CN102690859A - Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria - Google Patents
Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria Download PDFInfo
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Abstract
The invention discloses a method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria. The screening process of the method comprises the following steps: (1) preparation of alpha-ketone adipoyl-7-aminocephalosporanic acid and structural analogues thereof; (2) selection and culture of screening system indicator bacteria, enrichment culture of candidate bacteria, and selection and culture of positive and negative control strains; (3) preparation of a three-tier agar plate culture medium screening system; (4) judgement of the objective alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria. According to the method, simple operation is achieved, no expensive and complex experimental instrument is needed, 5000 candidate strains can be screened by one person per day, high screening efficiency is achieved and high throughput screening is obtained. Moreover, the method can be used in breeding of industrial strains.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the screening method that a kind of α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces bacterium.
Background technology
(7-aminocephalosporanic acid is that (Cephalosporin C, parent nucleus CPC) are to produce the antibiotic critical materials of clinical semi-synthetic cephalosporins more than 50 kinds to cephalosporin 7-ACA) to 7-amino-cephalosporanic acid.At present there are Austrian Biochemie company, gondola Antibioticos company and domestic main middle demulcen industry in main manufacturer, healthy unit, and good fortune is anti-, Shanxi Wei Qida, Harbin Pharmaceutical Group, companies such as anti-medicine in Shandong and federal pharmacy.The 7-ACA demand in the whole world in 2010 is at more than 4000 tons, and domestic 7-ACA actual output reaches about 5500 tons.Therefore, how environmental protection also prepares 7-ACA cost-effectively, reduces production costs, and improving enterprise competitiveness is the key issue that solution is needed in current national medicine development badly.
Current 7-ACA mainly obtains through chemical method or enzymatic cleavage CPC.Chemical cracking CPC prepares 7-ACA and has serious energy consumption and pollution treatment cost problem, is progressively replaced by the enzymatic lysis method at present.Industrialized both at home and abroad is two enzyme two-step approachs.About this operational path; At first; Utilize D-amino-acid oxidase (DAAO) under certain conditions with the CPC oxidative deamination, generate α-ketone hexanedioyl-7-amino-cephalosporanic acid (AKA-7-ACA) and katalase (CAT), this midbody of AKA-7-ACA is very unsettled; Be easy to by the hydrogen peroxide oxidation decarboxylation that generates simultaneously or add in addition generate glutaryl--7-amino-cephalosporanic acid (Glutaryl-7-amidocephalosporanic Acid, GL-7-ACA).This midbody of GL-7-ACA is sloughed side chain and is generated 7-ACA under the effect of GL-7-ACA acylase.Two enzyme two-step approachs have that production technique is simple, low in the pollution of the environment, working condition is gentle, low production cost, and advantage such as constant product quality.This arts demand relates to 2～3 reactor drums.Along with the increasingly serious international competition pressure of 7-ACA product, for shortening the production cost of producing and manufacturing technique with further reduction 7-ACA, people very pay close attention to the research that single stage method is produced 7-ACA (single jar of working system).
The one-step technology of being reported at present comprises two aspects: the first, single enzyme one-step method: transform CPC through a kind of single enzyme (cephalosporin C acrylase, Cephalosporin Cacylase) and produce 7-ACA.Be engaged in nearly 40 years research though dropped into a large amount of resources; Yet the cephalosporin C acrylase of finding so far exists enzyme activity low; Also can't satisfy industrial production requirement, and can't realize also that through gene clone technology this enzyme activity has substantial raising, this enzyme gene structure is very complicated.The second, two enzyme one-step method: utilize DAAO and Gl-7-ACA acylase synergy to transform the CPC single stage method and produce 7-ACA.This technology can obtain 7-ACA productive rate preferably, but because there is hydrogen peroxide in reaction system, makes that the stability of enzyme catalyst is had a strong impact on.
Recently; The technology that three enzymatic cleavage CPC produce 7-ACA is suggested; Its technology is exactly to produce AKA-7-ACA through DAAO and CAT synergy catalysis CPC; This intermediate product can be produced 7-ACA by the hydrolysis of Gl-7-ACA acylase under the condition of no pentanedioic acid, this has just been avoided the disadvantageous effect of hydrogen peroxide to reaction system.
The problem that three enzyme method techniques exist need to solve; Be exactly the enzymic activity lower (this enzyme to the milosevic constant km value of AKA-7-ACA be its to GL-7-ACA Km value 4 times) of Gl-7-ACA acylase to AKA-7-ACA; Make final 7-ACA productive rate have only 76～80%; Also have some distances with industrial production requirement, because industrialized pair of enzyme two-step approach 7-ACA productive rate >=86%.Like this, the acylase that obtains high vigor hydrolysis AKA-7-ACA will have huge economic interests, with starting a climax of research in the world.
