CN101381718A - Immobilization method of alcaligenes faecalis penicillin G acylase - Google Patents
Immobilization method of alcaligenes faecalis penicillin G acylase Download PDFInfo
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- CN101381718A CN101381718A CNA2007100127106A CN200710012710A CN101381718A CN 101381718 A CN101381718 A CN 101381718A CN A2007100127106 A CNA2007100127106 A CN A2007100127106A CN 200710012710 A CN200710012710 A CN 200710012710A CN 101381718 A CN101381718 A CN 101381718A
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Abstract
The invention relates to a process for immobilizing an enzyme in biological chemistry, in particular to a method for immobilizing a bacillus foecalis alkaligenes penicillin G acylase. The carrier for immobilizing the bacillus foecalis alkaligenes penicillin G acylase is ZH-EP. In a buffer solution system, when the ZH-EP is used to immobilize, the concentration of the enzyme is between 100 and 160 mg/ml, the immobilizing time is between 106 and 120 hours, the pH value of the buffer solution is between 8.5 and 10.0, the concentration of salt in the reaction system is between 1.0 and 1.5M, and the weight volume ratio of the carrier to the buffer solution is 1g : 4 to 6ml. The method improves the temperature and the pH stability of the immobilized enzyme compared with a free enzyme. Through continuous batches of hydrolysis of penicillin G kali salt, the immobilized bacillus foecalis alkaligenes penicillin G acylase shows the excellent operation stability and the superiority to the free enzyme.
Description
Technical field
The present invention relates in the biochemical industry enzyme be carried out immobilized technology the method for specifically a kind of novel epoxy group(ing) carrier ZH-EP immobilization alcaligenes faecalis penicillin G acylase.
Background technology
Since nineteen twenty-nine Fu Laiming found penicillin, penicillin had been made huge contribution for human medical and health care system.Use so that abuse yet be accompanied by, bacterium to the resistance of penicillin also in continuous increase.The development new antibiotic is the effective measure of dealing with problems.The sixties in 20th century, the scientific research personnel found penicillin G acylase (PGA; EC 3.5.1.11), and developed enzyme immobilization technology be used for hydrolyzing penicillin and cynnematin G produce 6-aminopenicillanic acid (6-APA) and 7-amino take off acetamido cephalosporanic acid (7-ADCA).And 6-APA and 7-ADCA are the important medicine intermediates of semisynthetic antibiotics.Immobilized penicillin acylated enzyme is the important means that the production of semi-synthetic beta-lactam antibiotics is carried out.And, increase industrial competitive power along with the better performance of the development need of social demand, more high efficiency catalyzer.
In recent years, people screen multiple penicillin acylase of different nature from nature, in the penicillin G acylase in all known sources, the gene of Bacillus foecalis alkaligenes (Alcaligenes faecalis) penicillin G acylase was just cloned (document 1 up to 1994, United States Patent (USP) 5695978), it has many characteristics that industrial value is arranged: alcaligenes faecalis penicillin G acylase has higher avidity to substrate, and its kcat is the highest in all penicillin G acylases to its Km than other penicillin acylases are all low; Alcaligenes faecalis penicillin G acylase is unique member who has disulfide linkage in primary structure in the penicillin G acylase family, and its thermostability and pH stability are all very high; To the recognition capability of chiral molecules, alcaligenes faecalis penicillin G acylase has higher selectivity; Alcaligenes faecalis penicillin G acylase has very high stability in organic solvent; Synthetic when the catalysis beta-lactam antibiotics is synthetic/percent hydrolysis is high.So alcaligenes faecalis penicillin G acylase has the better application prospect in semisynthetic antibiotics industry.
Immobilization can improve the character of enzyme, makes immobilized enzyme specific ionization enzyme have better stability and higher service efficiency, and endonuclease capable is recycled, and greatly saves production cost.Immobilization technology is that endonuclease capable is applied to one of industrial gordian technique.Research and development through decades, various fixation support of multiple performance and method have successively been developed, immobilization technology has been obtained significant progress, yet still can not satisfy all requirements of desirable biological catalyst, the immobilized enzyme that really can drop into industrial application is few, major cause is reagent and the carrier cost height that immobilization is used, and secondly is that immobilization efficiency is low, poor stability; And maintain secrecy for commerce, a lot of technological process details are not open.China's related work is started late, and still needs to rely on import to satisfy the production of semisynthetic antibiotics, studies therefore that immobilized penicillin acylated enzyme is significant efficiently.
