CN113462680A - Preparation of magnetic immobilized penicillin G acylase doped with divalent manganese ion - Google Patents
Preparation of magnetic immobilized penicillin G acylase doped with divalent manganese ion Download PDFInfo
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- CN113462680A CN113462680A CN202110857261.5A CN202110857261A CN113462680A CN 113462680 A CN113462680 A CN 113462680A CN 202110857261 A CN202110857261 A CN 202110857261A CN 113462680 A CN113462680 A CN 113462680A
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- 108010073038 Penicillin Amidase Proteins 0.000 title abstract description 51
- 229910001437 manganese ion Inorganic materials 0.000 title abstract description 51
- 238000002360 preparation method Methods 0.000 title abstract description 13
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 title abstract description 5
- 239000002122 magnetic nanoparticle Substances 0.000 abstract description 35
- 239000000243 solution Substances 0.000 abstract description 27
- 229920000858 Cyclodextrin Polymers 0.000 abstract description 25
- 229960004853 betadex Drugs 0.000 abstract description 25
- 102000004190 Enzymes Human genes 0.000 abstract description 23
- 108090000790 Enzymes Proteins 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 23
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 abstract description 18
- 229940056360 penicillin g Drugs 0.000 abstract description 15
- 238000011084 recovery Methods 0.000 abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 14
- 238000002156 mixing Methods 0.000 abstract description 13
- 238000006243 chemical reaction Methods 0.000 abstract description 12
- 239000008055 phosphate buffer solution Substances 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 11
- BPSIOYPQMFLKFR-UHFFFAOYSA-N trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CO[Si](OC)(OC)CCCOCC1CO1 BPSIOYPQMFLKFR-UHFFFAOYSA-N 0.000 abstract description 6
- 238000001291 vacuum drying Methods 0.000 abstract description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 abstract 2
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 abstract 2
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 abstract 2
- 229920001353 Dextrin Polymers 0.000 abstract 1
- 239000004375 Dextrin Substances 0.000 abstract 1
- 229910052797 bismuth Inorganic materials 0.000 abstract 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 abstract 1
- 235000019425 dextrin Nutrition 0.000 abstract 1
- 229910052742 iron Inorganic materials 0.000 abstract 1
- 239000000047 product Substances 0.000 description 24
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 19
- MTEZSDOQASFMDI-UHFFFAOYSA-N 1-trimethoxysilylpropan-1-ol Chemical compound CCC(O)[Si](OC)(OC)OC MTEZSDOQASFMDI-UHFFFAOYSA-N 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- NGHVIOIJCVXTGV-ALEPSDHESA-N 6-aminopenicillanic acid Chemical compound [O-]C(=O)[C@H]1C(C)(C)S[C@@H]2[C@H]([NH3+])C(=O)N21 NGHVIOIJCVXTGV-ALEPSDHESA-N 0.000 description 12
- NGHVIOIJCVXTGV-UHFFFAOYSA-N 6beta-amino-penicillanic acid Natural products OC(=O)C1C(C)(C)SC2C(N)C(=O)N21 NGHVIOIJCVXTGV-UHFFFAOYSA-N 0.000 description 12
- 239000001116 FEMA 4028 Substances 0.000 description 10
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 10
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000002245 particle Substances 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 8
- 238000011068 loading method Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 230000003197 catalytic effect Effects 0.000 description 6
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 5
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 239000011572 manganese Substances 0.000 description 5
- 239000011565 manganese chloride Substances 0.000 description 5
- 235000002867 manganese chloride Nutrition 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- 230000010355 oscillation Effects 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000001132 ultrasonic dispersion Methods 0.000 description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- 229910021577 Iron(II) chloride Inorganic materials 0.000 description 2