The acquisition of the acylase of high vigor hydrolysis AKA-7-ACA can be passed through two kinds of methods: the first, produce bacterium from nature screening enzyme; The second, pass through the orthogenesis technology to Gl-7-ACA acylase, CPC acylase or penicillin G acylase molecular structure alteration, obtain the stronger mutant enzyme of AKA-7-ACA hydrolysis vigor.But no matter which kind of strategy all will be referred to a technique means---the foundation of screening system.
AKA-7-ACA is very unsettled, is 28 ℃ in condition, during pH 8.0, and the transformation period is merely 5h.The present invention has prepared two analogs of AKA-7-ACA, i.e. hexanedioyl-7-amino-3-desacetoxycephalosporanic acid (adipoyl-7-ADCA) and ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid (Keto-7-ADCA).These three chemical structures have six carbon alkyl group side chains on the female ring of identical beta-lactam, the identical or close ring, all are the substrates of Gl-7-ACA acylase hydrolysis.Acylase is identical to the substrate specificity (or specificity combination) of these three kinds of chemical structures.The stability of two analogs is higher than 1～2 order of magnitude of AKA-7-ACA.Like this, these two analogs replace AKA-7-ACA to produce the substrate of inducing of bacterium as the screening enzyme.Bacillus cereus is insensitive to adipoyl-7-ADCA and Keto-7-ADCA, but responsive to 7-ADCA.
Summary of the invention
The object of the present invention is to provide a kind of α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase to produce the screening method of bacterium; Through setting up the screening system that three layers of agar plate is cultivated; Utilize the indicator of Bacillus cereus, from numerous candidate bacterium, filter out the AKA-7-ACA acylase efficiently and produce bacterium as screening system.The present invention is simple to operate, does not need the laboratory apparatus of expensive, and everyone can screen and surpass 5000 candidate strain in one day, and screening efficiency is high, possesses the characteristics of high flux screening, is fit to apply to the industrial strain breeding.
Technical scheme of the present invention:
A kind of α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium, and its screening process comprises:
(1) preparation of α-ketone hexanedioyl-7-amino-cephalosporanic acid (AKA-7-ACA) and analog thereof, said analog are hexanedioyl-7-amino-3-desacetoxycephalosporanic acid (adipoyl-7-ADCA) and/or ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid (Keto-7-ADCA);
The selection and the cultivation of (2) selection of screening system indicator and cultivation, candidate bacterium enrichment culture and positive and negative control strain;
The preparation of (3) three layers of Agar Plating screening system;
(4) target α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the judgement of bacterium, and the method for said judgement is that target α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces bacterium and cultivates through three layers of Agar Plating and have or not judging of inhibition zone that Bacillus cereus occurs.
The preparation of said α-ketone hexanedioyl-7-amino-cephalosporanic acid is to be 2% cephalosporin with the phosphoric acid buffer dissolving of 0.1～0.2mol/L with weight content; Phosphoric acid buffer pH value is 7.5; Put into reactor drum then, add the D-amino-acid oxidase of 40U and the katalase of 2000U, reaction process feeds pure oxygen; Start whipping appts; Temperature is controlled under 27～29 ℃, and centrifugal collection reaction solution behind reaction 2～2.5h carries out condensing crystal to reaction solution and obtains α-ketone hexanedioyl-7-amino-cephalosporanic acid.
The preparation of said ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid is to be 2% desacetoxycephalosporanic acid with the phosphoric acid buffer dissolving of 0.1～0.2mol/L with weight content; Phosphoric acid buffer pH value is 7.5; Put into reactor drum then, add the D-amino-acid oxidase of 40U and the katalase of 2000U, reaction process feeds pure oxygen; Start whipping appts; Temperature is controlled under 27～29 ℃, and centrifugal collection reaction solution behind reaction 2～2.5h carries out condensing crystal to reaction solution and obtains ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid.
The preparation of said hexanedioyl-7-amino-3-desacetoxycephalosporanic acid (adipoyl-7-ADCA) is a hexanedioyl chlorine (>=99.5%; Sigma company provides) to splash into the pH value lentamente be in 7-amino-3-desacetoxycephalosporanic acid basic soln of 8～9, under whipped state, adds sodium bicarbonate solid (NaHCO
3), the control reaction pH value does not change, and centrifugal collecting reaction product behind reaction 2～3h carries out drying, crystallization obtains adipoyl-7-ADCA.
The structure of AKA-7-ACA analog is identified and can be made LC-MS in the step (1), after perhaps the process crystallization obtains monomer, is identifying through simple appearance.
Said candidate bacterium derives from nature or candidate's two mutants, mainly is the pseudomonas class, and its enrichment culture is meant puts into the enrichment medium enrichment culture 16～24h that contains 10～12mmol/L adipoyl-7-ADCA with pseudomonas; Dilution is coated on the LB Agar Plating then, under 35～37 ℃ condition, cultivates 16～24h, obtains single bacterium colony cell of candidate bacterium.
