CN112695020B - Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide - Google Patents
Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide Download PDFInfo
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- CN112695020B CN112695020B CN202011508304.0A CN202011508304A CN112695020B CN 112695020 B CN112695020 B CN 112695020B CN 202011508304 A CN202011508304 A CN 202011508304A CN 112695020 B CN112695020 B CN 112695020B
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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)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- 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)
Abstract
The invention belongs to the technical field of polylactic acid production, and particularly discloses a chemically modified lipase for synthesizing L-lactide, and a preparation method of the lipase. The invention adopts epoxy isopropyl oleate to chemically modify lipase B from candida antarctica, and the chemically modified lipase is obtained. The chemically modified lipase has high enzyme activity, and the yield is obviously improved when the lipase is used for catalyzing and generating L-lactide.
Description
Technical Field
The invention relates to the technical field of polylactic acid production, in particular to synthesis of an important intermediate lactide for polylactic acid production, and especially relates to a lipase catalyst for synthesizing L-lactide.
Background
Polylactic acid (PLA) is a polymer material with wide application, and is one of the best choices for solving the problem of white pollution. At present, two-step method is mainly adopted in industry to synthesize PLA, and lactide is an important intermediate for synthesizing PLA. The quality of lactide is one of the key factors affecting the ring-opening polymerization of high molecular weight PLA. The optimization and control of the parameters of the reaction and purification processes in the lactide synthesis process directly determine the quality and yield of lactide, thereby affecting various properties of PLA.
The lactide is synthesized by a chemical two-step method, which is a relatively common method at present, but the polymerization degree of lactic acid oligomer is difficult to control in the chemical reaction process; meanwhile, the cracking process is carried out at high temperature, which may cause the generated lactide to have racemization reaction and may cause decomposition, carbonization, coking, oxidation and the like of materials, so the chemical synthesis method has many disadvantages.
Compared with the traditional chemical synthesis method, the lipase mediated catalytic reaction has the advantages of mild reaction conditions, high product specificity and the like, and can introduce the synthesis of polylactic acid. The Candida Antarctica Lipase B (CALB) is used as the most common lipase for enzymatic synthesis, is widely applied to organic synthesis reactions such as hydrolysis or alcoholysis, ester synthesis, ester exchange, lactone synthesis, polypeptide synthesis, high polymer synthesis, stereoisomer resolution and the like, and is one of the most widely used enzyme catalysts at present. However, the application of the natural CALB lipase has some disadvantages, such as easy inactivation in non-natural environments such as organic solvents, high temperature, extreme pH, etc., which limits the industrial application of the CALB lipase. For example, the literature (Jeon B W, lee J, kim H S, et al, lipase-catalyzed enzymatic synthesis of (R, R) -lactic from alkyl lactate to product PDLA (poly D-lactic acid) and stereocomplex PLA (poly lactic acid) [ J ]. Journal of Biotechnology,2013,168 (2): 201-207.) discloses a process for the preparation of lactide, which produces L-lactide as a product using L-methyl lactate as a starting material, methyl tert-butyl ether as a solvent, and Novozym 435, a commercially available enzyme for B, as a catalyst, but has a low yield of only about 54%, and is not suitable for industrial production.
Chemical modification is an important means to improve the performance of enzymes. Chemical modification has the advantages of low price, short period, feasibility in a laboratory, easy creation of novel enzymology properties and the like. Although some research progress has been made on chemically modified lipase, the current research is still limited to the research on common modifying agents such as fatty acid, amino acid, acid anhydride, polyethylene glycol (PEG) and polysaccharide, and the properties of the modified lipase, such as catalytic activity, stability and organic solvent resistance, cannot be effectively improved at the same time to meet the needs of the reaction.