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000004105 Penicillin G potassium Substances 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000007306 functionalization reaction Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 229910052748 manganese Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 235000019368 penicillin G potassium Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000004593 Epoxy Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000593 microemulsion method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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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)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
- C12N9/84—Penicillin amidase (3.5.1.11)
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase
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Abstract
本发明涉及一种经二价锰离子掺杂的磁性固定化青霉素G酰化酶的制备，该方法包括以下步骤：⑴制备掺杂二价锰离子的磁性纳米粒子四氧化三铁@β‑环糊精；⑵将3‑缩水甘油醚氧基丙基三甲氧基硅烷的乙醇溶液与掺杂二价锰离子的磁性纳米粒子四氧化三铁@β‑环糊精混合反应，即得掺杂二价锰离子的磁性纳米粒子四氧化三铁@β‑环糊精‑g‑3‑缩水甘油醚氧基丙基三甲氧基硅烷载体；⑶在磷酸盐缓冲溶液溶解的掺杂二价锰离子的磁性纳米粒子四氧化三铁@β‑环糊精‑g‑3‑缩水甘油醚氧基丙基三甲氧基硅烷载体加入游离青霉素G酰化酶溶液，振荡反应后真空干燥，即得掺杂二价锰离子的磁性固定化青霉素G酰化酶载体。本发明可有效提高酶活回收率。The invention relates to the preparation of a magnetically immobilized penicillin G acylase doped with manganese ions. The method comprises the following steps: (1) Preparation of magnetic nanoparticles doped with manganese ions, iron tetroxide@β-ring dextrin; (2) mixing and reacting the ethanol solution of 3-glycidyloxypropyltrimethoxysilane and the magnetic nanoparticle ferric tetroxide@β-cyclodextrin doped with divalent manganese ions to obtain the mixed reaction Magnetic nanoparticles of manganese ion ferric oxide@β-cyclodextrin-g-3-glycidyloxypropyltrimethoxysilane carrier; (3) doped manganese ions dissolved in phosphate buffer solution Magnetic nanoparticle ferric oxide@β-cyclodextrin-g-3-glycidyloxypropyltrimethoxysilane carrier was added to free penicillin G acylase solution, and the oscillating reaction was followed by vacuum drying to obtain doped bismuth Magnetically immobilized penicillin G acylase carrier for manganese ions. The invention can effectively improve the recovery rate of enzyme activity.
Description
Technical Field
The invention relates to the technical field of functional material preparation, in particular to preparation of magnetic immobilized penicillin G acylase doped with divalent manganese ions.
Background
At present, penicillin G acylase is discovered for the first time in the 60 th 20 th century, is an important biocatalyst, and has strong specificity and high catalytic efficiency. It can catalyze penicillin G potassium to generate 6-aminopenicillanic acid. 6-aminopenicillanic acid occupies a huge market in the field of antibiosis and antiphlogosis because the 6-aminopenicillanic acid is used as a key intermediate of semi-synthetic penicillin medicaments.
Although penicillin G acylases have many advantages in catalytic processes, the special structural features make the conformation of the free penicillin G acylase extremely unstable, and conformational changes and even separation of the two subunits easily occur. Therefore, the direct use of free penicillin G acylase in catalytic processes still has many disadvantages, such as poor environmental tolerance, susceptibility to temperature, pH, etc. and significant reduction or even loss of enzyme activity. Moreover, after catalytic hydrolysis, penicillin G acylase is difficult to separate from the substrate and the product, and continuous operation is difficult to realize in the production process, which not only increases the operation difficulty, prolongs the process route, increases the economic cost, but also causes pollution to the product. The above-mentioned problems of penicillin G acylases have limited their industrial application in the production of 6-aminopenicillanic acid.
At present, the problem to be solved in the research of immobilized penicillin G acylase is the balance of high-level immobilized penicillin G acylase load, high enzyme activity recovery rate, high response rate and high reusability, particularly high enzyme activity recovery rate. Therefore, how to design a carrier with excellent performance and alleviate the inherent contradiction among the performance indexes has important research significance for ensuring that the immobilized penicillin G acylase obtains ideal results in the four aspects.
Disclosure of Invention
The invention aims to solve the technical problem of providing the preparation of the magnetic immobilized penicillin G acylase doped with the divalent manganese ions, which can improve the recovery rate of enzyme activity.