Said screening system indicator is a Bacillus cereus, and its cultivation is that the Bacillus cereus of single bacterium colony is cultivated after the activation of LB liquid nutrient medium, under 35～37 ℃ condition, cultivates 15～17h.
Wherein the selection principle of screening system indicator is to inducing substrate (adipoyl-7-ADCA and Keto-7-ADCA) insensitive (minimal inhibitory concentration＞256 μ g/ml) in the step (2); But to 7-ADCA responsive (minimal inhibitory concentration 2～4 μ g/ml); And this bacterial strain is not pathogenic bacterium, can cultivate through simple.
Said positive and negative control strain is respectively nitroreduction pseudomonas and intestinal bacteria; Its cultivation is respectively the nitroreduction pseudomonas and the intestinal bacteria of single bacterium colony to be chosen; Streak culture on the LB Agar Plating; Under 35～37 ℃ condition, cultivate 16～24h, obtain single bacterium colony cell of nitroreduction pseudomonas and the single bacterium colony cell of intestinal bacteria.
The selection principle of positive and negative control strain in the step (2) wherein: over against require according to bacterial strain can secreting, expressing AKA-7-ACA acylase, and bacterial strain is not pathogenic bacterium, can be through simple cultivation.Negative control strain requires not secreting, expressing AKA-7-ACA acylase, and bacterial strain is not pathogenic bacterium, can cultivate through simple.
The preparation of the first layer Agar Plating in said three layers of Agar Plating screening system: 15～16mL LB substratum falls dull and stereotyped; Its agar content is 1.5%; The candidate bacterium is seeded in the plate culture medium through point; Simultaneously nitroreduction pseudomonas and intestinal bacteria also put and are seeded in the plate culture medium respectively as positive and negative contrast, and this plate culture medium is cultivated 12～24h under 35～37 ℃ of conditions grow bacterium colony.
The preparation of second layer Agar Plating in said three layers of Agar Plating screening system: semisolid medium covers on the first layer Agar Plating equably under 45 ℃ the condition being lower than; Its consumption is 3～5ml; The thickness that covers is 0.3～0.6mm, under 35～37 ℃ to strain culturing 4～5h; The agar that said semisolid medium contains is 0.5～0.6%, hexanedioyl-7-amino-3-desacetoxycephalosporanic acid or ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid 5～6mg/ml.
The preparation of the 3rd layer of Agar Plating in said three layers of Agar Plating screening system: be lower than under 45 ℃ the condition; To contain agar content is 0.5～0.6%; Bacillus cereus content is that 0.01～0.02% semisolid medium covers on the second layer Agar Plating equably; Its consumption is 3～5ml, and the thickness of covering is 0.3～0.6mm, under 28～32 ℃ condition, cultivates 11～12h.
Wherein in the step (3) in the preparation of three layers of Agar Plating screening system, the agar content of semisolid medium, consumption and the plate temperature that falls are critical processes, and the plate temperature that falls is no more than 45 ℃.
Wherein in the step (4), the judgement that target enzyme produces bacterium is the next preliminary judgement of size through the inhibition zone of Bacillus cereus, and final judgement need the enzyme activity determination result after fermentation culture be judged.
The nitroreduction pseudomonas of being adopted among the present invention (Pseudomonas nitroreducens), numbering ATCC 33634, original number JCM 2782 is preserved in Chinese common micro-organisms DSMZ (CGMCC); Intestinal bacteria (Escherichia coli), numbering ATCC 53323 is preserved in Microbial resources preservation center, Sichuan Province; Bacillus cereus (Bacillus cereus), numbering ATCC 13061, three step on the commercial purchase of Science and Technology Ltd. from Shanghai.
The nitroreduction pseudomonas, Gram-negative, amphitrichous, nonspore-bearing bacillus, bacterium colony is white, protuberance more greatly, regular edges, smooth surface, strict aerobic, chemoheterotrophy.
Intestinal bacteria are γ-distortion Gammaproteobacteria, enterobacteria order, enterobacteriaceae, Escherichia, the bacterium of intestinal bacteria kind.Intestinal bacteria are in Application in Biotechnology: intestinal bacteria are as the host of exogenous gene expression, and genetic background is clear, and technological operation is simple, and culture condition is simple, large scale fermentation economy, extremely genetic engineering expert's attention.Intestinal bacteria are to use the most extensive, the most successful expression system at present, often do the first-selected system that efficiently expresses.
Bacillus cereus is the bacillus order, Bacillaceae, and Bacillus, the bacterium of Bacillus cereus kind, it is a kind of region, the soil life, Gram-positive, the rod-shaped bacterium of β hemolytic, it can cause food poisoning.Bacillus cereus is an aerobic-type, and is identical with other bacilluss, and it can produce defensive endospore.
