and crosslinking agent, the aggregation of enzyme and albumen is carried out
After crosslinking, crosslinking agent is modified again on the cross-linking enzyme aggressiveness surface of formation, makes to carry out phase between biological enzyme and enzyme aggressiveness again
The biocatalyst being mutually cross-linked to form, the crosslinked bio enzyme catalyst being prepared is in Kinetic Resolution and Dynamic Kinetic Resolution
Excellent catalytic activity is shown in α-phenylethylamine, effectively improves traditional cross-linking enzyme aggressiveness and the mass transfer of macromolecule substrate is limited
It makes, shows the series of advantages such as stability height, separable recycling, repeatable utilization, simple for process, cheap.
Compared with prior art, the beneficial effects of the present invention are：
(1) crosslinked bio enzyme catalyst prepared by the present invention is made by crosslinked mode twice inside and outside enzyme aggressiveness obtained
The active sites that antarctic candidia lipase can be exposed, the table in Kinetic Resolution and Dynamic Kinetic Resolution α-phenylethylamine
Reveal excellent catalytic activity, improves mass transfer and limit of traditional cross-linking enzyme aggressiveness to macromolecule substrate, stability is high, separates back
It receives, reuses 5 times and remain to keep higher fractionation and Dynamic Kinetic Resolution efficiency.
(2) preparation process of the invention is simple, mild condition, raw material are easy to get, are cheap, is environmentally safe.
Description of the drawings
Fig. 1 is the SEM pictures of crosslinked bio enzyme catalyst prepared by embodiment 1.
Specific implementation mode
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
50mg bovine serum albumin(BSA)s are dissolved in 2mL phosphate buffers, and it is molten that 1mL antarctic candidia lipases are added
Liquid is stirring evenly and then adding into 30 μ L of ammonium sulfate 1.083g and glutaraldehyde (50wt%), after being stirred 2 hours under the conditions of ice-water bath from
The heart collects solid shaped bodies, is used in combination phosphate buffer to wash repeatedly, obtains cross-linking enzyme aggressiveness.Plus 3mL phosphate buffers pair then
Cross-linking enzyme aggressiveness is disperseed, and 50 μ L glutaraldehydes (50wt%) are added after stirring evenly, and continues 30 points of stirring under the conditions of ice-water bath
Antarctic candidia lipase solution 1mL is added in clock, is stirred under the conditions of ice-water bath and carries out within 1 hour being crosslinked for second, washed after centrifugation
It washs, is put into freeze drier and is lyophilized, sample is labeled as Com-CLEAs-1, and Fig. 1 is the crosslinked bio enzyme catalyst prepared
SEM pictures.
Embodiment 2
100mg bovine serum albumin(BSA)s are dissolved in 2mL phosphate buffers, and 1mL antarctic candidia lipases are added
Solution is stirring evenly and then adding into 30 μ L of ammonium sulfate 1.083g and glutaraldehyde (50wt%), after being stirred 2 hours under the conditions of ice-water bath from
The heart collects solid shaped bodies, is used in combination phosphate buffer to wash repeatedly, obtains cross-linking enzyme aggressiveness.Plus 3mL phosphate buffers pair then
Cross-linking enzyme aggressiveness is disperseed, and 50 μ L glutaraldehydes (50wt%) are added after stirring evenly, and continues 30 points of stirring under the conditions of ice-water bath
Antarctic candidia lipase solution 1mL is added in clock, is stirred under the conditions of ice-water bath and carries out within 1 hour being crosslinked for second, washed after centrifugation
It washs, is put into freeze drier and is lyophilized, sample is labeled as Com-CLEAs-2.
Embodiment 3
150mg bovine serum albumin(BSA)s are dissolved in 2mL phosphate buffers, and 1mL antarctic candidia lipases are added
Solution is stirring evenly and then adding into 30 μ L of ammonium sulfate 1.083g and glutaraldehyde (50wt%), after being stirred 2 hours under the conditions of ice-water bath from
The heart collects solid shaped bodies, is used in combination phosphate buffer to wash repeatedly, obtains cross-linking enzyme aggressiveness.Plus 3mL phosphate buffers pair then
Cross-linking enzyme aggressiveness is disperseed, and 50 μ L glutaraldehydes (50wt%) are added after stirring evenly, and continues 30 points of stirring under the conditions of ice-water bath
Antarctic candidia lipase solution 1mL is added in clock, is stirred under the conditions of ice-water bath and carries out within 1 hour being crosslinked for second, washed after centrifugation
It washs, is put into freeze drier and is lyophilized, sample is labeled as Com-CLEAs-3.
