CN1088755C - Fixed penicillin acidylated enzyme using agar-gelatin compounded microbeads as carrier and preparation method therefor - Google Patents
Fixed penicillin acidylated enzyme using agar-gelatin compounded microbeads as carrier and preparation method therefor Download PDFInfo
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- CN1088755C CN1088755C CN97116202A CN97116202A CN1088755C CN 1088755 C CN1088755 C CN 1088755C CN 97116202 A CN97116202 A CN 97116202A CN 97116202 A CN97116202 A CN 97116202A CN 1088755 C CN1088755 C CN 1088755C
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Abstract
The present invention relates to immobilized penicillin acylase using agar-gelatin compound micro balls as carriers, and a preparation method thereof. The present invention has the technical scheme that 2 to 15 wt % of agar solution is prepared, and 1 to 5 wt % of gelatin is added for dissolution; an organic solvent is added to form mocro-ball carriers which are combined with penicillin acylase through activation, and immobilized penicillin acylase is obtained. The method is simple and is suitable for industrial production; the present invention has the advantages of high product intensity, good activity and low price.
Description
The present invention relates to a kind of immobilized enzyme biotechnology, particularly relating to a kind of is the preparation immobilized penicillin acylated enzyme and preparation method thereof of carrier with the agar-gelatin compounded microbeads.
Immobilized enzyme (Immobilized enzyme) technology is a new bio technology that grows up in the sixties in this century, it is defined as " physical restriction or be positioned in the certain spatial areas enzyme that has kept catalytic activity and can repeat, use continuously ".
Natural enzyme widespread use in food, chemical industry and medicine industry.In the world wide, the enzyme sales volume that is used for food, washing agent, fine chemistry industry and diagnosis every year reaches 700,000,000 dollars.But because enzyme is a kind of protein of solubility, be difficult for reclaiming, can not reuse, caused the catalyzer waste; Enzyme addition albumen also can pollution products, causes difficulty to aftertreatment, and this problem is particularly serious in the production of pharmaceutical prod.
Early sixties, people such as Katchallski-Katzir are in order to study under the state of nature mechanism of action attached to the enzyme on the microbial film, at first be fixed in some proteolytic ferments on the insoluble carrier or be wrapped in the artificial rust, study the characteristic of its stability and heterogeneous catalysis effect, as document 1:Annu.Rev.Biochem.35,773-908,1966.They recognize that if artificial fixed enzyme stable, can design suitable reactor, realize reusing operate continuously.
People such as the Japanese Tanake Seiyaku Chibata of company successfully were used for enzyme immobilization technology industrial production first in 1967, and as document 2:Biotechnol.Bioeng.9,603-615 describes in 1967.They use immobilization amino acid acylase that the racemize DL-amino acid of synthetic is converted into corresponding L-type enantiomorph.Before and after 1970, trial production during two other immobilized enzyme system drops in succession.One is that Britain produces 6-amino-penicillanic acid (Proc.R.Soc.London Ser.B 179,321-334,1971) with immobilized penicillin acylated enzyme hydrolyzing penicillin G or V; Another is that the U.S. produces fructose (Applied Biochemistry and Bioengineering, Vol.1, pp222-327.Academic Press, 1976) with the fixed glucose isomerase transforming glucose.These successful industrial application have promoted the research extensively and profoundly of enzyme immobilization technology, make with the immobilized enzyme stable the increasing of quantity of the commercial run that is catalyzer.
Utilize immobilized enzyme to carry out industrial production, enzyme can be reclaimed, reuse, reduced the catalyzer cost, also improved the stability of enzyme, avoided zymoprotein, simplified aftertreatment the pollution that product causes.Its research and application develop rapidly, obtain widespread use in industrial production and biochemical analysis, become one of most active fields in the modern biotechnology.
At present, three kinds of immobilized enzyme that industrial output is the highest are glucose isomerase, nitrilase and penicillin acylase in the world, and annual production is respectively 80000 tons, 7500 tons, 1500 tons.
Enzyme immobilization method can reduce carrier combined techniques, crosslinking and entrapping method three major types.