Advantage of the present invention:
1, the present invention is simple to operate, does not need the laboratory apparatus of expensive simultaneously.
2, screening efficiency of the present invention is high, and everyone can screen and surpass 5000 candidate strain in one day, was fit to apply to the industrial strain breeding.
3, the present invention guarantees the growth of bacterial strain through agar content, consumption in the screening system of three layers of agar plates cultivation of strict control and the plate temperature that falls, thus the success ratio of control screening.
Specific embodiment
The present invention describes with the following example, but does not limit the scope of the invention.
Embodiment 1
1, the preparation of AKA-7-ACA and analog thereof:
Phosphoric acid buffer: Sodium phosphate, dibasic and SODIUM PHOSPHATE, MONOBASIC by pH be 7.5 proportional arrangement damping fluid to the utmost.
(1) preparation of AKA-7-ACA
It is the dissolving of 7.5 phosphoric acid buffer as 0.1mol/L, pH value that the 2%CPC of 100ml uses concentration; Put into reactor drum, the CAT of the DAAO of adding 3ml 40U and the 2000U of 3ml, reaction process feeds pure oxygen; And constantly stir; Temperature is controlled at 28 ℃, centrifugal collection reaction solution behind the 2h, and concentrating also to reaction solution, crystallization obtains AKA-7-ACA.
(2) preparation of Adipoyl-7-ADCA
It is in 8～9 the 7-ADCA basic soln that 5～10mL hexanedioyl chlorine (adipoyl chloride) splashes into 5 gram pH values lentamente; Add the sodium bicarbonate solid while stirring, keep pH not change centrifugal collecting reaction product behind the 2h; Crystallization is also carried out structure and is identified, adopts mass spectrometer configuration to identify.
Its operational path is following:
The selection and the cultivation of 2, selection of screening system indicator and cultivation, candidate bacterium enrichment culture and positive and negative control strain:
(1) bacterial classification situation
Nitroreduction pseudomonas (Pseudomonas nitroreducens), numbering ATCC 33634, original number JCM 2782 is preserved in Chinese common micro-organisms DSMZ (CGMCC); Intestinal bacteria (Escherichia coli), numbering ATCC53323 is preserved in Microbial resources preservation center, Sichuan Province; Bacillus cereus (Bacillus cereus), numbering ATCC13061, three step on the commercial purchase of Science and Technology Ltd. from Shanghai.
(2) substratum situation
Enrichment medium: 0.15% (NH
2) HPO
40.1%KH
2PO
40.05%MgSO
4.7H
2O; 0.001%FeCl
3.6H
2O; 0.001%MnCl
2.4H
2O; 0.001%NaCl,
LB liquid nutrient medium: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, deionized water configuration.
LB nutrient agar: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, agar 15g/l, deionized water configuration.
LB semisolid medium: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, agar 5g/l, deionized water configuration.
(3) selection of screening system indicator and cultivation
The screening system indicator is adopted Bacillus cereus, its cultivation: the Bacillus cereus of single bacterium colony is cultivated after the activation of LB liquid nutrient medium, under 35 ℃ condition, cultivates 16h.
(4) candidate bacterium enrichment culture
The screening of candidate bacterium is primarily aimed at pseudomonas and carries out enrichment culture, and enrichment medium contains 10mmol/L adipoyl-7-ADCA, and the candidate bacterium is through behind enrichment culture 16～20h, and dilution is coated on the LB Agar Plating.Under 35 ℃ condition, cultivate 16～20h, obtain single bacterium colony cell.
(5) selection of positive and negative control strain and cultivation
Nitroreduction pseudomonas and colibacillary cultivation: the bacterium of single bacterium colony is chosen, and is streak culture on the LB Agar Plating, under 35 ℃ condition, cultivates 16～20h, obtains single bacterium colony cell.
3, three layers of preparation that agar plate is cultivated screening system:
15mL LB substratum (agar content 1.5%) is petridish (specification: 9cm); The candidate bacterium is seeded to (100～200 candidate bacterium/individual flat boards) in the Agar Plating through point; Simultaneously nitroreduction pseudomonas (Pseudomonas nitroreducens) ATCC 33634 and intestinal bacteria (Escherichia coli) ATCC 53323 also are seeded in the flat board respectively as positive and negative contrast, and this flat board is cultivated 16h under 35～37 ℃ of conditions grow bacterium colony.3mL semisolid medium (agar content 0.5% contains keto-7-ADCA or the adipoyl-7-ADCA of 5mg/ml) covers above-mentioned flat board uniformly under 45 ℃ the condition and (cultivates 4h again under about 0.3～0.6mm), 35～37 ℃ of conditions of thickness being lower than then.In addition 3mL semisolid medium (agar content 0.5% contains 0.01% Bacillus cereus (Bacillus cereus) ATCC 13061) covers above-mentioned flat board being lower than under 45 ℃ the condition equally equably, under 28～32 ℃ condition, cultivates 12h again.
