CN113388604A - Synthetic immobilized lipase and novel method for applying same to resolution of 3-phenyllactic acid enantiomer - Google Patents
Synthetic immobilized lipase and novel method for applying same to resolution of 3-phenyllactic acid enantiomer Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 36
- 108090001060 Lipase Proteins 0.000 title claims abstract description 23
- 102000004882 Lipase Human genes 0.000 title claims abstract description 23
- 239000004367 Lipase Substances 0.000 title claims abstract description 23
- 235000019421 lipase Nutrition 0.000 title claims abstract description 23
- VOXXWSYKYCBWHO-UHFFFAOYSA-N 3-phenyllactic acid Chemical class OC(=O)C(O)CC1=CC=CC=C1 VOXXWSYKYCBWHO-UHFFFAOYSA-N 0.000 title claims description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 54
- 239000013215 MIL-88B Substances 0.000 claims abstract description 50
- 102000004190 Enzymes Human genes 0.000 claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 claims abstract description 36
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 21
- 101001003495 Pseudomonas fluorescens Lipase Proteins 0.000 claims abstract description 16
- 101001064559 Pseudomonas fluorescens Lipase Proteins 0.000 claims abstract description 16
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 5
- VOXXWSYKYCBWHO-UHFFFAOYSA-M 3-phenyllactate Chemical class [O-]C(=O)C(O)CC1=CC=CC=C1 VOXXWSYKYCBWHO-UHFFFAOYSA-M 0.000 claims abstract 3
- 238000005809 transesterification reaction Methods 0.000 claims abstract 3
- 239000000243 solution Substances 0.000 claims description 15
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical group COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 10
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 claims description 7
- 239000011942 biocatalyst Substances 0.000 claims description 6
- 239000003054 catalyst Substances 0.000 claims description 6
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- 239000012621 metal-organic framework Substances 0.000 abstract description 8
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- 241000894007 species Species 0.000 abstract 1
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- 239000000758 substrate Substances 0.000 description 12
- 239000008363 phosphate buffer Substances 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 238000000967 suction filtration Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 150000002148 esters Chemical group 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
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- 239000000377 silicon dioxide Substances 0.000 description 3
- VOXXWSYKYCBWHO-MRVPVSSYSA-N (R)-3-phenyllactic acid Chemical compound OC(=O)[C@H](O)CC1=CC=CC=C1 VOXXWSYKYCBWHO-MRVPVSSYSA-N 0.000 description 2
- GPNNOCMCNFXRAO-UHFFFAOYSA-N 2-aminoterephthalic acid Chemical compound NC1=CC(C(O)=O)=CC=C1C(O)=O GPNNOCMCNFXRAO-UHFFFAOYSA-N 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000007810 chemical reaction solvent Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000000696 magnetic material Substances 0.000 description 2
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- 229910001220 stainless steel Inorganic materials 0.000 description 2
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- VOXXWSYKYCBWHO-QMMMGPOBSA-M (S)-3-phenyllactate Chemical compound [O-]C(=O)[C@@H](O)CC1=CC=CC=C1 VOXXWSYKYCBWHO-QMMMGPOBSA-M 0.000 description 1
- VOXXWSYKYCBWHO-QMMMGPOBSA-N (S)-3-phenyllactic acid Chemical compound OC(=O)[C@@H](O)CC1=CC=CC=C1 VOXXWSYKYCBWHO-QMMMGPOBSA-N 0.000 description 1
- MVDXXGIBARMXSA-PYUWXLGESA-N 5-[[(2r)-2-benzyl-3,4-dihydro-2h-chromen-6-yl]methyl]-1,3-thiazolidine-2,4-dione Chemical compound S1C(=O)NC(=O)C1CC1=CC=C(O[C@@H](CC=2C=CC=CC=2)CC2)C2=C1 MVDXXGIBARMXSA-PYUWXLGESA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000507 anthelmentic effect Effects 0.000 description 1
- 229940124339 anthelmintic agent Drugs 0.000 description 1