The main subject of chemical modification is generally the free amino acid residues on the surface of the enzyme protein. It is described in the literature (Weber H K, zuegg J, faber K, equivalent. Molecular reasons for lipase-sensitive enzyme acetyl derivatives [ J ]. Journal of Molecular Catalysis B enzyme, 1997,3 (1): 131-138.) that CALB lipase has many free amino, hydroxyl, phenolic hydroxyl, carboxyl and sulfhydryl groups on its surface, especially 2-3% of its surface is covered by lysine residues, and these functional groups can react with carboxylic acid, ketone, aldehyde, etc. to perform acylation, alkylation, redox and aromatic ring substitution, thereby modifying CALB lipase.
Therefore, the molecular modification is carried out on the CALB lipase to strengthen the catalytic performance of the CALB lipase, a new enzyme variety with higher activity, better stability and stronger environmental tolerance is provided for industrial application, and the method has important practical application value.
Disclosure of Invention
The invention mainly solves the technical problem of providing a preparation method of chemically modified lipase, and also provides the chemically modified lipase obtained by the preparation method and application of the lipase. The yield of the L-lactide catalyzed and synthesized by the chemically modified lipase is obviously improved.
Specifically, in a first aspect, the present invention provides a process for producing a chemically modified lipase obtained by treating Candida antarctica lipase B with a solvent containing isopropyl epoxyoleate.
As a preferred embodiment of the present invention, the preparation method comprises the steps of: candida antarctica lipase B was added to the buffer and then mixed with a solvent containing isopropyl epoxy oleate.
Preferably, the solvent is an organic solvent; more preferably petroleum ether. And/or the buffer solution is a sodium phosphate buffer solution.
In a preferred embodiment of the present invention, the concentration of isopropyl oleate in the solvent containing isopropyl oleate is 0.1-0.5% by volume, preferably 0.1-0.3% by volume, and more preferably 0.2% by volume.
As a preferred embodiment of the invention, after the Candida antarctica lipase B is treated by a solvent containing epoxy isopropyl oleate, the method further comprises the step of adsorption treatment by using a solid phase carrier; the solid-phase carrier is preferably macroporous resin.
As a preferred embodiment of the present invention, the method for preparing the chemically modified lipase comprises the steps of:
(1) Adding the candida antarctica lipase B into a sodium phosphate buffer solution, and then uniformly mixing with a petroleum ether solution of epoxy isopropyl oleate;
(2) And (2) adding the solid-phase carrier macroporous resin into the mixed system obtained in the step (1), separating out solid and supernatant after oscillation adsorption, cleaning the solid, and then drying in vacuum to obtain a chemically modified immobilized lipase product.
Wherein the structural formula of the modifying reagent isopropyl epoxy oleate is as follows:
in a second aspect, the present invention provides a chemically modified lipase which is a Candida Antarctica Lipase B (CALB) enzyme modified with isopropyl epoxyoleate.
The chemically modified lipase is prepared by the preparation method.
Preferably, the chemically modified lipase is a chemically modified immobilized lipase.
In a third aspect, the invention provides the use of the chemically modified lipase or the lipase prepared by the preparation method in the catalytic synthesis of lactide; further preferred is the use in the catalytic synthesis of L-lactide.
In a fourth aspect, the invention provides a method for synthesizing lactide, which is catalyzed and synthesized by the chemically modified lipase or the lipase prepared by the preparation method.
In a preferred embodiment of the present invention, the lactide is enzymatically produced using lactic acid as a starting material, and more preferably produced using L-lactic acid as a starting material.
As a preferred embodiment of the present invention, the method for synthesizing lactide comprises the steps of:
mixing chemically modified lipase with L-lactic acid, molecular sieve and solvent for reaction;
then filtering, collecting reaction liquid, concentrating and removing the solvent;
and distilling the liquid obtained by concentration under a vacuum condition, collecting fractions at the temperature of 130 +/-5 ℃, and then cooling and crystallizing to obtain the L-lactide.
As a preferred embodiment, the mass ratio of the chemically modified lipase to L-lactic acid is 1: (15 to 25), more preferably 1: (15 to 20), more preferably 1:18.
as a preferred embodiment, the molecular sieve is a 5A molecular sieve.
As a preferred embodiment, the solvent is petroleum ether.
As a preferred embodiment, the reaction temperature is from 20 to 35 ℃ and preferably from 20 to 30 ℃.