The carrier combined techniques is that enzyme is incorporated into a class process for fixation on the water insoluble carrier.According to bonded form difference, can be divided into physisorphtion, ionic bond method and covalent attachment method etc. again.Physisorphtion be meant enzyme by physical adsorption on insoluble carrier.Carrier has activated carbon, sintered glass, aluminum oxide, silica gel, starch, synthetic resins etc.This method has enzyme active center and is difficult for advantage destroyed, that the enzymatic structure variation is little, but a little less than enzyme and the carrier interaction force, enzyme easily comes off.The ionic bond method is incorporated into enzyme on the water insoluble carrier with ion-exchange group by ionic linkage.The carrier of this method has polyose ion-exchanger and synthetic macromolecule ion exchange resin, for example DEAE-Mierocrystalline cellulose, CM-Mierocrystalline cellulose etc.This method is simple to operate, mild condition, and the higher structure of enzyme and the amino-acid residue in active centre are difficult for destroyed, can obtain the higher immobilized enzyme of enzyme yield alive.But the bonding force of carrier and enzyme a little less than, be subjected to the influence of damping fluid kind or pH easily; When reacting under the high condition of ionic strength, enzyme easily comes off from carrier.The covalent attachment method be with enzyme with covalent bonds on carrier.This method or with carrier related group activation, relevant with enzyme then group generation linked reaction; Or on carrier, connect a bifunctional reagent, then the enzyme coupling is got on.Amino, carboxyl, hydroxyl, phenolic group etc. can be arranged with the functional group of carrier-bound enzyme.Covalent attachment method reaction conditions is relatively harsher, complicated operation, and cause that easily the enzyme higher structure changes, and having destroyed the part active centre, enzyme yield alive is generally about 30%.But enzyme is in conjunction with firm, good stability.It is use in the carrier combined techniques maximum a kind of.
Crosslinking is to make the crosslinked process for fixation of generation between enzyme and the enzyme with difunctional or poly functional reagent.This method is the same with the covalency combined techniques also to be to utilize the covalent linkage immobilized enzyme, and different is that it does not use carrier.This method reaction conditions is fierce, enzyme is lived, and yield is low.
Entrapping method can be divided into and comprise two kinds of grid type and microcapsule-types.Enzyme is embedded in the grid type that is called in the trickle grid of high-molecular gel; Enzyme is embedded in the microcapsule-type that is called in the polymer semi-permeable membranes.Entrapping method generally seldom changes the higher structure of enzyme, and enzyme yield alive is higher.But entrapping method only is fit to act on the enzyme of small molecules substrate and product, because have only small molecules just can to spread by the grid of high-molecular gel.The macromolecular compound that is suitable for grid type has polyacrylamide, polyvinyl alcohol, starch, gelatin, Lalgine etc.The microcapsule-type immobilized enzyme particle is generally several microns spherules to the hundreds of micron of diameter, and is more much smaller than grid type particle.This helps the diffusion of substrate and product.But reaction conditions requires high, and preparation cost is also high.Preparation microcapsule-type immobilized enzyme interphase precipitate method commonly used, interfacial polymerization, secondary emulsion process and liposome embedded method etc.
In various process for fixation, entrapping method generally is applied to prepare immobilized cell.Industrial extensive employing covalent attachment legal system is equipped with immobilized enzyme.
Penicillin antibiotics is present clinical most popular microbiotic, and it is strong that it has an antimicrbial power, the curative effect height, and low toxin is important anti-infectives.Its anti-microbial effect mechanism is suppress bacteria cell wall synthetic.The integrity of its intramolecularly beta-lactam nucleus is the prerequisite of its anti-microbial effect.Natural penicillin, promptly the direct synthetic penicillin of microorganism has two kinds of penicillin G (PenG) and penicillin vs (PenV), and the life-time service of natural penicillin has produced tolerant bacteria.The resistance bacterium can produce penicillinase, and the intramolecular beta-lactam nucleus of hydrolyzing penicillin makes it inactivation.If the side-chain radical that is joined on manual change's natural penicillin intramolecularly beta-lactam nucleus promptly obtains semisynthetic penicillin, the change of side-chain radical can increase the stability of beta-lactam nucleus; It is wide that various semisynthetic penicillins have fungicidal spectrum, good stability, can be oral etc. advantage, thereby replaced natural penicillin clinically rapidly, at present, the natural penicillin in the world wide more than 80% all is the production that is used for semisynthetic penicillin.
6-amino-penicillanic acid (6-Aminopenicillanicacid is called for short 6-APA) is a key intermediate of producing semisynthetic penicillin, and at present, the semisynthetic penicillin of Shi Yonging is all from 6-amino-penicillanic acid clinically.