In order to solve the above problems, the preparation of the divalent manganese ion doped magnetic immobilized penicillin G acylase of the present invention comprises the following steps:
the preparation method comprises the following steps of preparing magnetic nano particle ferroferric oxide @ beta-cyclodextrin doped with bivalent manganese ions:
adding magnetic nano ferroferric oxide particles, beta-cyclodextrin and manganese dichloride into NaOH solution with the concentration of 4mol/L, mixing uniformly, and introducing N2Stirring and reacting for 1.5h at 80 ℃ to obtain a product A; vacuum drying the product A to constant weight to obtain bivalent manganese ion-doped magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin;
preparing a magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyl trimethoxy silane carrier doped with divalent manganese ions:
uniformly mixing an ethanol solution of 3-glycidyl ether oxypropyltrimethoxysilane and the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin doped with the divalent manganese ions according to the proportion of 1.30g/55mL, keeping the constant temperature of 20 ℃ under the protection of nitrogen, and stirring for reacting for 2.5 hours to obtain a product B; the product B is dried in vacuum to constant weight, and the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyl trimethoxy silane carrier doped with divalent manganese ions is obtained;
preparing a magnetic immobilized penicillin G acylase carrier doped with divalent manganese ions:
mixing the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-G-3-glycidyl ether oxypropyl trimethoxy silane carrier doped with the divalent manganese ions with 40.0 mM phosphate buffer solution with the pH = 8.0, adding 2.5 vol.% of free penicillin G acylase solution after ultrasonic dispersion, and drying a product C after oscillation reaction in vacuum to obtain the magnetic immobilized penicillin G acylase carrier doped with the divalent manganese ions.
The magnetic nano ferroferric oxide in the step is prepared by the following method: mixing 30.0 mL of OP-10, 44.0 mL of n-butanol and 100.0 mL of cyclohexane in sequence, fixing in a constant-temperature water bath at 20 ℃, and introducing nitrogen for 15 min; thereafter 1.2044 g FeCl were weighed3·6H2O、0.5262g FeCl2·4H2O and 50mL of distilled water are stirred uniformly, nitrogen is introduced for 30 min, and then 6.00 mL of ammonia water is added to continue reacting for 1 h to obtain a reactant; the reaction was dried in vacuo to constant weight.
The magnetic nano ferroferric oxide particles, the beta-cyclodextrin and the manganese dichloride in the step are mixed according to a mass ratio of 1: 5-6: 2-3; the ratio of the magnetic nano ferroferric oxide particles to the NaOH solution is 1 g: 100-110 mL.
The ethanol solution of the 3-glycidoxy propyl trimethoxy silane is obtained by diluting the 3-glycidoxy propyl trimethoxy silane to 16 g/L with absolute ethanol, adding distilled water according to 15-20 times of the volume of the mixed solution, and adjusting the pH value to 6 by successively adopting glacial acetic acid and ammonia water.
And the oscillation reaction in the step three is carried out under the conditions that the temperature is 30-40 ℃ and the time is 20-28 h.
The step three is that the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-G-3-glycidyl ether oxypropyl trimethoxy silane carrier doped with bivalent manganese ions, the phosphate buffer solution and the free penicillin G acylase solution are in a mass ratio of 1: 195: 5.
in the third step, the 2.5 vol.% free penicillin G acylase solution is prepared by adding 2.5 mL free penicillin G acylase enzyme solution into 100.00 mL 40.0 mM phosphate buffer solution with pH = 8.0 and mixing uniformly.
The 40.0 mM phosphate buffer solution with pH = 8.0 refers to 1.1000 g potassium dihydrogen phosphate (KH)2PO4) And 16.5200 g of dipotassium hydrogen phosphate (K)2HPO4·3H2O) is completely dissolved in a 100.00 mL beaker with 80 mL of distilled water, the solution is transferred to an 2000.00 mL volumetric flask, the beaker is washed with distilled water for a plurality of times, and the washing solution is transferred to the volumetric flask for constant volume.
The vacuum drying temperature is 50-60 ℃.
And the product A, the product B and the product C are obtained by utilizing magnet separation.
Compared with the prior art, the invention has the following advantages:
1. the magnetic nano ferroferric oxide with small particle size, high specific surface area and easy modification is used for immobilizing the penicillin G acylase, and the introduction of the magnetic nano ferroferric oxide is favorable for improving the response rate, the loading capacity and the reusability of the immobilized penicillin G acylase. Meanwhile, the introduction of the beta-cyclodextrin-based coating material with high hydrophilicity and biocompatibility is beneficial to maintaining the advantages of small particle size and high specific surface area of the magnetic nano ferroferric oxide, preventing the magnetic nano ferroferric oxide from being oxidized to keep higher magnetic saturation intensity and endowing the magnetic nano ferroferric oxide with the advantage of secondary functionalization, particularly, the introduction of the beta-cyclodextrin can construct a hydrophilic microenvironment for the immobilized penicillin G acylase, and is beneficial to improving the enzyme activity recovery rate of the immobilized penicillin G acylase.