The invention adopts epoxy isopropyl oleate to chemically modify lipase B (CALB) from candida antarctica, and the chemically modified lipase is obtained mainly by modifying amino of lysine on the surface of CALB zymoprotein.
The chemically modified lipase can directly catalyze L-lactic acid molecules to synthesize L-lactide in an organic solvent system. And compared with the unmodified CALB, the enzyme activity of the chemically modified lipase is obviously improved, and the yield of the L-lactide generated by catalysis is obviously improved.
Detailed Description
The technical solution of the present invention will be described in detail by specific examples.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the instruments and reagents used are commercially available instruments and reagents.
Example 1
The embodiment provides a chemically modified immobilized lipase, which is prepared by the following steps: weighing 1g of lipase (Candida antarctica lipase B, a product of Sigma company), adding 50mL of sodium phosphate buffer (50mM, pH7.5), adding 500mL of petroleum ether (which contains 1mL of epoxy isopropyl oleate), and stirring thoroughly for 24h;
then adding 50g of immobilized carrier macroporous resin ECR1030M (product of Purolite company), oscillating and adsorbing for 8h (30 ℃,120 r/min) by a water bath shaker, filtering and separating solid and supernatant, washing the immobilized enzyme by using a sodium phosphate buffer solution, and then drying for 3h in vacuum at 35 ℃ to obtain the chemically modified immobilized lipase CALB-M1, and storing for later use in a refrigerator at 4 ℃.
Comparative example 1
This example provides an immobilized lipase, which differs from example 1 in that no isopropyl epoxyoleate modification is performed, and is prepared by the following steps: weighing 1g of lipase (Candida antarctica lipase B, a product of Sigma), adding 50mL of sodium phosphate buffer (50mM, pH7.5), adding 500mL of petroleum ether, and stirring thoroughly for 24h;
then adding 50g of immobilized carrier macroporous resin ECR1030M (product of Purolite company), oscillating and adsorbing for 8h (30 ℃,120 r/min) by a water bath shaker, filtering and separating solid and supernatant, washing the immobilized enzyme by using a sodium phosphate buffer solution, and then drying for 3h in vacuum at 35 ℃ to obtain immobilized lipase CALB1, and storing for later use in a refrigerator at 4 ℃.
Example 2
The lipases obtained in example 1 and comparative example 1 were each subjected to activity measurement. The measurement method is as follows:
the immobilized lipase (20 mg each of CALB1 and CALB-M1) was weighed, 5mL of a sodium phosphate buffer (50mM, pH7.5) was added to a 50mL conical flask, the flask was preheated for 15min in a 37 ℃ constant temperature water bath shaker, then 5mL of tributyrin was added, the flask was shaken at 200rpm for 20min, and then 1mL of 95% ethanol was added to stop the reaction. Blank is to replace enzyme solution with buffer solution. After the reaction is terminated, the fatty acid produced by the reaction is titrated with 0.05mol/L sodium hydroxide standard solution, and phenolphthalein is used as an indicator.
The enzyme activity is defined as: under the above conditions, the amount of enzyme required to produce 1. Mu. Mol of fatty acid per minute was 1 activity unit expressed as LU/mg. The calculation formula is as follows:
wherein: V1-NaOH volume for titration, mL;
V2-NaOH volume for blank titration, mL;
c is the concentration of NaOH standard solution, mol/L;
50-0.05 mol/L sodium hydroxide solution 1ml is equivalent to fatty acid 50 mu mol;
n 1-dilution of sample;
0.05-conversion factor of NaOH standard solution concentration;
t-reaction time, min.
According to the formula, calculating the enzyme activity U of the lipase chemically modified by the epoxy oil isopropyl ester 1 72LU/mg lipase activity U without chemical modification of epoxy isopropyl oleate 0 It is 58LU/mg.
The enzyme activity determination data show that the enzyme activity of the lipase chemically modified by the epoxy oil isopropyl ester is obviously improved compared with that of unmodified lipase.