Embodiment 4
100mg bovine serum albumin(BSA)s are dissolved in 2mL phosphate buffers, and 1mL antarctic candidia lipases are added
Solution is stirring evenly and then adding into 30 μ L of ammonium sulfate 1.548g and glutaraldehyde (50wt%), after being stirred 2 hours under the conditions of ice-water bath from
The heart collects solid shaped bodies, is used in combination phosphate buffer to wash repeatedly, obtains cross-linking enzyme aggressiveness.Plus 3mL phosphate buffers pair then
Cross-linking enzyme aggressiveness is disperseed, and 50 μ L glutaraldehydes (50wt%) are added after stirring evenly, and continues 30 points of stirring under the conditions of ice-water bath
Antarctic candidia lipase solution 1mL is added in clock, is stirred under the conditions of ice-water bath and carries out within 1 hour being crosslinked for second, washed after centrifugation
It washs, is put into freeze drier and is lyophilized, sample is labeled as Com-CLEAs-4.
Embodiment 5
100mg bovine serum albumin(BSA)s are dissolved in 2mL phosphate buffers, and 1mL antarctic candidia lipases are added
Solution is stirring evenly and then adding into 50 μ L of ammonium sulfate 1.083g and glutaraldehyde (50wt%), after being stirred 2 hours under the conditions of ice-water bath from
The heart collects solid shaped bodies, is used in combination phosphate buffer to wash repeatedly, obtains cross-linking enzyme aggressiveness.Plus 3mL phosphate buffers pair then
Cross-linking enzyme aggressiveness is disperseed, and 50 μ L glutaraldehydes (50wt%) are added after stirring evenly, and continues 30 points of stirring under the conditions of ice-water bath
Antarctic candidia lipase solution 1mL is added in clock, is stirred under the conditions of ice-water bath and carries out within 1 hour being crosslinked for second, washed after centrifugation
It washs, is put into freeze drier and is lyophilized, sample is labeled as Com-CLEAs-5.
Embodiment 6
100mg bovine serum albumin(BSA)s are dissolved in 2mL phosphate buffers, and 1mL antarctic candidia lipases are added
Solution is stirring evenly and then adding into 70 μ L of ammonium sulfate 1.083g and glutaraldehyde (50wt%), after being stirred 2 hours under the conditions of ice-water bath from
The heart collects solid shaped bodies, is used in combination phosphate buffer to wash repeatedly, obtains cross-linking enzyme aggressiveness.Plus 3mL phosphate buffers pair then
Cross-linking enzyme aggressiveness is disperseed, and 50 μ L glutaraldehydes (50wt%) are added after stirring evenly, and continues 30 points of stirring under the conditions of ice-water bath
Antarctic candidia lipase solution 1mL is added in clock, is stirred under the conditions of ice-water bath and carries out within 1 hour being crosslinked for second, washed after centrifugation
It washs, is put into freeze drier and is lyophilized, sample is labeled as Com-CLEAs-6.
Catalyst performance is tested：
The crosslinked bio enzyme catalyst of gained in example 1-6 is used in Kinetic Resolution α-phenylethylamine, specifically used step
Suddenly it is：Sequentially add the dry toluene of crosslinked bio enzyme catalyst 100mg, 2mL in 25mL single-necked flasks, 80 μ L ± α-phenylethylamines,
60 μ L ethyl methoxyacetates clog bottleneck with rubber stopper, open and are stirred to react 2 hours under the conditions of 70 DEG C of oil baths.Reaction product
Add internal standard compound normal heptane after being filtered with organic phase filter, the gas chromatographic detection equipped with CP7503 chromatographic columns is used in combination.Institute is active
Data be repeated three times it is above, error range within 5%, Kinetic Resolution highest theoretical yield be 50%, catalytic performance number
According to being shown in Table 1.
1 embodiment 1-6 catalytic performance data of table
| Catalyst | Reaction times | R- α-phenylethylamines conversion ratio (%) | R- amide selectivity (%) |
| Com-CLEAs-1 | 1 | 40 | 99 |
| Com-CLEAs-2 | 1 | 46 | 99 |
| Com-CLEAs-3 | 1 | 45 | 99 |
| Com-CLEAs-4 | 1 | 44 | 99 |
| Com-CLEAs-5 | 1 | 50 | 99 |
| Com-CLEAs-6 | 1 | 37 | 99 |
In order to test the service life of catalyst, 1 Com-CLEAs-5 will be used to be centrifuged, use phosphoric acid
It is reused after w salt buffer washes, remains to keep higher fractionation efficiency, specific data as shown in table 2 using 5 times.