2. The magnetic nano ferroferric oxide prepared by the reversed-phase microemulsion method is used as a basic carrier, so that the response rate and the loading capacity of the immobilized penicillin G acylase are improved, and the immobilized penicillin G acylase can be recycled and reused for multiple times by adding a magnetic field.
3. In the process of carrying out secondary functionalization on beta-cyclodextrin by using magnetic nano ferroferric oxide, the activity recovery rate and reusability of immobilized penicillin G acylase can be obviously improved by doping metal divalent manganese ions, and the recovery rate of a carrier is further improved to a certain extent.
4. According to the invention, a silane coupling agent 3-glycidyl ether oxypropyltrimethoxysilane is grafted on the surface of the magnetic nano-particle ferroferric oxide @ beta-cyclodextrin, an epoxy functional group is introduced, covalent fixation of penicillin G acylase is realized through click reaction, and the enzyme activity recovery rate and reusability are improved to a certain extent.
5. The method fixes the free penicillin G acylase on the carrier, and utilizes the advantage of good reusability of the carrier, thereby improving the environmental tolerance (namely the stability) of the penicillin G acylase on the one hand, and realizing the recoverability of the penicillin G acylase on the other hand, thereby realizing the green production with low cost.
6. The immobilized penicillin G acylase obtained by the invention has the enzyme activity of 31462U/G, the enzyme activity recovery rate of 94 percent and the loading amount of 102mg/G, and after being repeatedly used for 11 times, the enzyme activity retention rate is 72 percent and the carrier recovery rate is 89 percent.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a superparamagnetic diagram of a magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyloxypropyltrimethoxysilane carrier doped with divalent manganese ions in example 1 of the present invention. Wherein: (a) magnetic nano-particle ferroferric oxide; (b) the magnetic nano particle ferroferric oxide @ beta-cyclodextrin doped with bivalent manganese ions is prepared; (c) the magnetic nano particle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyltrimethoxysilane doped with divalent manganese ions.
FIG. 2 is an EDS diagram of the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin doped with divalent manganese ions in example 1 of the present invention.
FIG. 3 is an EDS diagram of the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidoxypropyltrimethoxysilane doped with divalent manganese ions in example 1 of the present invention.
FIG. 4 is a standard curve of 6-aminopenicillanic acid in example 1 of the present invention.
FIG. 5 shows the effect of the feed ratio of divalent manganese ions to beta-cyclodextrin on the loading capacity, enzyme activity and recovery rate of immobilized penicillin G acylase in example 1.
FIG. 6 is a graph showing the reusability of the carrier-immobilized penicillin G acylase of example 1 of the present invention.
Detailed Description
The preparation of magnetic immobilized penicillin G acylase doped with divalent manganese ions comprises the following steps:
the preparation method comprises the following steps of preparing magnetic nano particle ferroferric oxide @ beta-cyclodextrin doped with bivalent manganese ions:
adding magnetic nano ferroferric oxide particles, beta-cyclodextrin and manganese dichloride into NaOH solution with the concentration of 4mol/L, mixing uniformly, and introducing N2Stirring and reacting for 1.5h at 80 ℃, and separating by using a magnet to obtain a product A; and (3) drying the product A at 50-60 ℃ in vacuum to constant weight to obtain the magnetic nano particle ferroferric oxide @ beta-cyclodextrin doped with the divalent manganese ions.
Wherein: the mass ratio (g/g) of the magnetic nano ferroferric oxide particles, the beta-cyclodextrin and the manganese dichloride is 1: 5-6: 2-3; the proportion of the magnetic nano ferroferric oxide particles to the NaOH solution is 1 g: 100-110 mL.
The magnetic nano ferroferric oxide is prepared by the following method: mixing 30.0 mL of OP-10, 44.0 mL of n-butanol and 100.0 mL of cyclohexane in sequence, fixing in a constant-temperature water bath at 20 ℃, and introducing nitrogen for 15 min; thereafter 1.2044 g FeCl were weighed3·6H2O、0.5262g FeCl2·4H2O and 50mL of distilled water are stirred uniformly, nitrogen is introduced for 30 min, and then 6.00 mL of ammonia water is added to continue reacting for 1 h to obtain a reactant; washing the reactant, and drying the reactant in vacuum at 50-60 ℃ to constant weight to obtain the catalyst.
