CN1320688A - Carrier for immobilizing penicillin amidase - Google Patents
Carrier for immobilizing penicillin amidase Download PDFInfo
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- CN1320688A CN1320688A CN 00106228 CN00106228A CN1320688A CN 1320688 A CN1320688 A CN 1320688A CN 00106228 CN00106228 CN 00106228 CN 00106228 A CN00106228 A CN 00106228A CN 1320688 A CN1320688 A CN 1320688A
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- 108010073038 Penicillin Amidase Proteins 0.000 title claims abstract description 14
- 230000003100 immobilizing effect Effects 0.000 title 1
- 102000004190 Enzymes Human genes 0.000 claims abstract description 75
- 108090000790 Enzymes Proteins 0.000 claims abstract description 75
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims description 30
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 12
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 239000004382 Amylase Substances 0.000 claims description 2
- 102000013142 Amylases Human genes 0.000 claims description 2
- 108010065511 Amylases Proteins 0.000 claims description 2
- 102000018832 Cytochromes Human genes 0.000 claims description 2
- 108010052832 Cytochromes Proteins 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 102000004882 Lipase Human genes 0.000 claims description 2
- 108090001060 Lipase Proteins 0.000 claims description 2
- 239000004367 Lipase Substances 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 235000019418 amylase Nutrition 0.000 claims description 2
- 238000009826 distribution Methods 0.000 claims description 2
- 235000019421 lipase Nutrition 0.000 claims description 2
- 230000003993 interaction Effects 0.000 claims 1
- 239000000969 carrier Substances 0.000 abstract description 11
- 239000013335 mesoporous material Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 238000011068 loading method Methods 0.000 abstract description 2
- 229930182555 Penicillin Natural products 0.000 description 14
- 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 description 13
- 229940049954 penicillin Drugs 0.000 description 13
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- 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 9
- 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 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 8
- 125000000524 functional group Chemical group 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 108010091086 Recombinases Proteins 0.000 description 5
- 102000018120 Recombinases Human genes 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002808 molecular sieve Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 229910052710 silicon Inorganic materials 0.000 description 3
- 239000010703 silicon Substances 0.000 description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007306 functionalization reaction Methods 0.000 description 2
- -1 highly basic Substances 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- ULGJWNIHLSLQPZ-UHFFFAOYSA-N 7-[(6,8-dichloro-1,2,3,4-tetrahydroacridin-9-yl)amino]-n-[2-(1h-indol-3-yl)ethyl]heptanamide Chemical compound C1CCCC2=NC3=CC(Cl)=CC(Cl)=C3C(NCCCCCCC(=O)NCCC=3C4=CC=CC=C4NC=3)=C21 ULGJWNIHLSLQPZ-UHFFFAOYSA-N 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- 239000004115 Sodium Silicate Substances 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- ANBBXQWFNXMHLD-UHFFFAOYSA-N aluminum;sodium;oxygen(2-) Chemical compound [O-2].[O-2].[Na+].[Al+3] ANBBXQWFNXMHLD-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 108010003977 aminoacylase I Proteins 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001354 calcination Methods 0.000 description 1
- RTYJTGSCYUUYAL-YCAHSCEMSA-L carbenicillin disodium Chemical compound [Na+].[Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)C(C([O-])=O)C1=CC=CC=C1 RTYJTGSCYUUYAL-YCAHSCEMSA-L 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000002444 silanisation Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910001388 sodium aluminate Inorganic materials 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 229910052911 sodium silicate Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
本发明涉及一种可用作酶固定化载体的介孔材料,该介孔材料的结构特征及表面性质均具有可调变性。将该介孔材料作为酶,特别是青霉素酰化酶的固定化载体,利用采用适宜的负载方式在载体内孔表面成功地实现了酶的固定化,获得了高于游离酶和固定于其它载体表面的固定化酶酶活。The invention relates to a mesoporous material which can be used as an enzyme immobilization carrier, and the structural characteristics and surface properties of the mesoporous material are adjustable. The mesoporous material is used as the immobilized carrier of enzyme, especially penicillin acylase, and the enzyme is successfully immobilized on the surface of the inner hole of the carrier by adopting a suitable loading method, which is higher than that of free enzyme and other carriers. Immobilized enzyme activity on the surface.