Bionic technical progress in modern age can be carried out the structure function that transformation on the molecular level improve enzyme to enzyme and obtain the higher enzyme of vigor, and relies on the progress of enzyme engineering enzyme immobilization to be obtained to be more suitable for the biological catalyst of large-scale commercial production.We are with the alcaligenes faecalis penicillin G acylase gene; conversion has made up the genetic engineering bacterium of high expression level alcaligenes faecalis penicillin G acylase in intestinal bacteria; need not to induce and to carry out composing type high expression level (document 2; first-class among Yuan; People's Republic of China's patent, ZL200310108792.6).On the basis that obtains alcaligenes faecalis penicillin G acylase, the applicant has carried out the research of alcaligenes faecalis penicillin G acylase immobilized enzyme again.
Investigate a kind of fixing condition of novel vector, its technical process is evaluation index for investigating conditions such as enzyme concn, pH, salt concn, temperature with immobilized enzyme vigor, temperature stability, pH stability and stability in use.
Summary of the invention
The objective of the invention is to screen the immobilization that new fixation support is applied to alcaligenes faecalis penicillin G acylase, obtain the immobilized enzyme product; And be model with the alcaligenes faecalis penicillin G acylase, set up and use novel vector enzyme is carried out immobilized general technology flow process.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of method of immobilization alcaligenes faecalis penicillin G acylase; the carrier of immobilization alcaligenes faecalis penicillin G acylase is ZH-EP; in buffer solution system; when carrying out immobilization with ZH-EP; enzyme concn is 100-160mg/ml, immobilization time 106-120h, buffered soln pH8.5-10.0; the concentration 1.0-1.5M of salt in the reaction system, the bulking value ratio of carrier and buffered soln is the 1g carrier: 4-6ml buffered soln.
The concentration of buffered soln is lower than 1.0-1.5M in system, and insufficient ionic strength can be replenished with soluble salt in the system, make salt in the reaction system concentration 1.0-1.5M (as, adopt the buffered soln of concentration 0.05M, add 1.0-1.5M NaCl.)。
It is the immobilization of the epoxy carrier of active group to alcaligenes faecalis penicillin G acylase that the present invention has set up epoxide group.
The present invention also adopts industrialization universal support Eupergit C to carry out immobilization to alcaligenes faecalis penicillin G acylase; and its fixing condition studied; when having determined to carry out immobilization with Eupergit C; its top condition is 80-160mg/ml; immobilization 200-320h; buffered soln pH7.0-9.5, buffer concentration 0.4-0.8M.
The present invention has following characteristics:
1. the present invention adopts commercialization carrier Eupergit C that the less alcaligenes faecalis penicillin G acylase of present research has been carried out immobilization first, has obtained the immobilized enzyme that performance is greatly improved than free enzyme; And filtered out a kind of carrier ZH-EP that is suitable for the immobilization alcaligenes faecalis penicillin G acylase; the price of ZH-EP is lower than Eupergit C; adopt this kind carrier that enzyme is carried out immobilization and not only greatly reduce production cost; required immobilization time ratio also greatly reduces than Eupergit C, and the immobilized enzyme vigor of acquisition is also higher.The processing method of setting up is applicable to carries out immobilization to all proteins molecule, improves performance, improves its stability and service efficiency.
2. the present invention has determined the optimal fixed condition by the investigation to conditions such as enzyme concn, immobilization time, pH and buffer concentrations, has set up the immobilized technical process of employing novel vector.The temperature of immobilized enzyme, pH stability have all had improvement than free enzyme.Through continuous many batches of hydrolyzing penicillin G sylvite, the immobilization alcaligenes faecalis penicillin G acylase demonstrates good operational stability, shows the advantage than free enzyme.
Description of drawings
Fig. 1 is the influence to carrier Eupergit C immobilization alcaligenes faecalis penicillin G acylase of enzyme concn and immobilization time; Fixing condition: pH7.5, the 1M phosphoric acid buffer; Room temperature.(◇), 10mg protein/ml; (), 40mg protein/ml; (△), 80mg protein/ml; (*), 120mg protein/ml; (+), 160mg protein/ml.