6-amino-penicillanic acid is to obtain by the side chain that cuts penicillin G or penicillin v.With natural penicillin G or penicillin v is initiator, falls its side chain with chemical method or enzymatic hydrolysis, and what wherein enzyme process adopted is penicillin acylase.Chemical method and enzyme process start to walk to develop simultaneously, all have been used for industrial production.The two respectively has advantage.But, along with enzyme immobilization technology develop rising steadily of application and petroleum chemicals and energy prices rapidly, enzyme process shows bigger economy; In addition, enzyme process is free from environmental pollution, operates also more convenient.Therefore, the industrial production of 6-APA mainly realizes by employing penicillin acylase hydrolyzing penicillin G or penicillin v now.
Industrially about nineteen sixty, begin to start to walk with enzymatic hydrolysis penicillin production 6-amino-penicillanic acid.Original adoption be the cell suspension of active bacteria, only can use once, productivity is very low, be approximately 0.5-1Kg6-aminopenicillanic acid/Kg E.coli suspension, afterwards, by immobilization E.coli cell, productivity can be increased to 50Kg 6-amino-penicillanic acid/Kg immobilized catalyst.At present, mainly use immobilized enzyme, productivity is generally 100-1000Kg 6-amino-penicillanic acid/Kg immobilized catalyst.
At present, the consumption of annual immobilized penicillin acylated enzyme is the 10-30 ton in the world.All there be fixation support and the immobilized enzyme of oneself in each big drugmaker.
The research of China's enzyme immobilization technology has obtained certain achievement, has developed some unique immobilization technologies, as bifunctional reagent right-use of beta-sulfuric ester ethyl sulfone aniline (SESA), be China investigator initiative.But the research of fixation support aspect seldom.So far still there is not commercialization immobilized enzyme dedicated carrier.In the research of enzyme immobilization technology, adopt through the natural materialss of simple processing and some industrial by-products or tankage as carrier poor-performing more.Though reduced the cost of carrier like this, influenced the performance of immobilized enzyme function, and seriously limited the development and the application of enzyme immobilization technology in some cases.
At present, China produces the used 6-amino-penicillanic acid of semisynthetic penicillin mainly by import.Also there are producer's import immobilized penicillin acylated enzyme and supporting technology to produce 6-amino-penicillanic acid.And country will spend the key intermediate (as 6-APA) that semisynthetic penicillin is produced in a large amount of foreign exchange imports every year, not only consumes a large amount of man power and materials, also brings problems such as serious environmental pollution simultaneously, has a strong impact on and restricting the development of China's medicine industry.
Purpose of the present invention: the purpose of invention is industrialization fixation support and the immobilized enzyme that is fit to China's national situation in order to develop, and promotes the development of enzyme immobilization technology and the production domesticization of semisynthetic antibiotics raw material; A kind of agar-gelatin compounded microbeads carrier is provided; not only has the premium properties of each side but also compare cheap, as to be fit to industrial application fixed enzyme vector; penicillin acylase is fixed thereon; the preparation immobilized penicillin acylated enzyme; the industrial production that is used for 6-amino-penicillanic acid, the raw material problem of solution China semisynthetic penicillin.
The object of the present invention is achieved like this:
Gelatin embedding in agar, is made the plural gel pearl.But utilize agar strength height, easy-formation and many, the easily-activated advantage of gelatin activating group, make carrier not only have higher intensity but also be easy to activation.Agar is more cheap than agarose, but activable group is few, and itself is not suitable as fixed enzyme vector, in plural gel, mainly plays the support effect, makes carrier have higher intensity and stability.And the effect of gelatin is to introduce to be easy to activatory amino in carrier.Complex carrier can live the with glutaraldehyde one step, with penicillin acylase in conjunction with getting on the preparation immobilized penicillin acylated enzyme.And,, keep sterile state in helping producing because glutaraldehyde itself or a kind of sterilizing agent in activated carrier, are sterilized to carrier.Ultimate principle:
Preparation process:
1) agar is dissolved in the hot water, is made into the agar-agar soln of 2～15% (weight),, make it dissolving again to the gelatin that wherein adds 1-5% (weight).