Preparing a magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyl trimethoxy silane carrier doped with divalent manganese ions:
uniformly mixing an ethanol solution of 3-glycidyl ether oxypropyltrimethoxysilane and magnetic nano particle ferroferric oxide @ beta-cyclodextrin doped with divalent manganese ions according to the proportion of 1.30g/55mL, keeping the temperature of 20 ℃ constant under the protection of nitrogen, stirring for reacting for 2.5 h, and separating by using a magnet to obtain a product B; and (3) drying the product B at 50-60 ℃ in vacuum to constant weight to obtain the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyl trimethoxysilane carrier doped with the divalent manganese ions.
Wherein: the ethanol solution of the 3-glycidoxypropyltrimethoxysilane is obtained by diluting the 3-glycidoxypropyltrimethoxysilane to 16 g/L with absolute ethanol, adding distilled water according to 15-20 times of the volume of the mixed solution, and adjusting the pH value to 6 with glacial acetic acid and ammonia water successively.
Preparing a magnetic immobilized penicillin G acylase carrier doped with divalent manganese ions:
mixing the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-G-3-glycidyl ether oxypropyltrimethoxysilane doped with divalent manganese ions with 40.0 mM phosphate buffer solution with the pH = 8.0, adding 2.5 vol.% of free penicillin G acylase solution after ultrasonic dispersion, carrying out oscillation reaction at 37 ℃ for 24 hours, washing for a plurality of times by using the phosphate buffer solution, recycling the magnet until the absorbance of the product C is less than 0.005 before and after two times measured by a PDAB color development method, and carrying out vacuum drying on the product C at 50-60 ℃ to obtain the magnetic immobilized penicillin G acylase doped with the divalent manganese ions.
Wherein: the mass ratio (G/G) of the ferroferric oxide @ beta-cyclodextrin-G-3-glycidyl ether oxypropyl trimethoxysilane carrier, the phosphate buffer solution and the free penicillin G acylase solution doped with the bivalent manganese ions is 1: 195: 5.
the 2.5 vol.% free penicillin G acylase solution is prepared by adding 2.5 mL free penicillin G acylase enzyme solution to 100.00 mL 40.0 mM phosphate buffer solution with pH = 8.0 and mixing well.
40.0 mM phosphate buffer solution with pH = 8.0 means that 1.1000 g potassium dihydrogen phosphate (KH)2PO4) And 16.5200 g of dipotassium hydrogen phosphate (K)2HPO4·3H2O) is completely dissolved in a 100.00 mL beaker with 80 mL of distilled water, the solution is transferred to an 2000.00 mL volumetric flask, the beaker is washed with distilled water for a plurality of times, and the washing solution is transferred to the volumetric flask for constant volume.
Example 1 preparation of a divalent manganese ion doped magnetic immobilized penicillin G acylase comprising the following steps:
the preparation method comprises the following steps of preparing magnetic nano particle ferroferric oxide @ beta-cyclodextrin doped with bivalent manganese ions:
adding 2.30 g of magnetic nano ferroferric oxide particles, 12.8g of beta-cyclodextrin and 6.69g of manganese dichloride into 250mL of NaOH solution with the concentration of 4mol/L, mixing uniformly, and introducing N2And the reaction was stirred at 80 ℃ for 1.5 h. After the reaction is finished, N is continuously introduced2Cooling, and separating by using a magnet to obtain a product A; and washing the product A, and drying the product A in vacuum at 55 ℃ to constant weight to obtain the magnetic nano particle ferroferric oxide @ beta-cyclodextrin doped with the divalent manganese ions.
Preparing a magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyl trimethoxy silane carrier doped with divalent manganese ions:
55mL of ethanol solution of 3-glycidyl ether oxypropyltrimethoxysilane and 1.30g of magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin doped with divalent manganese ions are uniformly mixed, the constant temperature of 20 ℃ is kept under the protection of nitrogen, and the mixture is stirred and reacts for 2.5 hours. After the reaction is finished, separating by using a magnet to obtain a product B; and washing and vacuum drying the product B at 50-60 ℃ to constant weight to obtain the magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyl trimethoxysilane carrier doped with the divalent manganese ions.