- 239000000921 anthelmintic agent Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
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- 239000008346 aqueous phase Substances 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 238000004130 chiral capillary electrophoresis Methods 0.000 description 1
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- 239000002178 crystalline material Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229950002375 englitazone Drugs 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940126904 hypoglycaemic agent Drugs 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
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- 238000000746 purification Methods 0.000 description 1
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- 239000002461 renin inhibitor Substances 0.000 description 1
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- 239000007787 solid Substances 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N9/14—Hydrolases (3)
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- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
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Abstract
MOFs具有合成简单、种类丰富、稳定性好等优点，是一种潜在的固定化酶载体，在工业应用中具有广阔的前景。本专利保护了一种以荧光假单胞菌脂肪酶为催化活性组分，以NH2‑MIL‑88B/Fe3O4磁性MOFs为最佳载体，通过共价键法合成磁性固定化酶PFL@NH2‑MIL‑88B/Fe3O4的新方法。该材料的酶负载量为241.79 mg PFL/g NH2‑MIL‑88B/Fe3O4，在酯交换拆分3‑苯基乳酸对映体反应中展现出优异的催化活性，反应24 h，总转化率为39.55%，产物对映体过剩量为96.76%；与游离脂肪酶相比，固定化酶具有更优异的耐受性及重复使用性，在重复使用6次后，其催化活性为初始的46.75%。MOFs have the advantages of simple synthesis, abundant species, and good stability. They are potential immobilized enzyme carriers and have broad prospects in industrial applications. This patent protects a method for synthesizing magnetic immobilized enzyme PFL by covalent bond method with Pseudomonas fluorescens lipase as catalytic active component and NH 2 ‑MIL‑88B/Fe 3 O 4 magnetic MOFs as optimal carrier A new method for @ NH2 ‑MIL‑88B/ Fe3O4 . The enzyme loading of the material was 241.79 mg PFL/g NH 2 ‑MIL‑88B/Fe 3 O 4 , and it exhibited excellent catalytic activity in the transesterification and separation of 3‑phenyllactate enantiomers. The reaction was performed for 24 h. The total conversion rate was 39.55%, and the product enantiomeric excess was 96.76%. Compared with free lipase, the immobilized enzyme had better tolerance and reusability. After 6 times of repeated use, its catalytic activity was Initial 46.75%.
Description
Technical Field
The invention belongs to the technical field of biocatalysis, and provides a method for preparing an optical homochiral compound by using a biocatalysis method, and a method for fixing Pseudomonas Fluorescens Lipase (PFL) on magnetic NH by using a covalent bond method2-MIL-88B/Fe3O4Preparation of immobilized enzyme (PFL @ NH)2-MIL-88B/Fe3O4). Reacting PFL @ NH2-MIL-88B/Fe3O4The chiral ligand is used as a biocatalyst for stereoselectively catalyzing ester exchange to split a 3-phenyllactic acid enantiomer.
Background
The 3-phenyl lactic acid is a synthetic precursor of a series of important compounds and is widely applied to the fields of food, biology, medicine, chemical industry and the like. The pharmacological effects produced by two different configuration of enantiomeric molecules are also different, and (R) -3-phenyllactic acid can be used to synthesize hypoglycemic agents, such as: englitazone, anthelmintics, and the like; the (S) -3-phenyl lactic acid can be used for synthesizing various chiral intermediates of antiviral and anticancer compounds and non-peptide renin inhibitors for treating hypertension. Therefore, the selective resolution of (R, S) -3-phenyl lactic acid has important significance in organic synthesis and medicinal chemistry. The traditional resolution method mainly adopts an asymmetric synthesis method and a chiral capillary electrophoresis method for resolution, however, the resolution method has the defects of low separation concentration, complex operation or difficult preparation of reagents and the like.