Fig. 2 is the influence to carrier ZH-EP immobilization alcaligenes faecalis penicillin G acylase of enzyme concn and immobilization time; Fixing condition: pH7.5, the 1M phosphoric acid buffer; Room temperature.(◇), 10mg protein/ml; (), 40mg protein/ml; (△), 80mg protein/ml; (*), 120mg protein/ml; (+), 160mg protein/ml.
Fig. 3 is the influence of pH to carrier Eupergit C and ZH-EP immobilization alcaligenes faecalis penicillin G acylase; Fixing condition: 100mg protein/g carrier; Room temperature; The immobilization time is respectively 150h, 48h to Eupergit C, ZH-EP.(◇)，Eupergit?C；(□)，ZH-EP
Fig. 4 is the influence of ionic strength to carrier Eupergit C and ZH-EP immobilization alcaligenes faecalis penicillin G acylase; Fixing condition: 100mg protein/g carrier; Room temperature; PH8.0; The immobilization time is respectively 150h, 48h to Eupergit C, ZH-EP.(◇)，Eupergit?C；(□)，ZH-EP
Fig. 5 pH is to free alcaligenes faecalis penicillin G acylase and the influence that is immobilized in carrier Eupergit C, the last alcaligenes faecalis penicillin G acylase vigor of ZH-EP; (◇), free AfPGA; (), immobilization AfPGA/Eupergit C; (△), immobilization AfPGA/ZH-EP.
Fig. 6 temperature is to free alcaligenes faecalis penicillin G acylase and the influence that is immobilized in the last alcaligenes faecalis penicillin G acylase vigor of carrier EupergitC, ZH-EP; (◇), free AfPGA; (), immobilization AfPGA/Eupergit C; (△), immobilization AfPGA/ZH-EP.
Free alcaligenes faecalis penicillin G acylase of Fig. 7 and the pH stability that is immobilized in carrier Eupergit C, the last alcaligenes faecalis penicillin G acylase of ZH-EP; (◇), free AfPGA; (), immobilization AfPGA/Eupergit C; (△), immobilization AfPGA/ZH-EP
Free alcaligenes faecalis penicillin G acylase of Fig. 8 and the temperature stability that is immobilized in carrier Eupergit C, the last alcaligenes faecalis penicillin G acylase of ZH-EP; (◇), free AfPGA; (), immobilization AfPGA/Eupergit C; (△), immobilization AfPGA/ZH-EP
Fig. 9 is immobilized in the stability in use of carrier Eupergit C, the last alcaligenes faecalis penicillin G acylase of ZH-EP.(), immobilization AfPGA/Eupergit C; (*), immobilization AfPGA/ZH-EP
Embodiment
Cultivate the thalline that produces alcaligenes faecalis penicillin G acylase at first according to a conventional method, cytoclasis is purified, and obtains alcaligenes faecalis penicillin G acylase.Be the research of the carrier of selecting being carried out fixing condition then, the immobilized enzyme of acquisition carries out property research to estimate immobilization effect and process application potentiality thereof to it.
Embodiment
Carry out preliminary experiment earlier and filter out potential carrier, again the carrier of selecting is carried out detailed fixing condition research, its character of research and solution enzyme are relatively behind the acquisition immobilized enzyme.
The selection of 1 carrier is through the test to multiple candidate's carrier, and finishing screen is selected the carrier ZH-EP of Hong Kong GeneRadBiotech Laboratory company limited exploitation.ZH-EP, the carrier Eupergit C of similar industrial applications has epoxy-activated group.In contrast, Eupergit C also is used to immobilization.
The investigation of 2 fixing conditions
2.1 the concentration of alcaligenes faecalis penicillin G acylase and immobilization time
Generally, covalent immobilization is followed two-step approach mechanism: at first being physical adsorption fast between carrier and the albumen, is between the activating group of the albumen that is adsorbed and carrier covalent reaction to take place then.The first step means that enzyme concn is an important factor for immobilization.Its suitableeest enzyme concn difference of different properties carrier adopts the suitableeest enzyme concn immobilization can obtain the immobilized enzyme of high vigor under the situation of enzyme that avoids waste.