Catalytic performance data are used for multiple times in 2 Com-CLEAs-5 of table
| Catalyst | Reaction times | R- α-phenylethylamines conversion ratio (%) | R- amide selectivity (%) |
| Com-CLEAs-5 | 1 | 50 | 99 |
| Com-CLEAs-5 | 2 | 49 | 99 |
| Com-CLEAs-5 | 3 | 50 | 99 |
| Com-CLEAs-5 | 4 | 49 | 99 |
| Com-CLEAs-5 | 5 | 47 | 99 |
During the catalyst Com-CLEAs-5 of 5 gained of example is used for Dynamic Kinetic Resolution α-phenylethylamine, specifically
It is using step：
The dry toluene of 70mg Pd/C, 100mg Com-CLEAs-5,2mL, 40 μ L ± α-are added in 15mL low-voltage hydrogenation kettles
Phenyl ethylamine, 60 μ L ethyl methoxyacetates lead to the H of 0.015mPa after tightening2And gas displacement is repeatedly carried out, in 70 DEG C of oil bath items
It opens and is stirred to react 4 hours under part.Reaction product adds internal standard compound normal heptane after being filtered with organic phase filter, is used in combination and is furnished with CP7503
The gas chromatographic detection of chromatographic column.All activity datas are repeated three times above, and error range is within 5%, Dynamic Kinetic
It is 100% to split highest theoretical yield, and catalytic performance data are shown in Table 3.
3 Com-CLEAs-5 of table is used for Dynamic Kinetic Resolution α-phenylethylamine
| Catalyst | Reaction times | R- α-phenylethylamines conversion ratio (%) | R- amide selectivity (%) |
| Com-CLEAs-5, Pd/C | 1 | 99 | 99 |
In order to test the service life of catalyst, 1 Pd/C and Com-CLEAs-5 mixed catalyst will be used to carry out
It centrifuges, is reused after being washed with phosphate buffer, remain to keep higher fractionation efficiency, specific data using 5 times
As shown in table 4.
4 Com-CLEAs-5 of table and Pd/C is used in mixed way catalytic effect
From table 1-4 as it can be seen that crosslinked bio enzyme catalyst prepared by the present invention is urged in Kinetic Resolution and in conjunction with Industrial Metal
Excellent catalytic activity is shown in the reaction of agent Pd/C Dynamic Kinetic Resolution α-phenylethylamines, stability is high, separates back
It receives, reuses 5 times and remain to keep higher fractionation and Dynamic Kinetic Resolution efficiency, and the substep crosslinking of enzyme more can be effective
Improve traditional cross-linking enzyme aggressiveness to the mass transfer and limit of macromolecule substrate, shows good prospects for commercial application.
The above is preferred embodiments of the present invention, but the present invention should not be limited to the example disclosure of that.
So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the scope of protection of the invention is both fallen within.
Claims (10)
1. a kind of crosslinked bio enzyme catalyst, which is characterized in that the crosslinked bio enzyme catalyst is prepared by the following method to obtain：
(1) bovine serum albumin(BSA) is dissolved in phosphate buffer, obtains bovine serum albumin solution, add South Pole vacation silk ferment
Female lipase B solution stirring, obtains enzyme-mixed liquid of protein；
(2) precipitating reagent and crosslinking agent are added into enzyme-mixed liquid of protein, is stirred under the conditions of ice-water bath, collects solid shaped bodies, uses phosphorus
Phthalate buffer washs solid shaped bodies, after removing extra precipitating reagent and crosslinking agent, obtains cross-linking enzyme aggressiveness；
(3) phosphate buffer is added into cross-linking enzyme aggressiveness to be disperseed, adds crosslinking agent, is stirred under the conditions of ice-water bath
It mixes, obtains aldehyde group modified cross-linking enzyme aggressiveness；
(4) candida antarctica lipase B solution is added into aldehyde group modified cross-linking enzyme aggressiveness, is stirred under the conditions of ice-water bath
It mixes, is collected by centrifugation product, to get to product after washing, freeze-drying.
2. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (1) described ox blood is pure
A concentration of 25-75mg/mL of albumen.
3. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (1) described phosphate is slow
Fliud flushing a concentration of 20mM, pH=7.5.
4. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (1) and step (4) are described
A concentration of 8mg/mL of candida antarctica lipase B, the volume of candida antarctica lipase B and the matter of bovine serum albumin(BSA)
The ratio of amount is 1L/50-150g.
5. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (1) described ox blood is pure
The quality of albumen and the volume ratio of phosphate buffer are 50-150g/2L, the quality of step (3) described bovine serum albumin(BSA) with
The volume ratio of phosphate buffer is 50-150g/3L.
6. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (2) described precipitating reagent is
Ammonium sulfate, the quality of ammonium sulfate and the mass ratio of bovine serum albumin(BSA) are 7-22.
7. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (2) and step (3) are described
Crosslinking agent is glutaraldehyde, and the addition volume of step (2) crosslinking agent and the mass ratio of bovine serum albumin(BSA) are 3-7 μ L/5-15mg, step
Suddenly the mass ratio of the addition volume of (3) crosslinking agent and bovine serum albumin(BSA) is 1 μ L/1-3mg.
8. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (2) is in ice-water bath condition
The time that lower stirring carries out cross-linking reaction is 2h.
9. a kind of crosslinked bio enzyme catalyst according to claim 1, which is characterized in that step (4) is in ice-water bath condition
The time that lower stirring carries out cross-linking reaction is 1h.
10. a kind of a kind of application of crosslinked bio enzyme catalyst as described in claim 1, which is characterized in that the crosslinking life
Object enzyme catalyst is applied to Kinetic Resolution α-phenylethylamine or Dynamic Kinetic Resolution α-phenylethylamine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735525A (en) * | 2018-12-28 | 2019-05-10 | 浙江工业大学 | A kind of preparation method of cross-linking enzyme aggressiveness |
| CN113214984A (en) * | 2021-04-19 | 2021-08-06 | 北京化工大学 | Preparation and use method of hollow fiber membrane-CLEAs enzyme membrane reactor |
| CN113444706A (en) * | 2020-03-25 | 2021-09-28 | 万华化学集团股份有限公司 | Imprinted lipase and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002061067A1 (en) * | 2001-02-01 | 2002-08-08 | Technische Universiteit Delft | A method of preparing cross-linked enzyme aggregates |
| CN1914316A (en) * | 2004-01-28 | 2007-02-14 | Csir公司 | Stabilization of enzymes |
| CN104593353A (en) * | 2015-01-12 | 2015-05-06 | 上海师范大学 | Biological enzyme-chemical compound catalyst and preparation method and application thereof |
| CN104911222A (en) * | 2015-06-24 | 2015-09-16 | 中国科学院广州能源研究所 | Method for preparing biodiesel by catalyzing vector-free immobilized lipase in ionic liquid system |
-
2018
- 2018-05-30 CN CN201810542157.5A patent/CN108753755B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002061067A1 (en) * | 2001-02-01 | 2002-08-08 | Technische Universiteit Delft | A method of preparing cross-linked enzyme aggregates |
| CN1914316A (en) * | 2004-01-28 | 2007-02-14 | Csir公司 | Stabilization of enzymes |
| CN104593353A (en) * | 2015-01-12 | 2015-05-06 | 上海师范大学 | Biological enzyme-chemical compound catalyst and preparation method and application thereof |
| CN104911222A (en) * | 2015-06-24 | 2015-09-16 | 中国科学院广州能源研究所 | Method for preparing biodiesel by catalyzing vector-free immobilized lipase in ionic liquid system |
Non-Patent Citations (1)
| Title |
|---|
| 何平: "无载体固定化猪胰脂肪酶", 《中国优秀硕士学位论文基础科学辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735525A (en) * | 2018-12-28 | 2019-05-10 | 浙江工业大学 | A kind of preparation method of cross-linking enzyme aggressiveness |
| CN109735525B (en) * | 2018-12-28 | 2021-05-07 | 浙江工业大学 | Preparation method of cross-linked enzyme polymer |
| CN113444706A (en) * | 2020-03-25 | 2021-09-28 | 万华化学集团股份有限公司 | Imprinted lipase and application thereof |
| CN113444706B (en) * | 2020-03-25 | 2023-03-03 | 万华化学集团股份有限公司 | Imprinted lipase and application thereof |
| CN113214984A (en) * | 2021-04-19 | 2021-08-06 | 北京化工大学 | Preparation and use method of hollow fiber membrane-CLEAs enzyme membrane reactor |
| CN113214984B (en) * | 2021-04-19 | 2022-05-27 | 北京化工大学 | Preparation and use method of hollow fiber membrane-CLEAs enzyme membrane reactor |
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