Description
Enzyme is a kind of biological catalyst, and its chemical nature is a protein.Because it has the catalytic activity height, selects characteristics such as specificity is strong, reaction conditions gentleness, enzyme has been widely used in fields such as biochemical industry, medication chemistry, foodstuffs industry, light industry.At present, though the enzyme of having found reaches thousands of kinds, be used for have only tens kinds of suitability for industrialized production, the major cause of restriction enzyme industrial applications is:
1) because resolvase is water miscible catalyzer, microbiological deterioration or gathering take place easily and inactivation, and the existence of heat or strong acid, highly basic, organic solvent etc. also may cause the forfeiture of enzymic activity;
2) owing to enzyme behind the homogeneous catalytic reaction is difficult to separate with reactant and product, therefore,
(a) enzyme recovery difficulty can not repeat to utilize again, thereby causes the loss of enzyme, and is unreasonable economically;
(b) enzyme is difficult to reclaim and causes realizing operate continuously, can only disposable periodical operation;
(c) residual a large amount of enzyme species in the product, the impure product performance that have a strong impact on of product.
Research starts from the beginning of the 60 ' s about enzyme immobilization; 60 ' s end; immobilized aminoacylase is used for the amino acid whose optical resolution of DL-and realizes industrialization, becomes the beginning that immobilized enzyme is used for industry, has from then on also stimulated the research about immobilization technology and the immobilized enzyme character and the catalytic property of enzyme greatly.With enzyme immobilization, because the change of the change of enzyme molecule microenvironment of living in and enzyme molecular configuration etc. is compared with resolvase, immobilized enzyme has with appropriate carriers:
(1) thermostability of enzyme, chemical stability improve greatly, and immobilized enzyme also may produce the new feature [1] that is different from resolvase;
(2) immobilized enzyme is easy to separate with reactant, product, helps the repeated use of enzyme;
(3), and quality product is improved because therefore immobilized enzyme and product separate easily have simplified purifying technique;
(4) can realize operate continuously.
The development of enzyme immobilization technology is achieved the large-scale industrial application of enzyme.Therefore, the immobilization research of enzyme becomes an important directions of zymochemistry research in recent years.
Up to now, the preparation method of immobilized enzyme is broadly divided into four kinds, i.e. absorption method, covalent attachment method, crosslinking and entrapping method.Absorption method is divided into physisorphtion and ionic adsorption method; Combine with ionic linkage, Van der Waals force etc. between enzyme and carrier; Bonding force between the two a little less than, so enzyme easily runs off, and is difficult for destroyed and the enzyme higher structure changes advantages such as less but this kind method has enzyme active center.The covalent attachment method be with enzyme with covalent bonds on carrier, during concrete operations or with the relevant group activation of carrier surface, then with the enzyme molecule in relevant group coupling, or connect bifunctional reagent at carrier surface, then the enzyme coupling is got on.The immobilized enzyme that this method makes combines with carrier firmly, difficult drop-off, but this method severe reaction conditions, complicated operation, and violent reaction conditions causes that easily the zymoprotein higher structure changes, destroy the part active centre, therefore often can not obtain active higher immobilized enzyme.Crosslinking is meant and earlier enzyme is adsorbed on the insoluble carrier, uses difunctional or poly functional reagent then, makes and carries out intermolecular cross-linking between the enzyme molecule, forms cross-linked structure and makes the method for enzyme immobilization.Also there are deficiencies such as reaction conditions is violent, enzyme loss alive in this kind method.Entrapping method is that enzyme is embedded in the micro-capsule.The inapplicable macromolecular reaction thing of immobilized enzyme (substrate) of this method preparation, and the easy inactivation of enzyme, essential ingenious design reaction conditions generally is used to prepare immobilized cell.Four kinds of methods respectively have relative merits, generally speaking, absorption method/covalent method are used in combination; Industrial widely used be the covalent attachment method.
By several common methods of above-mentioned preparation immobilized enzyme as can be known, active higher, stability better, can be used for industrialized immobilized enzyme and be the requirement of used carrier:
1) carrier (host) surface have can with interactional functional groups is taken place by load enzyme molecule (guest), maybe can give functional groups by surface modification;
2) functional groups of carrier surface quantity and distributing suitably not only, and approaching easily;
3) carrier must be a porous material, has higher specific surface area;
4) carrier has bigger aperture, to reduce the diffusional resistance that exists in the reaction process as far as possible;
5) carrier should have enough chemical stabilities and mechanical stability, and (as enzymic catalytic reaction) carrier surface group does not participate in reaction in chemical reaction process, and keeps structure constant under reaction conditions;
6) the carrier granularity is suitable, is convenient to react separating of back and product.