Generally do not need to adopt the high enzyme of purity to carry out immobilization, adopt among the present invention and carry out immobilization than the proteic enzyme of 334U/g alive.The immobilization system is 1g carrier, 4-6ml buffered soln.
Adopt different enzyme concn systems to carry out immobilization, according to investigating result (Fig. 1, Fig. 2), for obtaining the highest immobilized enzyme vigor, determine to adopt carrier Eupergit C, its suitableeest protein concentration is 120mg/ml, immobilization 260h; Adopt protein concentration 120mg/ml, immobilization 116h for ZH-EP.
2.2pH
Therefore buffered soln buffering range deficiencies such as phosphoric acid commonly used, Tris hydrochloric acid adopt Britton-Robinson buffered soln (mixed solution of 1.0M boric acid-1.0M acetate-1.0M phosphoric acid, its pH is regulated by NaOH).
In the system of different pH, enzyme is carried out immobilization, by the immobilized enzyme vigor that obtains is compared (Fig. 3), for using carrier Eupergit C immobilization alcaligenes faecalis penicillin G acylase, its suitableeest immobilization pH scope 7.5-9.0; For ZH-EP, its suitableeest immobilization pH scope 8.5-10.0.
2.3 ionic strength
The two-step approach immobilization mechanism the first step of enzyme on carrier requires albumen and carrier surface to have stronger interaction force, and the reactive force between albumen and the carrier is subjected to causing the salt concn of albumen and carrier surface hydrophobic interaction or the influence of buffer concentration.
In the system of different ionic strength, enzyme is carried out immobilization, by the immobilized enzyme vigor that obtains is compared (Fig. 4), for using carrier Eupergit C immobilization alcaligenes faecalis penicillin G acylase, its suitableeest buffer concentration 0.5M; For ZH-EP, its suitableeest buffer concentration 1.5M.
2.4 temperature
Through investigating, temperature is not very remarkable for immobilized influence, from economy angle consideration easily, adopts room temperature.Can consider the temperature that it is suitable for the responsive to temperature type enzyme.
The character of 3 immobilization alcaligenes faecalis penicillin G acylases
3.1 optimal pH
Adopt Britton-Robinson buffered soln (0.05M boric acid-0.05M Glacial acetic acid-0.05M phosphoric acid, NaOH regulates pH) of different pH to measure the influence of pH to solution enzyme and immobilized enzyme vigor.The optimal pH of free enzyme is 7.5, and skew has taken place the equal alkalitropism scope of immobilized enzyme, and the AfPGA/EupergitC optimal pH is 8.0, and the AfPGA/ZH-EP optimal pH is 10.0-11.0 (Fig. 5).
3.2 optimum temperuture
At the different temperature measuring solution enzymes and the vigor of immobilized enzyme.The optimum temperuture of free enzyme is 50 ℃, and the optimum temperuture of immobilized enzyme all increases, and is 55～60 ℃ (Fig. 6).
3.3pH stability
Sample is measured its remaining vigor behind the room temperature insulation 1h in the buffered soln of different pH, and compare without the sample vigor of insulation, in whole acidity to the pH stability of alkaline range (pH4.0-10.0) immobilized enzyme obviously than good (Fig. 7) of free enzyme.Because penicillin G acylase can not only can also be in the acidic catalyst building-up reactions at catalytic hydrolysis reaction under the alkaline environment, so in acidity and alkaline range, improved its stability after the immobilization significance has been arranged.
3.4 temperature stability
In industry, adopt higher temperature of reaction, following advantages are arranged: higher speed of reaction, change molecular balance, better substrate solvability, lower reaction medium viscosity and the reduction of microbial contamination possibility etc.Therefore the enzyme of thermostability is significant for industrial application.
Sample is measured its remaining vigor behind differing temps insulation 1h, and compare without the sample of insulation, estimates its temperature stability.Solution enzyme and immobilized enzyme have all shown good temperature stability, and immobilized enzyme is showing its stabilization (Fig. 8) more than 55 ℃.