4, target enzyme produces the judgement of bacterium:
In the candidate bacterium, there is the AKA-7-ACA acylase to produce bacterium and (is mainly pseudomonas; Belong to external secretion and express acylase); Can decompose Adipoyl-7-ADCA generation 7-ADCA by this acylase of secretion; Thereby suppress the growth of system's indicator Bacillus cereus, and form inhibition zone, tentatively infer the height of acylase vigor through the big I of antibacterial circle diameter.As over against the nitroreduction pseudomonas of photograph owing to produce the AKA-7-ACA acylase, hydrolysis substrate generates 7-ADCA, makes the bacterium colony periphery inhibition zone of a Bacillus cereus occur.On the contrary, as the intestinal bacteria of negative contrast owing to there is not acylase not produce 7-ADCA by hydrolysis substrate, and can normal growth at its bacterium colony periphery Bacillus cereus.By this method, a people can screen in two days time and surpass 5000 candidate bacterium.Experimental result such as table 1
Table 1
The MV of 3 experimental results
Embodiment 2
1, the preparation of AKA-7-ACA and analog thereof:
Phosphoric acid buffer: Sodium phosphate, dibasic and SODIUM PHOSPHATE, MONOBASIC by pH be 7.5 proportional arrangement damping fluid to the utmost.
(1) preparation of AKA-7-ACA
It is the dissolving of 7.5 phosphoric acid buffer as 0.1mol/L, pH value that the 2%CPC of 100ml uses concentration; Put into reactor drum, the CAT of the DAAO of adding 3ml 40U and the 2000U of 3ml, reaction process feeds pure oxygen; And constantly stir; Temperature is controlled at 27～29 ℃, centrifugal collection reaction solution behind the 2h, and concentrating also to reaction solution, crystallization obtains AKA-7-ACA.
(2) preparation of keto-7-ADCA
It is the dissolving of 7.5 phosphoric acid buffer as 0.1mol/L, pH value that the 2% deacetylate CPC of 100ml uses concentration; Put into reactor drum, the CAT of the DAAO of adding 2ml 40U and the 2000U of 4ml, reaction process feeds pure oxygen; And constantly stir; Temperature is controlled at 29 ℃, centrifugal collection reaction solution behind the 2.5h, and concentrating also to reaction solution, crystallization obtains keto-7-ADCA.
The selection and the cultivation of 2, selection of screening system indicator and cultivation, candidate bacterium enrichment culture and positive and negative control strain:
(1) bacterial classification situation
Nitroreduction pseudomonas (Pseudomonas nitroreducens), numbering ATCC 33634, original number JCM 2782 is preserved in Chinese common micro-organisms DSMZ (CGMCC); Intestinal bacteria (Escherichia coli), numbering ATCC53323 is preserved in Microbial resources preservation center, Sichuan Province; Bacillus cereus (Bacillus cereus), numbering ATCC13061, three step on the commercial purchase of Science and Technology Ltd. from Shanghai.
(2) substratum situation
Enrichment medium: 0.15% (NH
2) HPO
40.1%KH
2PO
40.05%MgSO
4.7H
2O; 0.001%FeCl
3.6H
2O; 0.001%MnCl
2.4H
2O; 0.001%NaCl,
LB liquid nutrient medium: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, deionized water configuration.
LB nutrient agar: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, agar 15g/l, deionized water configuration.
LB semisolid medium: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, agar 5g/l, deionized water configuration.
(3) selection of screening system indicator and cultivation
The screening system indicator is adopted Bacillus cereus, its cultivation: the Bacillus cereus of single bacterium colony is cultivated after the activation of LB liquid nutrient medium, under 36 ℃ condition, cultivates 17h.
(4) candidate bacterium enrichment culture
The screening of candidate bacterium is primarily aimed at pseudomonas and carries out enrichment culture, and enrichment medium contains 12mmol/L adipoyl-7-ADCA, and the candidate bacterium is through behind enrichment culture 20～24h, and dilution is coated on the LB Agar Plating.Under 36 ℃ condition, cultivate 20～24h, obtain single bacterium colony cell.
(5) selection of positive and negative control strain and cultivation
Nitroreduction pseudomonas and colibacillary cultivation: the bacterium of single bacterium colony is chosen, and is streak culture on the LB Agar Plating, under 36 ℃ condition, cultivates 20～24h, obtains single bacterium colony cell.