At present, use more enzyme immobilization carrier to mainly contain superpolymer and porous inorganic material.As enzyme immobilization carrier, the structure of superpolymer itself and character have determined it to have following weakness:
1) though synthesising macromolecule copolymer is not so difficult, if will control pore structure and the quantity of surface-functional group and the non-easy thing that distributes thereof of superpolymer;
2) some functional groups is in the height interlinkage net of superpolymer, although make moderate progress through situation after the swelling of solvent, but still may be difficult to by reactant molecule approaching, thereby reduce the utilization ratio of carrier surface;
3) thermostability of polymer support, mechanical stability and solvent resistance a little less than.
Though at the weakness of polymer support, people develop and have used some mineral-type carriers, as amorphous compounds such as diatomite, silica gel, aluminum oxide, sintered glasses.Compare with organic class carrier, inorganic carrier has remarkable advantages undoubtedly aspect stable, but because therefore the structure weakness of amorphous carrier itself still exists certain limitation.For example, because the aperture of amorphous carrier is excessive, therefore not only to molecular size and shape non-selectivity, and gathering inactivation that still can not inhibitory enzyme; If adopt molecular sieve then can overcome the limitation of amorphous carrier as carrier with fixed orifices size.Molecular sieve not only can screen molecular size and shape, but also has many interesting character, is rich in functional groups monohydroxy etc. as high-specific surface area, hydrophilic or hydrophobic nature, solvent resistance, surface.But, the aperture of micro porous molecular sieve is generally below 2nm, because the restriction of port size, not only the accessibility of functional groups is limited, and some large volume species are difficult to enter in duct or the cage, therefore, or at all can't load large volume species, or charge capacity is too low, also can cause collapsing of structure sometimes.
The invention provides a kind of enzyme immobilization carrier material with unique texture, but the constructional feature of functionalization slightly acidic hydroxyl is rich on the wide aperture, high-ratio surface and the surface that utilize this solid support material, with the fixation support of this solid support material as enzyme, adopt suitable mode of loading, not only in its holes of nano size, successfully realize the immobilization of enzyme, but also can obtain to be higher than the immobilized enzyme enzyme work of other carrier.
The invention provides the solid support material that can be used for enzyme immobilization.One of feature of carrier of the present invention is that it has distribution homogeneous and adjustable nano-scale aperture (seeing embodiment 1).Therefore, its bigger aperture makes surface active groups have bigger accessibility, allows that the large volume guest molecule enters in the hole.
Two of the feature of carrier of the present invention is, oh group is rich on its surface, with the probe molecule method sign of carrier surface character is found, adsorb alkaline probe molecule after, the FT-IR bands of a spectrum of carrier surface hydroxyl of the present invention disappear, and show that these hydroxyls show acid (see figure 1).
Three of the feature of carrier of the present invention is that the number of its surface acidity group can carry out modulation according to target.As shown in table 1, in carrier of the present invention, introduce aluminium element, carrier surface acid amount increases.
The carrier absorption pyridine amount that table 1.Al content is different
Si/Al?ratio 25 50 ∝
A(μVs) 3.2×10
6 1.4×10
6 0.33×10
6
Annotate: the pyridine desorption peaks area that the A-chromatogram detects, the pyridine amount of its expression carrier surface absorption
Four of the feature of carrier of the present invention is that the strength of acid of its surface hydroxyl also can be carried out modulation by changing the skeleton composition in building-up process.
Five of the feature of carrier of the present invention is that its surface hydroxyl can directly use as the immobilization functional group;
Six of the feature of carrier of the present invention is that its surface hydroxyl is functionalization further.As shown in Figure 2, after the silylating reagent processing, the FT-IR bands of a spectrum of carrier surface hydroxyl of the present invention disappear, and illustrate that these hydroxyls with silylating reagent reaction have taken place.
Seven of the feature of carrier of the present invention is that it has up to 1000m
2The specific surface area of/g helps improving the charge capacity of functional groups.
Eight of the feature of carrier of the present invention is that it has good thermostability, acid resistance, solvent resistance and certain alkali resistance (except the highly basic).
Nine of the feature of carrier of the present invention is that its surface shows inertia in enzyme-catalyzed reaction.