Adopt different agar concentration 2-15% (weight) preparation carrier and immobilized penicillin acylated enzyme.When agar concentration was too low, the support strength of preparation was little, and the gelatin amount of energy embedding is few.The activity of the immobilized enzyme and the intensity of preparation are all lower.When agar concentration is too high, because mother liquor viscosity is big, the preparation difficulty, the carrier granule sphericity of preparation is poor.Through test, agar concentration 8-12% (weight) scope, the support strength of preparation is better, and the gelatin amount of energy embedding is big, brings bigger difficulty and be unlikely to preparation process.When agar concentration is 8-12% (weight), change the consumption of gelatin, preparation carrier and penicillin acylation enzyme-fixing.When gelatin concentration was too low, the immobilized penicillin acylated enzyme activity of preparation was lower.When gelatin concentration is too high, also can bring difficulty, and cause the carrier stability decreases to preparation.Tested the scope of gelatin concentration 1～5%, the result shows, gelatin concentration 2-4% (weight), and activity of the immobilized enzyme is higher, and stability is better, and easy to prepare.
2) in round-bottomed flask with agar-gelatin mixing solutions and isopyknic organic solvent (as: chloroform, toluene, gasoline, butylacetate etc., consider from the toxicity aspect of solvent, we advise using the butylacetate nontoxic to human body) mix, start stirring (device is as shown in Figure 1), rotating speed is 300 rev/mins; In beaker, add frozen water after 30 seconds, continue to stir 5 minutes.At this moment agar-gelatin forms and mixes the microballon carrier.
3) mixture is filtered, remove organic solvent; Successively with acetone and distilled water wash.
4) activation: get the microballon carrier of certain volume (stacking volume), add isopyknic 2.5% (v/v) glutaraldehyde solution, at room temperature shook 4 hours; Remove by filter glutaraldehyde, with distilled water wash.
5) combine with penicillin acylase: the carrier microballon that will activate mixes through rare free penicillin acylase solution (10-50 units per ml) of releasing with equal-volume, shakes under 4 ℃ 12 hours; Solution is removed in suction, and with the phosphoric acid buffer washing of 0.5M NaCl solution and pH8.0, obtain with the agar-gelatin compounded microbeads is the immobilized penicillin acylated enzyme of carrier to product successively.
Effect of the present invention: the immobilized penicillin acylated enzyme with the present invention's preparation is compared with the immobilized enzyme for preparing with the external agarose 6B microballon that adopts usually, and intensity is higher, and activity is in identical level, but product price is cheap.And the preparation method is simple, is suitable for industrial production, solves the raw material of China's semisynthetic penicillin.
The immobilized penicillin acylated enzyme of the present invention's preparation compares with the activity of the immobilized enzyme of the carrier for preparing with other method:
Carrier activation method immobilized enzyme alive relatively
The property
(％)
Agarose 6B NalO
4100
(uncrosslinked) bisglycidyl ether 17.6
Three chlorotriazines 71.5
Benzoquinones 77.4
Agarose 6B NalO
477.9
(crosslinked) bisglycidyl ether 42.0 complex carriers (gelatin 2%, agar 8～12%) glutaraldehyde 90.8～92.7 complex carriers (gelatin 3%, agar 8～12%) glutaraldehyde 106～110 complex carriers (gelatin 4%, agar 8～12%) glutaraldehyde 138～148
The present invention is described in detail below in conjunction with drawings and Examples:
Fig. 1 is the setting drawing that the present invention prepares the agar-gelatin compounded microbeads carrier.
Fig. 2 be of the present invention be the stability diagram of the immobilized penicillin acylated enzyme of preparing carriers with the agar-gelatin compounded microbeads.
Embodiment 1:
1. get agar 2g and put into 95 ℃ of hot water of 100ml, it is fully dissolved, be made into the agar-agar soln that concentration is 2% (weight); And then adding the 1g gelatin, insulation makes it dissolving, is made into the agar-gelatin mixing solutions.
2. in round-bottomed flask with agar-gelatin mixing solutions and isopyknic organic solvent butylacetate, mix, start stirring, rotating speed is 300 rev/mins; In beaker, add frozen water after 30 seconds, continue to stir 5 minutes.At this moment agar-gelatin forms and mixes the microballon carrier.
3. gained agar-gelatin compounded microbeads carrier is filtered with usual method, remove ethyl acetate, more successively with acetone and distilled water wash.
4. activation: the glutaraldehyde solution of getting 100ml compounded microbeads carrier and 100ml 2.5% (v/v) mixes, and at room temperature shakes 4 hours; Remove by filter glutaraldehyde, with distilled water wash.