Preparing a magnetic immobilized penicillin G acylase carrier doped with divalent manganese ions:
mixing 0.3000G of magnetic nanoparticle ferroferric oxide @ beta-cyclodextrin-G-3-glycidyloxypropyltrimethoxysilane doped with divalent manganese ions with 30.0 mL of 40.0 mM phosphate buffer solution with pH = 8.0, removing the supernatant by using a magnet after ultrasonic dispersion, adding 60.0 mL of 2.5 vol.% free penicillin G acylase solution, carrying out oscillation reaction at 37 ℃ for 24h, washing for a plurality of times by using the phosphate buffer solution, recovering the magnet until the absorbance of the product C measured by a PDAB color development method and the absorbance of the product C measured at the front and back times are less than 0.005, and carrying out vacuum drying on the product C at 50-60 ℃ to obtain the magnetic immobilized penicillin G acylase doped with divalent manganese ions.
[ test for determining enzyme Activity, enzyme Loading amount, and recovery Rate of enzyme Activity ]
0.05G of the obtained divalent manganese ion-doped magnetic immobilized penicillin G acylase support was reacted with 10% (wt%) of 50 times the amount of penicillin G potassium for 5 minutes, and the reaction solution was diluted 50 times. 0.5mL of the supernatant was added to 3.5mL of PDAB solution to develop the color for 3 minutes, and then the content of 6-aminopenicillanic acid was determined by the PDAB color development method. The enzyme activity, the enzyme loading amount and the enzyme activity recovery rate are respectively calculated according to the following formula, and the test results are shown in figures 1-6.
In the formula:EA(U/g) is the enzyme activity of the immobilized PGA;EAR(%) is the recovery rate of enzyme activity of the immobilized PGA;ELC(mg/g) represents the supported amount of immobilized PGA;V(mL) represents the volume of 6-aminopenicillanic acid in the reaction system;C(mM) represents the concentration of 6-aminopenicillanic acid in the catalytic hydrolysis reaction system; 5 (min) represents the catalytic hydrolysis reaction time;m 2 (g) represents the dry weight of immobilized PGA after immobilization;m 1 (g) represents the dry weight of the composite carrier before immobilization.
As can be seen from FIG. 1, the magnetic regression lines of the magnetic nano-particle ferroferric oxide, the magnetic nano-particle ferroferric oxide @ beta-cyclodextrin doped with the divalent manganese ions and the magnetic nano-particle ferroferric oxide @ beta-cyclodextrin-g-3-glycidyl ether oxypropyltrimethoxysilane doped with the divalent manganese ions are uniform and pass through the origin, which indicates that the magnetic nano-particle ferroferric oxide, the magnetic nano-particle ferroferric oxide @ beta-cyclodextrin doped with the divalent manganese ions have superparamagnetism.
As can be seen from fig. 2, Mn accounts for about 10% (wt%) of the total mass, indicating that Mn ions were successfully doped.
As can be seen from FIG. 3, about 10% (wt%) Mn and about 0.18% (wt%) Si based on the total mass indicate that the modification with manganese ions and 3-glycidoxypropyltrimethoxysilane was successful.
FIG. 4 is a standard curve of 6-aminopenicillanic acid. When the concentration of 6-aminopenicillanic acid is in the range of 0.05-0.35mM, the absorbance and the concentration have good linear relation, and the linear formula is
As can be seen from fig. 5, with the increase of the doping of the divalent manganese ions, the enzyme activity increases first and then decreases, which indicates that the optimal charge ratio of the divalent manganese ions to the beta-cyclodextrin is 3: 1, the immobilized penicillin G acylase activity is 31462U/G, the enzyme activity recovery rate is 94 percent, and the loading capacity is 102 mg/G.
The enzyme activity retention of the obtained immobilized penicillin G acylase after 11 times of repeated use was 72% (see FIG. 6).
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| CN110760496A (en) * | 2019-05-07 | 2020-02-07 | 宁波大学 | A kind of co-crosslinking immobilization method of penicillin G acylase |
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| CN110760496A (en) * | 2019-05-07 | 2020-02-07 | 宁波大学 | A kind of co-crosslinking immobilization method of penicillin G acylase |
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