Solid support material of the present invention can be used for the immobilization of the immobilization, particularly penicillin acylase of trypsinase, papoid, amylase, horseradish peroxidase, cytochrome C, lipase etc.Penicillin acylase is the catalyzer that the cracking of catalysis penicillin prepares 6-amino-penicillanic acid (6-APA).6-APA is a key intermediate of producing semisynthetic penicillin, on the amino of 6-APA, connect some side chain, can obtain the semisynthetic penicillin of efficient, wide spectrum, easy administration,, amoxycillin blue or green zero plain and Pyocianil etc. as penbritin, methoxy benzyl.Because the widespread use clinically of these semisynthetic penicillins makes the requirement of 6-APA growing.At present, the preparation method of 6-APA mainly contains chemical cracking method and enzyme process.Because there is severe reaction conditions in chemical method, yield is low, side reaction is many and problem such as three-waste pollution, shows in the world mainly with Production by Enzymes 6-APA.But,, then, can't compete with chemical method economically owing to the weakness of resolvase if use the hydrolytic process of resolvase catalysis penicillin.In recent years, because the development of enzyme immobilization technology, people utilize the immobilized penicillin acylated enzyme hydrolyzing penicillin to produce 6-APA.Because immobilized penicillin acylated enzyme has stability height, separate easily, can realize characteristics such as operate continuously, on technology, all can compete economically with chemical method.From 1993, the U.S., Britain began to produce 6-APA with immobilized penicillin acylated enzyme, and states such as France, Japan, Italy, Germany, Hungary also all actively develop relevant research work.The research work of China's this respect is started late; though owing to working in concert of each side; aspect immobilized enzyme, obtain gratifying results, but also be in the main present situation of leaning on import fixation support or immobilized penicillin acylase to satisfy semisynthetic antibiotics production demand.The present invention is expected to change this present situation that is limited by foreign products or technology at present.
The process of the carrier immobilized penicillin acylase of the present invention can be described below: get a certain amount of carrier of the present invention; with predetermined enzyme liquid/carrier enzyme liquid more an amount of than adding; under predetermined temperature, pH value, vibrate the regular hour, realize the immobilization of enzyme with direct immobilization way.Enzyme liquid of the present invention/carrier is than between 1-30, between the preferred 5-25, and more preferably 15-25; The immobilization temperature is 0-45 ℃, preferred 15-45 ℃, and more preferably 33-40 ℃; Immobilization pH is 6.0-8.0, preferred 6.0-7.6, more preferably 6.3; The immobilization time is 1-60 hour, preferred 2-24 hour, and more preferably 2-4 hour.
In preferred embodiments, the enzyme of the carrier immobilized penicillin acylase of the present invention is lived and is 511U/g/min.
Carrier of the present invention can and reclaim by simple filtration and reactants separate.
Embodiment 1
With hexadecyl trimethyl ammonium bromide (aqueous solution of 25wt%) is template, is the silicon source with the Na/Si=0.75 sodium silicate solution, the synthesis of silica-base mesoporous material.The ratio of components of reaction mixture is 4SiO
2: 1.5Na
2O: CTABr: 250H
2O.Under the magnetic agitation, the silicon source is added template solution by ratio of components, stir 1.0h under the room temperature.After putting into 98 ℃ of baking oven stationary water thermal crystallisation 24h, this solution is reduced to room temperature, regulate pH value to 10-11, and then at 98 ℃ of stationary water thermal crystallisation 120h.Filter, with the deionized water thorough washing.After 98 ℃ of dryings,, obtain solid support material at 500 ℃ of constant temperature calcinings 10h at least.The most probable aperture of this carrier is 3.3nm, and specific surface area is 800m
2/ g.
4 parts of 0.6g carriers mixed with 3ml penicillin acylase solution respectively, and the pH value of mixed solution is transferred to 6.30,7.26,7.58 and 7.70 respectively, put into shaking table, 25 ℃ of vibrations 24 hours.Filter,, get immobilized penicillin acylated enzyme with the deionized water thorough washing.
Table 2 is listed in the enzyme work of said fixing enzyme.
Fixed immobilized enzyme enzyme is lived under the different pH of table 2.
Immobilization pH value immobilized enzyme enzyme (U/g.min) alive
6.30 194
7.26 177
7.58 163
7.70 146
Embodiment 2
The carrier of embodiment 1.
4 parts of 0.6g carriers mix with 3ml penicillin acylase solution respectively, under pH=6.30, put into the shaking table of constant temperature in differing temps respectively, vibrate 24 hours.Filter,, get immobilized penicillin acylated enzyme with the deionized water thorough washing.
Table 3 is listed in the enzyme work of said fixing enzyme.
Fixed immobilized enzyme enzyme is lived under table 3. differing temps
Fixed temperature (℃) immobilized enzyme enzyme (U/g.min) alive
25 194
33 199
40 236
45 213
Embodiment 3
The carrier of embodiment 1.
6 parts of 0.6g carriers mix with 3ml penicillin acylase solution respectively, under pH=6.30, put into constant temperature at 25 ℃ shaking table, vibrate respectively 2,4,8,12,24 and 48 hours.Filter,, get immobilized penicillin acylated enzyme with the deionized water thorough washing.