5. combine with penicillin acylase: the carrier microballon that will activate mixes with the diluted free penicillin acylase solution of equal-volume (10-50 units per ml), shakes under 4 ℃ 12 hours; Solution is removed in suction, and with the phosphoric acid buffer washing of 0.5M NaCl solution and pH=8.0, obtain with the agar-gelatin compounded microbeads is the immobilized penicillin acylated enzyme of carrier to product successively.
Embodiment 2: press the operation of embodiment 1 preparation process fully, agar is got 15g, and gelatin is got 5g, and organic solvent uses chloroform.
Embodiment 3: experiment condition is pressed embodiment 1 fully.Use agar concentration to be made into 8%, gelatin concentration is made into 3% (weight)
Fixedly the penicillin acylase of the bacillus megaterium of Institute of Micro-biology of Chinese Academy of Sciences screening prepares immobilized penicillin acylated enzyme, enzyme yield average out to 70～83% alive.When reusing for 37 ℃, it is 0.85～0.93% that enzyme activity is criticized loss, with close with the immobilized enzyme of other method preparation.As shown in Figure 2.
Claims (3)
1. one kind is the immobilized penicillin acylated enzyme of carrier with the agar-gelatin compounded microbeads, and its structure is:
-N＝CH(CH
2)
3CH＝N-R
R=penicillin acylase wherein
One kind to prepare claim 1 described be the immobilized penicillin acylated enzyme method of carrier with the agar-gelatin compounded microbeads, it is characterized in that following these steps to successively carry out: 1) agar is dissolved in the hot water, be made into the agar-agar soln of 2-15% (weight %) concentration, to the gelatin that wherein adds 1-5% (weight %), make it dissolving again; 2) in round-bottomed flask with agar-gelatin mixing solutions and isopyknic organic solvent, mix and to stir the companion, in beaker, add frozen water after 30 seconds, continue to stir 5 minutes, at this moment agar-gelatin forms and mixes the microballon carrier; 3) mixture is filtered, remove organic solvent; Successively with acetone and distilled water wash; 4) the agar-gelatin compounded stacking volume microballon carrier that obtains activation: get isopyknic 3) adds isopyknic 2.5% (v/v) glutaraldehyde solution, at room temperature shakes 4 hours; Removing by filter glutaraldehyde, with distilled water wash: 5) combine: with 4 with penicillin acylase) the carrier microballon of the activation that obtains mixes through diluting free penicillin acylase solution (10-50 units per ml) with equal-volume, shook under 4 ℃ 12 hours; Solution is removed in suction, and with the phosphoric acid buffer washing of 0.5M NaCl solution and pH=8.0, obtain with the agar-gelatin compounded microbeads is the immobilized penicillin acylated enzyme of carrier to product successively.
3. be the method for the immobilized penicillin acylated enzyme of carrier with the agar-gelatin compounded microbeads by the described preparation of claim 2, it is characterized in that employed organic solvent comprises: toluene, chloroform, gasoline, butylacetate.
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| Application Number | Priority Date | Filing Date | Title |
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| CN97116202A CN1088755C (en) | 1997-08-25 | 1997-08-25 | Fixed penicillin acidylated enzyme using agar-gelatin compounded microbeads as carrier and preparation method therefor |
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| CN97116202A CN1088755C (en) | 1997-08-25 | 1997-08-25 | Fixed penicillin acidylated enzyme using agar-gelatin compounded microbeads as carrier and preparation method therefor |
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| CN1209454A CN1209454A (en) | 1999-03-03 |
| CN1088755C true CN1088755C (en) | 2002-08-07 |
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| CN104528953B (en) * | 2015-01-10 | 2016-09-07 | 福州创源同方水务有限公司 | A kind of biofilm system for sewage disposal and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0125105A2 (en) * | 1983-05-09 | 1984-11-14 | Pfizer Inc. | Immobilization of catalytically active microorganisms in agar gel fibers |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0125105A2 (en) * | 1983-05-09 | 1984-11-14 | Pfizer Inc. | Immobilization of catalytically active microorganisms in agar gel fibers |
Non-Patent Citations (3)
| Title |
|---|
| 丝绸,1996年第9期 1996.1.1 黄晨等,丝素膜作为固定化酶载体的研究 * |
| 福州大学学报(自然科学版),第24卷第6期 1996.12.1 林耀辉等,固定化果胶酶的酶学性质研究 * |
| 福州大学学报(自然科学版),第24卷第6期 1996.12.1 林耀辉等,固定化果胶酶的酶学性质研究;丝绸,1996年第9期 1996.1.1 黄晨等,丝素膜作为固定化酶载体的研究 * |
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