Example 3
The method for synthesizing the L-lactide by using the lipase with chemical modification catalysis comprises the following steps: the reaction system is carried out in a 1000mL conical flask with a plug, and 10.0g of chemically modified immobilized lipase CALB-M, 180.0g (2.0 mol) of L-lactic acid, 20.0g of 5A molecular sieve and 450mL of petroleum ether which are prepared in the example 1 are added into the reaction flask; the reaction was carried out at 25 ℃ and 400rpm for 48h.
After the reaction is finished, filtering and removing the 5A molecular sieve, the immobilized enzyme catalyst and the like, and collecting reaction liquid; vacuum concentrating the collected reaction liquid at 60 +/-2 ℃ to remove a solvent petroleum ether to obtain a light yellow viscous liquid; distilling the viscous liquid under the vacuum condition of-0.09 MPa, discarding low-temperature front fraction, collecting 130 + -5 deg.C fraction, cooling, and crystallizing to obtain 109.4g of L-lactide white crystal with yield of 76% (109.4/180) and content of 99.5%.
Comparative example 2
Adopts chemically unmodified lipase to catalyze and synthesize L-lactide, and comprises the following steps: the reaction system was carried out in a 1000mL conical flask with a stopper, and 10.0g of the immobilized lipase CALB1 prepared in comparative example 1, 180.0g (2.0 mol) of L-lactic acid, 20.0g of 5A molecular sieve, and 450mL of petroleum ether were added to the reaction flask. The reaction was carried out at 25 ℃ and 400rpm for 48h.
After the reaction is finished, filtering and removing the 5A molecular sieve, the immobilized enzyme catalyst and the like, and collecting reaction liquid; the collected reaction liquid is vacuum concentrated at 60 +/-2 ℃ to remove the solvent petroleum ether, and light yellow viscous liquid is obtained. Distilling the viscous liquid under-0.09 MPa vacuum, discarding low-temperature front fraction, collecting 130 + -5 deg.C fraction, cooling, and crystallizing to obtain L-lactide white crystal 77.8g with yield of 54% (77.8/180) and content of 99.0%.
Although theoretically 100% of L-lactic acid is synthesized as L-lactide, 2 molecules of L-lactic acid are dehydrated to form one molecule of L-lactide, and the theoretical yield is 80%.
As can be seen from the results of example 3 and comparative example 2, compared with CALB1, the lipase chemically modified by isopropyl epoxy oleate has the advantages that the enzyme activity is obviously improved, the yield of L-lactide generated by catalysis is improved by 22%, and the yield is improved to 76% from 54%.
Example 4
The content of L-lactide obtained in example 3 and comparative example 2 was measured. The detection method comprises the following steps:
(1) Weighing 0.15g L-lactide standard sample, dissolving with dichloromethane, diluting to 25mL volumetric flask, filtering with organic phase filter membrane in 2mL sample flask, and determining lactide peak area A by gas-phase chromatograph Sign (ii) a Chromatographic conditions are as follows: and (3) chromatographic column: HP-5 column (30 m.times.0.32 mm. Times.0.32 mm), flow rate: 1mL/min; the calculation method comprises the following steps: external standard method;
(2) Respectively weighing 0.15g of L-lactide finished product obtained by catalyzing CALB1 and CALB-M1, dissolving with dichloromethane, fixing the volume to 25mL volumetric flask, filtering with organic phase filter membrane in 2mL sample bottle, and determining lactide peak area A by gas-phase chromatograph Sample (II) (ii) a Chromatographic conditions are as follows: a chromatographic column: HP-5 column (30 m.times.0.32 mm.times.0.32 mm), flow rate: 1mL/min; the calculation method comprises the following steps: and (4) an external standard method. The calculation formula is as follows:
in the formula: m is a unit of Sign board -weighing the mass of the standard sample, g;
m sample (A) -weighing the mass of the sample, g;
W sign board -mass fraction of standard sample (i.e. amount of L-lactide contained).