In recent years, enzymatic kinetic resolution has the advantages of mild reaction conditions, high catalytic efficiency, high stereoselectivity and the like, and is widely applied to resolution of racemates. The industrial application of lipase has a series of problems, which can be mainly summarized into the following aspects: (1) the separation and purification difficulty of the free enzyme is large: and the reaction substrate is difficult to separate from the reaction system, the reuse and the recovery are difficult to realize, the industrial cost is increased, and the industrial automation and the continuity are not facilitated. (2) Poor stability of free enzyme: is easy to be deactivated in high temperature, strong acid, strong alkali and organic solvent. (3) The activity of the free enzyme is to be improved: agglomeration or degradation is easy to occur in the reaction process to influence the enzyme activity, so that the enzyme catalysis performance is reduced. After the free enzyme is immobilized, the defects are effectively overcome, and the spatial structure and the active center of the enzyme can be basically maintained. However, since the immobilized carrier and the immobilization mode may interact with the enzyme, the structure of the enzyme, particularly the structure of an essential group of the catalytic reaction active center, is changed. Therefore, the mode of immobilization and the choice of the immobilization carrier have a significant influence in industrial production.
The carrier plays a decisive role in the preparation process of the immobilized enzyme, and in the process of synthesizing the immobilized enzyme, a proper carrier is selected according to the characteristics of free enzyme and the immobilization method. In general, immobilized enzyme carriers need to have several characteristics: (1) stability: the material has stable structure, certain acid and alkali resistance, and does not react with a substrate, a product and a medium in the process of enzyme catalytic reaction; (2) has biocompatibility: the three-dimensional structure of the enzyme can not be damaged, and the enzyme activity can be favorably exerted; (3) good permeability: is beneficial to the permeation of the substrate and the product. Immobilization of enzymes has been widely reported, and many carriers are available, such as sol-gel matrix, hydrogel, organic microparticles, mesoporous silica, and the like. However, these carriers have certain defects, such as easy enzyme denaturation caused by sol-gel matrix and limited mass transfer of the substrate in the sol-gel matrix; because the hydrogel and the organic particles are easy to expand and degrade, the enzyme fixed on the hydrogel and the organic particles is easy to lose and denature, and the mass transfer efficiency is low; although the mesoporous silica has various advantages, the mesoporous silica also has the problems that the structure cannot be reasonably designed, the surface is easy to change, and thus enzyme is denatured or lost, and the like.
With the rapid development of material science, the diversity of carrier materials is also developed. In recent years, MOFs have been gaining increasing attention as a new class of immobilized carriers. MOFs are crystalline materials formed by coordination of metal ions and organic ligands. Due to the diversity of the geometric connection between MOFs nodes and ligands, the structure of the MOFs is rich and diverse. In addition, the MOFs also have the advantages of high specific surface area, pore volume which can be designed and adjusted according to requirements, mild synthesis conditions, good water stability and thermal stability and the like. These advantages make MOFs a very potential class of enzyme-immobilized vectors.
The invention utilizes a covalent bond method to fix the pseudomonas fluorescens lipase on a magnetic material NH2-MIL-88B/Fe3O4Above (PFL @ NH)2-MIL-88B/Fe3O4) Using PFL @ NH2-MIL-88B/Fe3O4Excellent catalytic activity and stereospecificitySelectively, catalyzing ester exchange in an organic phase to split raceme 3-phenyl lactic acid, and preparing (R) -3-phenyl lactic acid and (S) -3-phenyl lactate with high optical activity. The method converts PFL @ NH2-MIL-88B/Fe3O4High temperature tolerance comparison with free PFL, PFL @ NH2-MIL-88B/Fe3O4Has higher thermal stability. PFL @ NH2-MIL-88B/Fe3O4Has excellent dispersibility and stability in the organic phase, is easy to separate and reuse, and obviously reduces the production cost. The technology solves the problems that the free enzyme can not be repeatedly used in an enzyme splitting method, the product and the enzyme are difficult to separate, and the reaction time is long.