3, three layers of preparation that agar plate is cultivated screening system:
16mL LB substratum (agar content 1.5%) is petridish (specification: 9cm); The candidate bacterium is seeded to (150～200 candidate bacterium/individual flat boards) in the Agar Plating through point; Simultaneously nitroreduction pseudomonas (Pseudomonas nitroreducens) ATCC 33634 and intestinal bacteria (Escherichia coli) ATCC 53323 also are seeded in the flat board respectively as positive and negative contrast, and this flat board is cultivated 24h under 35～36 ℃ of conditions grow bacterium colony.5mL semisolid medium (agar content 0.6% contains keto-7-ADCA or the adipyl-7-ADCA of 6mg/ml) covers above-mentioned flat board uniformly under 45 ℃ the condition and (cultivates 5h again under about 0.3～0.5mm), 35～36 ℃ of conditions of thickness being lower than then.In addition 5mL semisolid medium (agar content 0.5% contains 0.02% Bacillus cereus (Bacillus cereus) ATCC 13061) covers above-mentioned flat board being lower than under 45 ℃ the condition equally equably, under 30～32 ℃ condition, cultivates 11h again.
4, target enzyme produces the judgement of bacterium:
In the candidate bacterium, there is the AKA-7-ACA acylase to produce bacterium and (is mainly pseudomonas; Belong to external secretion and express acylase); Can decompose keto-7-ADCA generation 7-ADCA by this acylase of secretion; Thereby suppress the growth of system's indicator Bacillus cereus, and form inhibition zone, tentatively infer the height of acylase vigor through the big I of antibacterial circle diameter.As over against nitroreduction pseudomonas (Pseudomonas nitroreducens) ATCC of photograph 33634 owing to produce the AKA-7-ACA acylase, hydrolysis substrate generates 7-ADCA, makes the bacterium colony periphery inhibition zone of a Bacillus cereus occur.On the contrary, as intestinal bacteria (Escherichia coli) ATCC 53323 of negative contrast owing to there is not acylase not produce 7-ADCA by hydrolysis substrate, and can normal growth at its bacterium colony periphery Bacillus cereus.By this method, one can be screened in two days time above 5000 candidate bacterium.Experimental result such as table 2
Table 2
The MV of 3 experimental results
Embodiment 3
1, the preparation of AKA-7-ACA and analog thereof:
Phosphoric acid buffer: Sodium phosphate, dibasic and SODIUM PHOSPHATE, MONOBASIC by pH be 7.5 proportional arrangement damping fluid to the utmost.
(1) preparation of AKA-7-ACA
It is the dissolving of 7.5 phosphoric acid buffer as 0.2mol/L, pH value that the 2%CPC of 100ml uses concentration; Put into reactor drum, the CAT of the DAAO of adding 4ml 40U and the 2000U of 2ml, reaction process feeds pure oxygen; And constantly stir; Temperature is controlled at 27～29 ℃, centrifugal collection reaction solution behind the 2.5h, and concentrating also to reaction solution, crystallization obtains AKA-7-ACA.
(2) preparation of Adipoyl-7-ADCA
It is in 8～9 the 7-ADCA basic soln that 5～10mL hexanedioyl chlorine (adipoyl chloride) splashes into 5 gram pH lentamente; Add the sodium bicarbonate solid while stirring, keep pH constant, centrifugal collecting reaction product behind the 3h; Crystallization is also carried out structure and is identified, adopts mass spectrograph to carry out structure and identifies.
Its operational path is following:
The selection and the cultivation of 2, selection of screening system indicator and cultivation, candidate bacterium enrichment culture and positive and negative control strain:
(1) bacterial classification situation
Nitroreduction pseudomonas (Pseudomonas nitroreducens), numbering ATCC 33634, original number JCM 2782 is preserved in Chinese common micro-organisms DSMZ (CGMCC); Intestinal bacteria (Escherichia coli), numbering ATCC53323 is preserved in Microbial resources preservation center, Sichuan Province; Bacillus cereus (Bacillus cereus), numbering ATCC13061, three step on the commercial purchase of Science and Technology Ltd. from Shanghai.
(2) substratum situation
Enrichment medium: 0.15% (NH
2) HPO
40.1%KH
2PO
40.05%MgSO
4.7H
2O; 0.001%FeCl
3.6H
2O; 0.001%MnCl
2.4H
2O; 0.001%NaCl,
LB liquid nutrient medium: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, deionized water configuration.
LB nutrient agar: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, agar 15g/l, deionized water configuration.
LB semisolid medium: Tryptones 10g/l; Yeast extract paste 5g/l, NaCl5g/l, agar 5g/l, deionized water configuration.
(3) selection of screening system indicator and cultivation
The screening system indicator is adopted Bacillus cereus, its cultivation: the Bacillus cereus of single bacterium colony is cultivated after the activation of LB liquid nutrient medium, under 37 ℃ condition, cultivates 15h.