It was calculated from the above formula that the content of L-lactide produced in example 3, i.e., L-lactide produced by CALB-M1, was 99.5%, and the content of L-lactide produced in comparative example 2, i.e., L-lactide produced by CALB1, was 99.0%. Thus, the purity of the L-lactide prepared by the chemically modified lipase is further improved.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (17)
1. A preparation method of chemically modified lipase is characterized in that the chemically modified lipase is prepared by treating Candida antarctica lipase B with a solvent containing epoxy isopropyl oleate;
the preparation method comprises the following steps: adding candida antarctica lipase B into a buffer solution, and then mixing with a solvent containing epoxy isopropyl oleate; the solvent is petroleum ether, and the buffer solution is a sodium phosphate buffer solution;
after the candida antarctica lipase B is treated by a solvent containing epoxy isopropyl oleate, the method also comprises the step of adsorption treatment by using a solid phase carrier, wherein the solid phase carrier is macroporous resin.
2. The method according to claim 1, wherein the concentration of isopropyl oleate in the solvent containing isopropyl oleate is 0.1-0.5% by volume.
3. The method according to claim 2, wherein the concentration of isopropyl oleate in the solvent containing isopropyl oleate is 0.1-0.3% by volume.
4. The production method according to any one of claims 1 to 3, characterized by comprising the steps of:
(1) Adding the candida antarctica lipase B into a sodium phosphate buffer solution, and then uniformly mixing with a petroleum ether solution of epoxy isopropyl oleate;
(2) And (2) adding solid-phase carrier macroporous resin into the mixed system obtained in the step (1), separating out solid and supernatant after oscillation adsorption, cleaning the solid, and then drying in vacuum to obtain a chemically modified immobilized lipase product.
5. A chemically modified lipase, wherein said chemically modified lipase is a candida antarctica lipase B enzyme modified with isopropyl epoxyoleate.
6. The chemically modified lipase according to claim 5, characterized in that the chemically modified lipase is a chemically modified immobilized lipase.
7. A lipase produced by the production method according to any one of claims 1 to 4.
8. Use of the chemically modified lipase of any of claims 5-7 for the catalytic synthesis of lactide.
9. The use according to claim 8, wherein the chemically modified lipase is used for the catalytic synthesis of L-lactide.
10. A method for synthesizing lactide, which is characterized in that the lactide is prepared by catalytic synthesis of the chemically modified lipase of any one of claims 5 to 7.
11. The synthesis method according to claim 10, characterized in that the lactide is enzymatically produced using lactic acid as starting material.
12. The synthesis method according to claim 11, wherein the lactide is enzymatically produced using L-lactic acid as a starting material.
13. The method of synthesis according to claim 10, comprising the steps of:
mixing chemically modified lipase with L-lactic acid, molecular sieve and solvent for reaction;
then filtering, collecting reaction liquid, concentrating and removing the solvent;
and distilling the liquid obtained by concentration under vacuum condition, collecting fractions with the temperature of 130 +/-5 ℃, and then cooling and crystallizing to obtain the L-lactide.
14. The method of claim 13, wherein the mass ratio of the chemically modified lipase to L-lactic acid is 1: (15-25); the molecular sieve is a 5A molecular sieve; the solvent is petroleum ether; the reaction temperature is 20-35 ℃.
15. The method of synthesizing according to claim 14, wherein the mass ratio of the chemically modified lipase to L-lactic acid is 1: (15 to 20).
16. The method of synthesizing according to claim 15, wherein the mass ratio of the chemically modified lipase to L-lactic acid is 1:18.
17. the synthesis method according to claim 14, wherein the reaction temperature is 20 to 30 ℃.
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| WO2012036973A2 (en) * | 2010-09-15 | 2012-03-22 | Beeler, Nicole M. | Process for immobilization of candida antarctica lipase b |
| WO2015113848A1 (en) * | 2014-01-28 | 2015-08-06 | Ernst-Moritz-Arndt-Universität Greifswald | Process for the enzymatic production of oligo-/polyesters |
| CN107709457A (en) * | 2015-06-12 | 2018-02-16 | 卡比奥斯公司 | biodegradable polyester composition and application thereof |
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