Disclosure of Invention
The invention provides a method for fixing pseudomonas fluorescens lipase on NH by adopting a covalent bond method2-MIL-88B/Fe3O4Immobilized enzyme PFL @ NH prepared on2-MIL-88B/Fe3O4And further PFL @ NH2-MIL-88B/Fe3O4The method is applied to the catalytic ester exchange resolution of the 3-phenyl lactic acid enantiomer, and excellent yield and purity are obtained. After the free lipase passes through the immobilization reaction, compared with the free lipase, the thermal stability and reusability of the free lipase are remarkably provided.
The technical scheme of the invention is as follows: the invention uses FeCl3·6H2O and Fe3O4Preparing NH by taking 2-amino-1, 4-terephthalic acid as an organic ligand and N, N-dimethylformamide as a reaction solvent in the center of metal ions2-MIL-88B/Fe3O4A crystal; then with NH2-MIL-88B/Fe3O4For immobilizing carrier, in phosphate buffer solution with pH of 5.0-9.0, activating NH by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide2-MIL-88B/Fe3O4The activated carboxyl group reacts with the amino group of the lipase at the temperature of 5-65 DEG C oC, reacting for 4-30 h to fix the free lipaseAt NH2-MIL-88B/Fe3O4The above. In the ester exchange resolution 3-phenyl lactic acid enantiomer reaction, methyl tert-butyl ether is used as a reaction medium, racemic 3-phenyl lactic acid and vinyl acetate are used as substrates, the concentration of the 3-phenyl lactic acid enantiomer is 5-20 mmol/L, the concentration of the vinyl acetate is 100-600 mmol/L, and 5-60 mg PFL @ NH is added2-MIL-88B/Fe3O4As biocatalyst at a temperature of 30-75 deg.C oAnd C, stirring and heating for reaction for a certain time. After the reaction is finished, a certain amount of samples are taken to carry out qualitative and quantitative detection on the product through a high performance liquid chromatograph, and the substrate conversion rate and the enantiomeric excess of the product are calculated.
Compared with the prior art, the invention has the following advantages:
the invention utilizes magnetic material NH2-MIL-88B/Fe3O4As an immobilized carrier, PFL @ NH is successfully prepared by a covalent bond method2-MIL-88B/Fe3O4. Due to PFL @ NH2-MIL-88B/Fe3O4Has the characteristics of magnetism, simple separation, high separation efficiency and the like. The immobilized enzyme is synthesized by a covalent bond method, the interaction between the lipase and a carrier is enhanced, the tolerance of the lipase is improved, the immobilized enzyme has higher stereoselectivity compared with free enzyme through reaction in a high-temperature and long-time organic reagent, and PFL @ NH is used2-MIL-88B/Fe3O4When the lipase is used as a reaction catalyst, the defects of instability of lipase, difficulty in separation from a product and the like can be overcome, and the target product with high yield and high purity can be obtained. At the same time, due to PFL @ NH2-MIL-88B/Fe3O4Is insoluble in both aqueous and organic phases and can be separated by simple centrifugation after reaction. The method is simple and convenient to implement and operate, green and environment-friendly, products are easy to separate, and the reuse rate of the catalyst is high.
[ detailed description ] according to the present embodiment
The method comprises the following specific steps:
first, testing and analyzing
Liquid phase: agilent 1260, chromatography column: a Daicel Chiralpak AD-H column (250 mm. times.4.6 mm, 5 μm) was set at a wavelength of 210 nm and a column temperature of 25 ℃. The mobile phase is a mixed solution of normal hexane and isopropanol with the proportion of 90:10, contains 0.1 percent of trifluoroacetic acid and has the flow rate of 1 mL/min; the amount of sample was 10. mu.L.
Enzyme content: the immobilization efficiency was measured by the bicinchoninic acid method (BCA method) using bovine serum albumin as a standard protein. And (4) analyzing and detecting the concentration of the protein in the enzyme solution by using a microplate reader. The immobilization yield of lipase was indirectly determined by comparing the difference in protein concentration between the solution before and after immobilization.