3.5 stability in use
Immobilized enzyme catalytic hydrolysis potassium penicillin G in column reactor produces 6-APA and toluylic acid, and NaOH solution adds automatically and keeps reaction system pH is 8.0, and the initial wear rate of the vigor of immobilized enzyme and NaOH is directly proportional.In reaction process, the hydrolysis rate of potassium penicillin G reduces gradually, the also corresponding reduction of the adding speed of NaOH, and reaction finishes when final NaOH stops to consume, and repetitive operation begins the next batch reaction then.Through surplus the successive reaction 40 batch, the immobilized enzyme vigor does not have significantly sacrificing, has shown good stability in use (Fig. 9).
Adopt than the proteic alcaligenes faecalis penicillin G acylase of 334U/g alives, enzyme is dissolved in the phosphoric acid buffer of pH8.0, and is concentrated or dilute enzyme liquid to 120mg/ml.The wet carrier Eupergit C of 1g adds 5ml enzyme liquid, and the reaction of uniform mixing ground is 260 hours under the room temperature.Use the sintered filter funnel suction filtration, gained immobilization product washs 3-5 times with phosphoric acid buffer, and immobilization finishes.
Embodiment 2 is with carrier ZH-EP immobilization alcaligenes faecalis penicillin G acylase
Adopt than the proteic alcaligenes faecalis penicillin G acylase of 334U/g alives, enzyme is dissolved in the phosphoric acid buffer of pH9.0, and is concentrated or dilute enzyme liquid to 120mg/ml.Wet carrier ZH-EP of 1g adds 5ml enzyme liquid, and uniform mixing ground reacted 116 hours under the room temperature.Use the sintered filter funnel suction filtration, gained immobilization product washs 3-5 times with phosphoric acid buffer, and immobilization finishes.
Claims (2)
- The method of 1 one kinds of immobilization alcaligenes faecalis penicillin G acylases; it is characterized in that: the carrier of immobilization alcaligenes faecalis penicillin G acylase is ZH-EP; in buffer solution system; when carrying out immobilization with ZH-EP; enzyme concn is 100-160mg/ml, immobilization time 106-120h, buffered soln pH8.5-10.0; the concentration 1.0-1.5M of salt in the reaction system, the bulking value ratio of carrier and buffered soln is the 1g carrier: 4-6ml buffered soln.
- 2. according to the method for the described immobilization alcaligenes faecalis penicillin G acylase of claim 1; it is characterized in that: the concentration of buffered soln is lower than 1.0-1.5M in system; insufficient ionic strength can be replenished with soluble salt in the system, makes the concentration 1.0-1.5M of salt in the reaction system.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104120120A (en) * | 2014-06-27 | 2014-10-29 | 浙江工业大学 | Immobilized recombinant penicillin G acylase and application thereof |
| CN104962543A (en) * | 2015-06-26 | 2015-10-07 | 上海理工大学 | Oriented immobilization method for bacillus subtilis neutral protease |
| CN107858312A (en) * | 2017-12-07 | 2018-03-30 | 北京理工大学 | One plant of beta-lactam class Degradation of Antibiotics bacterial strain PGB1 and its application |
| CN115181691A (en) * | 2022-06-21 | 2022-10-14 | 中国科学院烟台海岸带研究所 | Bacillus DB-Q3 and application thereof |
-
2007
- 2007-09-05 CN CNA2007100127106A patent/CN101381718A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104120120A (en) * | 2014-06-27 | 2014-10-29 | 浙江工业大学 | Immobilized recombinant penicillin G acylase and application thereof |
| CN104962543A (en) * | 2015-06-26 | 2015-10-07 | 上海理工大学 | Oriented immobilization method for bacillus subtilis neutral protease |
| CN104962543B (en) * | 2015-06-26 | 2017-11-03 | 上海理工大学 | A kind of oriented immobilization method of Bacillus subtilis neutral protease |
| CN107858312A (en) * | 2017-12-07 | 2018-03-30 | 北京理工大学 | One plant of beta-lactam class Degradation of Antibiotics bacterial strain PGB1 and its application |
| CN115181691A (en) * | 2022-06-21 | 2022-10-14 | 中国科学院烟台海岸带研究所 | Bacillus DB-Q3 and application thereof |
| CN115181691B (en) * | 2022-06-21 | 2024-04-19 | 中国科学院烟台海岸带研究所 | Bacillus DB-Q3 and application thereof |
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