(4) candidate bacterium enrichment culture
The screening of candidate bacterium is primarily aimed at pseudomonas and carries out enrichment culture, and enrichment medium contains 11mmol/L adipoyl-7-ADCA, and the candidate bacterium is through behind enrichment culture 20～24h, and dilution is coated on the LB Agar Plating.Under 37 ℃ condition, cultivate 20～24h, obtain single bacterium colony cell.
(5) selection of positive and negative control strain and cultivation
Nitroreduction pseudomonas and colibacillary cultivation: the bacterium of single bacterium colony is chosen, and is streak culture on the LB Agar Plating, under 34 ℃ condition, cultivates 18～22h, obtains single bacterium colony cell.
3, three layers of preparation that agar plate is cultivated screening system:
15mL LB substratum (agar content 1.5%) is petridish (specification: 9cm); The candidate bacterium is seeded to (100～150 candidate bacterium/individual flat boards) in the Agar Plating through point; Simultaneously nitroreduction pseudomonas (Pseudomonas nitroreducens) ATCC 33634 and intestinal bacteria (Escherichia coli) ATCC 53323 also are seeded in the flat board respectively as positive and negative contrast, and this flat board is cultivated 12h under 36 ℃ of conditions grow bacterium colony.4mL semisolid medium (agar content 0.5% contains keto-7-ADCA or the adipyl-7-ADCA of 5mg/ml) covers above-mentioned flat board uniformly under 45 ℃ the condition and (cultivates 4h again under about 0.3～0.4mm), the 36 ℃ of conditions of thickness being lower than then.In addition 4mL semisolid medium (agar content 0.6% contains 0.01% Bacillus cereus (Bacillus cereus) ATCC 13061) covers above-mentioned flat board being lower than under 45 ℃ the condition equally equably, under 30～32 ℃ condition, cultivates 11h again.
4, target enzyme produces the judgement of bacterium:
In the candidate bacterium, there is the AKA-7-ACA acylase to produce bacterium and (is mainly pseudomonas; Belong to external secretion and express acylase); Can decompose Adipoyl-7-ADCA generation 7-ADCA by this acylase of secretion; Thereby suppress the growth of system's indicator Bacillus cereus, and form inhibition zone, tentatively infer the height of acylase vigor through the big I of antibacterial circle diameter.As over against the nitroreduction pseudomonas of photograph owing to produce the AKA-7-ACA acylase, hydrolysis substrate generates 7-ADCA, makes the bacterium colony periphery inhibition zone of a Bacillus cereus occur.On the contrary, as the intestinal bacteria of negative contrast owing to there is not acylase not produce 7-ADCA by hydrolysis substrate, and can normal growth at its bacterium colony periphery Bacillus cereus.By this method, one can be screened in two days time above 5000 candidate bacterium.Experimental result such as table 3
Table 3
The MV of 3 experimental results
Experiment shows, through such agar plate screening system, can filter out target enzyme efficiently and produce bacterium, and the screening method simple and sensitive, possess high-throughout character.
Claims (10)
1. α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium, and it is characterized in that: its screening process comprises:
(1) preparation of α-ketone hexanedioyl-7-amino-cephalosporanic acid and analog thereof, said analog are hexanedioyl-7-amino-3-desacetoxycephalosporanic acid and/or ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid;
The selection and the cultivation of (2) selection of screening system indicator and cultivation, candidate bacterium enrichment culture and positive and negative control strain;
The preparation of (3) three layers of Agar Plating screening system;
(4) target α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the judgement of bacterium, and the method for said judgement is that target α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces bacterium and cultivates through three layers of Agar Plating and have or not judging of inhibition zone that Bacillus cereus occurs.
2. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium; It is characterized in that: the preparation of said α-ketone hexanedioyl-7-amino-cephalosporanic acid is to be 2% cephalosporin with the phosphoric acid buffer dissolving of 0.1～0.2mol/L with weight content, and phosphoric acid buffer pH value is 7.5, puts into reactor drum then; Add the D-amino-acid oxidase of 40U and the katalase of 2000U; Reaction process feeds pure oxygen, starts whipping appts, and temperature is controlled under 27～29 ℃; Centrifugal collection reaction solution behind reaction 2～2.5h carries out condensing crystal to reaction solution and obtains α-ketone hexanedioyl-7-amino-cephalosporanic acid.
3. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium; It is characterized in that: the preparation of said ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid is to be 2% desacetoxycephalosporanic acid with the phosphoric acid buffer dissolving of 0.1～0.2mol/L with weight content; Phosphoric acid buffer pH value is 7.5; Put into reactor drum then, add the D-amino-acid oxidase of 40U and the katalase of 2000U, reaction process feeds pure oxygen; Start whipping appts; Temperature is controlled under 27～29 ℃, and centrifugal collection reaction solution behind reaction 2～2.5h carries out condensing crystal to reaction solution and obtains ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid.
4. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium; It is characterized in that: the preparation of said hexanedioyl-7-amino-3-desacetoxycephalosporanic acid is that to splash into the pH value lentamente be in 7-amino-3-desacetoxycephalosporanic acid basic soln of 8～9 to hexanedioyl chlorine; Under whipped state, add the sodium bicarbonate solid; The control reaction pH value is constant; Centrifugal collecting reaction product behind reaction 2～3h carries out drying, crystallization obtains hexanedioyl-7-amino-3-desacetoxycephalosporanic acid.
5. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium, and it is characterized in that: said candidate bacterium enrichment culture is meant puts into the enrichment medium enrichment culture 16～24h that contains 10～12mmol/L hexanedioyl-7-amino-3-desacetoxycephalosporanic acid with pseudomonas; Dilution is coated on the LB Agar Plating then, under 35～37 ℃ condition, cultivates 16～24h, obtains single bacterium colony cell of candidate bacterium.
6. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium; It is characterized in that: said screening system indicator is a Bacillus cereus; Its cultivation is that the Bacillus cereus of single bacterium colony is cultivated after the activation of LB liquid nutrient medium, under 35～37 ℃ condition, cultivates 15～17h.
7. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium; It is characterized in that: said positive and negative control strain is respectively nitroreduction pseudomonas and intestinal bacteria; Its cultivation is respectively the nitroreduction pseudomonas and the intestinal bacteria of single bacterium colony to be chosen; Streak culture on the LB Agar Plating, under 34～36 ℃ condition, cultivate 16～24h, obtain single bacterium colony cell of nitroreduction pseudomonas and the single bacterium colony cell of intestinal bacteria.
8. produce the screening method of bacterium according to claim 1 or 5 described α-ketone hexanedioyl-7-amino-cephalosporanic acid acylase; It is characterized in that: the preparation of the first layer Agar Plating in said three layers of Agar Plating screening system: 15～16mL LB substratum falls dull and stereotyped; Its agar content is 1.5%; The candidate bacterium is seeded in the plate culture medium through point; Simultaneously nitroreduction pseudomonas and intestinal bacteria also put and are seeded in the plate culture medium respectively as positive and negative contrast, and this plate culture medium is cultivated 12～24h under 35～37 ℃ of conditions grow bacterium colony.
9. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium; It is characterized in that: the preparation of second layer Agar Plating in said three layers of Agar Plating screening system: semisolid medium covers on the first layer Agar Plating equably under 45 ℃ the condition being lower than; Its consumption is 3～5ml; The thickness that covers is 0.3～0.6mm, under 35～37 ℃ to strain culturing 4～5h; The agar that said semisolid medium contains is 0.5～0.6%, hexanedioyl-7-amino-3-desacetoxycephalosporanic acid or ketone hexanedioyl-7-amino-3-desacetoxycephalosporanic acid 5～6mg/ml.
10. α according to claim 1-ketone hexanedioyl-7-amino-cephalosporanic acid acylase produces the screening method of bacterium; It is characterized in that: the preparation of the 3rd layer of Agar Plating in said three layers of Agar Plating screening system: be lower than under 45 ℃ the condition; To contain agar content is 0.5～0.6%; Bacillus cereus content is that 0.01～0.02% semisolid medium covers on the second layer Agar Plating equably; Its consumption is 3～5ml, and the thickness of covering is 0.3～0.6mm, under 28～32 ℃ condition, cultivates 11～12h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436521A (en) * | 2013-06-28 | 2013-12-11 | 山东鲁抗医药股份有限公司 | High cephalosporin C yield bacterial breeding method |
| CN107937471A (en) * | 2017-11-24 | 2018-04-20 | 广西中医药大学 | A kind of method that three enzyme system prepares 7 amino-cephalo-alkanoic acids |
| CN115232856A (en) * | 2022-07-27 | 2022-10-25 | 河南省健康元生物医药研究院有限公司 | High-throughput screening method for acremonium chrysogenum based on solid fermentation |
-
2012
- 2012-04-16 CN CN2012101091831A patent/CN102690859A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| SONGCHENG ZHU等: "A rapid and specific method to screen environmental microorganisms for cephalosporin acylase activity", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436521A (en) * | 2013-06-28 | 2013-12-11 | 山东鲁抗医药股份有限公司 | High cephalosporin C yield bacterial breeding method |
| CN107937471A (en) * | 2017-11-24 | 2018-04-20 | 广西中医药大学 | A kind of method that three enzyme system prepares 7 amino-cephalo-alkanoic acids |
| CN115232856A (en) * | 2022-07-27 | 2022-10-25 | 河南省健康元生物医药研究院有限公司 | High-throughput screening method for acremonium chrysogenum based on solid fermentation |
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