Second, example
Example 1
Weighing 300 mg of Fe3O4Adding 90 mL of N, N-dimethylformamide, and carrying out ultrasonic treatment until the N, N-dimethylformamide is completely dissolved; 240 mg of 2-aminoterephthalic acid and 715.5 mg of FeCl are weighed respectively3·6H2O, dissolved in 45 mL of N, N-dimethylformamide respectively; further adding the above-mentioned Fe3O4、FeCl3·6H2Mixing the O solution in N, N-dimethylformamide, adding the 2-amino terephthalic acid solution, ultrasonically mixing for 15 minutes, filling the mixed solution into a 300 mL stainless steel autoclave lined with Teflon, and putting the stainless steel autoclave into an oven to react for 3-24 hours at 170 ℃. Finally, carrying out suction filtration, and washing with water and methanol for 3 times respectively to obtain NH2-MIL-88B/Fe3O4And (4) crystals.
Example 2
60 mg of NH are weighed2-MIL-88B/Fe3O4Adding into a 50 mL centrifuge tube, adding 3.6 mL phosphate buffer (50 mM, pH = 6.0), adding 200 μ L20 mg/mL EDC solution, and placing the centrifuge tube into a shaker (15 ℃, 200 rpm) and shaking for 1.5 h; then, 200 μ L of 20 mg/mL NHS solution was added, and the shaking was continued for 1.5 hours; finally, 40 mg of lipase was added and shaking was continued in a shaker (25 ℃, 200 rpm) for 24 h. The product was isolated by suction filtration and washed 3 times with phosphate buffer (50 mM, pH = 6.0) to isolate PFL @ NH2-MIL-88B/Fe3O4，PFL@NH2-MIL-88B/Fe3O4The enzyme loading was 182.33 mg PFL/g NH2-MIL-88B/Fe3O4。
Example 3
60 mg of NH are weighed2-MIL-88B/Fe3O4To a 50 mL centrifuge tube, 3.6 mL phosphate buffer (50 mM, pH = 7.5) was added, 200. mu.L of 20 mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution was added, and the centrifuge tube was placed in a shaker (15 ℃, 200 rpm) and shaken for 1.5 h; then, 200 μ L of 20 mg/mL N-hydroxysuccinimide solution was added, and the shaking was continued for 1.5 hours; finally, 40 mg of lipase was added and shaking was continued in the shaker (5 ℃ C., 200 rpm) for 24 h. The product was isolated by suction filtration and washed 3 times with phosphate buffer (50 mM, pH = 7.5) to isolate PFL @ NH2-MIL-88B/Fe3O4，PFL@NH2-MIL-88B/Fe3O4The enzyme loading was 237.89 mg PFL/g NH2-MIL-88B/Fe3O4。
Example 4
60 mg of NH are weighed2-MIL-88B/Fe3O4To a 50 mL centrifuge tube, 3.6 mL phosphate buffer (50 mM, pH = 7.5) was added, 200. mu.L of 20 mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution was added, and the centrifuge tube was placed in a shaker (15 ℃, 200 rpm) and shaken for 1.5 h; then, 200 μ L of 20 mg/mL N-hydroxysuccinimide solution was added, and the shaking was continued for 1.5 hours; finally, 20 mg of lipase was added and shaking was continued in a shaker (15 ℃, 200 rpm) for 24 h. The product was isolated by suction filtration and washed 3 times with phosphate buffer (50 mM, pH = 7.5) to isolate PFL @ NH2-MIL-88B/Fe3O4，PFL@NH2-MIL-88B/Fe3O4The enzyme loading was 195.13 mg PFL/g NH2-MIL-88B/Fe3O4。
Example 5
60 mg of NH are weighed2-MIL-88B/Fe3O4To a 50 mL centrifuge tube, 3.6 mL phosphate buffer (50 mM, pH = 7.5) was added, 200. mu.L of 20 mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution was added, and the centrifuge tube was placed in a shaker (15 ℃, 200 rpm) and shaken for 1.5 h; then, 200. mu.L of the mixture was addedContinuing to shake the N-hydroxysuccinimide solution of 20 mg/mL for 1.5 hours; finally, 30 mg of lipase was added and shaking was continued in the shaker (15 ℃, 200 rpm) for 16 h. The product was isolated by suction filtration and washed 3 times with phosphate buffer (50 mM, pH = 7.5) to isolate PFL @ NH2-MIL-88B/Fe3O4，PFL@NH2-MIL-88B/Fe3O4The enzyme loading was 213.01 mg PFL/g NH2-MIL-88B/Fe3O4。
Example 6