Table 4 is listed in the enzyme work of said fixing enzyme.
Fixed immobilized enzyme enzyme is lived under table 4. differing temps
Set time (hour) immobilized enzyme enzyme (U/g.min) alive
2 300
4 320
8 303
12 297
24 194
48 169
Embodiment 4
The carrier of embodiment 1.
4 parts of 0.6g carriers mix with 3,6,9 and the penicillin acylase solution of 15ml respectively, under pH=6.30, put into constant temperature at 25 ℃ shaking table, vibrate 24 hours.Filter,, get immobilized penicillin acylated enzyme with the deionized water thorough washing.
Table 5 is listed in the enzyme work of said fixing enzyme.
The different enzyme liquid/carriers of table 5. are lived than following fixed immobilized enzyme enzyme
Enzyme liquid/carrier is than (ml/g) immobilized enzyme enzyme (U/g.min) alive
5 194
10 200
15 336
25 376
Embodiment 5
Similar process described in the preparation of carrier and the embodiment 1, just in the silicon source with after template solution mixes, press Si/Al mol ratio=100,50,25 more respectively and measure the adding sodium aluminate solutions.Obtain the carrier that difference contains the Al amount.The structural characteristic parameter of these carriers is listed in table 6.
Get above-mentioned solid support material 0.6g respectively, mix, under pH=6.30, put into shaking table, 25 ℃ of vibrations 24 hours with 3ml penicillin acylase solution.Filter,, get immobilized penicillin acylated enzyme with the deionized water thorough washing.
Table 7 is listed in the enzyme work of said fixing enzyme.
The different Si/Al of table 6. are than the structural parameter of carrier
Si/Al is than most probable aperture ratio pore volume (cm
3/ g) specific surface area
(nm) (m
2/g)
∝ 3.32 0.83 800
100 3.26 0.97 1000
50 3.26 0.98 1092
Immobilized enzyme enzyme on the different Si/Al carriers of 25 3.24 0.87 985 tables 7. is lived
Carrier S i/Al is than immobilized enzyme enzyme (U/g.min) alive
∝ 364
100 475
50 491
25 511
Description of drawings
Fig. 1 is the hydroxyl FT-IR spectrogram of carrier of the present invention before and after the absorption pyridine
(a) before the absorption pyridine; (b) behind the absorption pyridine
Fig. 2 handles the hydroxyl FT-IR spectrogram of back carrier of the present invention for silanization.
Claims (10)
1. enzyme immobilization carrier with unique texture feature and surface properties.
2. the carrier of claim 1, its constitutional features is to have allows that the large volume guest molecule enters the nano-scale aperture in the hole.
3. the carrier of claim 1, its constitutional features are that the surface is rich in the slightly acidic oh group.
4. the carrier of claim 1, its constitutional features is the quantity of surface hydroxyl group and can carries out modulation according to target in the distribution on surface.
5. the carrier of claim 1, its constitutional features is to have up to 1000m
2The specific surface area of/g.
6. the carrier of claim 1 is characterized in that showing inertia in enzyme-catalyzed reaction.
7. the carrier of claim 1, it can be used as the fixation support of trypsinase, papoid, amylase, horseradish peroxidase, cytochrome C, lipase etc.
8. the immobilized enzyme of claim 7, wherein said enzyme is a penicillin acylase.
9. the immobilized enzyme of claim 8, its immobilization process can be by the interaction realization of carrier surface hydroxyl with the enzyme molecule.
10. the immobilized enzyme of claim 8, its enzyme work is 511U/g.min greatly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00106228 CN1320688A (en) | 2000-04-27 | 2000-04-27 | Carrier for immobilizing penicillin amidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00106228 CN1320688A (en) | 2000-04-27 | 2000-04-27 | Carrier for immobilizing penicillin amidase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1320688A true CN1320688A (en) | 2001-11-07 |
Family
ID=4578219
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00106228 Pending CN1320688A (en) | 2000-04-27 | 2000-04-27 | Carrier for immobilizing penicillin amidase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1320688A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207466A (en) * | 2018-11-08 | 2019-01-15 | 山东鲁抗医药股份有限公司 | A kind of process for fixation and immobilised enzymes of PA ase |
-
2000
- 2000-04-27 CN CN 00106228 patent/CN1320688A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207466A (en) * | 2018-11-08 | 2019-01-15 | 山东鲁抗医药股份有限公司 | A kind of process for fixation and immobilised enzymes of PA ase |
| CN109207466B (en) * | 2018-11-08 | 2021-01-08 | 山东鲁抗医药股份有限公司 | Immobilization method of penicillin acylase and immobilized enzyme |
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