Using 20 mM of 3-phenyllactic acid, 120 mM of vinyl acetate as a reaction substrate and 2 mL of methyl tert-butyl ether as a reaction solvent, and adding 5 mg of free lipase or 25 mg of PFL @ NH2-MIL-88B/Fe3O4As a biocatalyst, carrying out constant-temperature water bath oscillation reaction for 12 h in a sealed reaction tube of 25 mL, wherein the temperature is 55 ℃, and after the reaction is finished, analyzing the substrate conversion rate and the enantiomeric excess of the product by using a high performance liquid chromatograph. In the reaction taking free lipase as a catalyst, the total conversion rate is 12.11 percent, and the enantiomeric excess value of the product is 95.62 percent; in the reaction using immobilized enzyme as catalyst, the total conversion rate is 14.71%, and the enantiomeric excess value of the product is 97.52%.
Example 7
Using 20 mM of 3-phenyl lactic acid, 120 mM of vinyl acetate as a reaction substrate and 2 mL of methyl tert-butyl ether as a reaction medium, and adding 10 mg of free lipase or 50 mg of PFL @ NH2-MIL-88B/Fe3O4As a biocatalyst, carrying out constant-temperature water bath oscillation reaction for 32 h in a sealed reaction tube of 25 mL, wherein the temperature is 50 ℃, and after the reaction is finished, analyzing the substrate conversion rate and the enantiomeric excess of the product by using a high performance liquid chromatograph. In the reaction taking free lipase as a catalyst, the total conversion rate is 45.52 percent, and the enantiomeric excess value of the product is 91.32 percent; in the reaction using immobilized enzyme as catalyst, the total conversion rate is 43.37%, and the enantiomeric excess value of the product is 95.53%.
Example 8
Using 20 mM of 3-phenyl lactic acid, 120 mM of vinyl acetate as a reaction substrate and 2 mL of methyl tert-butyl ether as a reaction medium, and adding 50 mg of PFL @ NH2-MIL-88B/Fe3O4As a biocatalyst, carrying out constant-temperature water bath oscillation reaction for 20 h in a sealed reaction tube of 25 mL, wherein the temperature is 50 ℃, and after the reaction is finished, analyzing the substrate conversion rate and the enantiomeric excess of the product by using a high performance liquid chromatograph. Then the solid-liquid separation is carried out on the reaction system, the immobilized enzyme PFL @ NH is recovered after the solid part is freeze-dried2-MIL-88B/Fe3O4And then carrying out catalytic reaction under the same conditions. The analysis result shows that: after the reaction is repeated twice, the total conversion rate is 31.63 percent, and the enantiomeric excess value of the product is 96.82 percent; after the reaction is repeated for four times, the total conversion rate is 24.98%, and the enantiomeric excess value of the product is 97.16%; after six repetitions of the reaction, the total conversion was 17.96% and the enantiomeric excess of the product was 97.31%.
The above examples merely express several embodiments of the present invention, and the description thereof is more specific and detailed, but the technical scope thereof is not limited to the above embodiments. It will be apparent to those skilled in the art that various modifications and embodiments can be made without departing from the spirit of the invention, and these are within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (4)
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| CN111662898A (en) * | 2020-06-30 | 2020-09-15 | 湖南理工学院 | New lipase immobilization technology and method for applying same to enantiomer resolution |
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| CN108642119A (en) * | 2018-03-24 | 2018-10-12 | 湖南理工学院 | A kind of method that stereoselectivity lipase-catalyzed esterification splits 2- (4- aminomethyl phenyls) propionic acid enantiomer |
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