CN105543332A - DAB color development-based high-throughput screening method for CPC histone deacetylases - Google Patents
DAB color development-based high-throughput screening method for CPC histone deacetylases Download PDFInfo
- Publication number
- CN105543332A CN105543332A CN201610002076.7A CN201610002076A CN105543332A CN 105543332 A CN105543332 A CN 105543332A CN 201610002076 A CN201610002076 A CN 201610002076A CN 105543332 A CN105543332 A CN 105543332A
- Authority
- CN
- China
- Prior art keywords
- cpc
- acetylase
- aca
- aqueous solution
- mixture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a DAB color development-based high-throughput screening method for CPC histone deacetylases. According to the method, on the basis of the principle that a compound of paradimethylaminobenzaldehyde and 7-ACA has the maximum absorption peak at 405 nm, low-temperature low-concentration inducer-induced expression is performed on a recombinant bacterium through a sterile 96-well plate, bacterium splitting decomposition is performed with a dilute alkaline solution, and then neutralizing is performed with sodium dihydrogen phosphate; substrate hydrolysis is performed with the 96-well plate, after p-DAB color development is performed, a product is rapidly quantified through a microplate reader, and then high-throughput screening of CPC histone deacetylases mutants is achieved. The method has the advantages of being rapid to screen, easy and convenient to operate, high in screening throughput and accurate and the like, the CPC histone deacetylases transforming speed can be increased, and the great application potential and economic value are achieved.
Description
Technical field
The present invention relates to technical field of bioengineering, specifically a kind of method of low temperature CPC acetylase (the i.e. cephalosporin acetylase) high flux screening based on DAB colour developing.
Background technology
The chemical name of described 7-ACA is 3-acetyl-o-methyl-5-sulphur-7-amino-8-oxygen-1-azabicyclic oct-2-ene-2 carboxylic acid, and being corn steep liquor to be fermented the cephalosporin obtained by cephalo bacterium, and at amido linkage place, hydrolysis obtains 7-AcA to cephalosporin, and its structure is as the right side:
.7-ACA is the important intermediate of synthesis cephalosporin analog antibiotic; what it was traditional prepares approach is by cephalosporin (CephalosporinC; CPC) formed by the deacetylated process of chemical process, chemical method needs harsh reaction conditions, and can produce some poisonous materials.Compared with traditional chemical process, novel biological catalysis has that security is high, environmental friendliness, selectivity are strong and the advantage such as equipment investment is low.What biological catalysis was common mainly contains two step enzyme methods and a step enzyme method.The key enzyme that two step enzyme methods use is D-AAO and Glularyl-7-amino-cephalo-alkanoic acid acetylase, and CPC can change into 7-ACA under the priority effect of two enzymes.Through development for many years, the technique of two step enzyme methods reaches its maturity, and is widely applied in the industrial production, but two step enzyme method by products are many, the preparation of enzyme and the defect of production technique relative complex still exist.One step enzyme method is that CPC directly can change into 7-ACA under the effect of cephalosporin (i.e. CPC acetylase), and this approach more can simplify production technique, reduce production cost, more attractive in the industrial production.Due to 7-ACA extremely unstable, easily other by product of generation more than 20 DEG C, in order to obtain high-quality 7-ACA, its synthesis often needs to carry out at low ambient temperatures, and the production general control of industrial 7-ACA is at about 13 DEG C.And existing CPC acetylase optimum temperuture is higher, catalytic activity is lower at low ambient temperatures, must must improves enzyme concentration under low temperature environment, add production cost.At present, all also successfully do not develop optimum temperuture both at home and abroad to report lower than the research of the low temperature CPC acetylase of 20 DEG C.
At present, because the understanding of enzyme being fitted to cold still lacks unified final conclusion, the method that initiative cold-adapted enzyme is commonly used is still based on orthogenesis or some saturation mutation technology.At present, the measuring method of conventional CPC acetylase activity mainly contains alkali titration, p-DAB colorimetry and HPLC method, and wherein alkali titration mainly uses activity that is simple, coarse, assessment cephalosporin acetylase rapidly in the industrial production.HPLC method can measure the amount of 7-ACA very exactly and then calculate the activity of cephalosporin acetylase exactly, but limit by instrument, and finding speed is comparatively slow and be difficult to realize high-throughout mensuration.Traditional p-DAB colorimetry, because the stop buffer described in forefathers is to the termination limited efficiency of reaction, is also not too applicable to high-throughout mensuration.
Summary of the invention
The present invention seeks to the deficiency for solving the problems of the technologies described above, provide a kind of method of the low temperature CPC acetylase high flux screening based on DAB colour developing, it can obtain low temperature CPC acetylase fast, accurately from the mutated library of magnanimity.
The present invention for solving the problems of the technologies described above adopted technical scheme is: a kind of method of the low temperature CPC acetylase high flux screening based on DAB colour developing, the method is based on paradimethy laminobenzaldehyde (p-dimethylaminobenzaldehyde, p-DAB) principle of maximum absorption band is had at 405nm with the mixture of 7-ACA, 96 aseptic orifice plates are adopted to carry out low-temperature and low-concentration inductor abduction delivering to recombinant bacterium, SODIUM PHOSPHATE, MONOBASIC is utilized to neutralize after carrying out cellular lysate with dilute alkaline soln, and then the hydrolysis of substrate is carried out with 96 orifice plates, after p-DAB colour developing, utilize microplate reader to carry out the fast quantification of product, and then realize the high flux screening of CPC acetylase mutant.
Based on a method for the low temperature CPC acetylase high flux screening of DAB colour developing, working method more specifically, is preferably following scheme; Comprise the steps:
The high-throughput of step one, CPC acetylase is expressed: from the mutated library of spore rhzomorph C acetylase saturation mutation, the muton of picking individual colonies is seeded to that to be equipped with containing concentration be in the sterile polystyrene plate of 200 μ LLB substratum of 100 μ g/mL penbritins, at being placed in 37 DEG C, 200rpm overnight incubation; Be inoculated into by the product pipettor of incubated overnight in the LB liquid nutrient medium of 300 μ L, inoculum size is 4% of the LB liquid nutrient medium volume of 300 μ L; Then, at being placed in 37 DEG C, 200rpm cultivates 2.5h, finally adds the IPTG aqueous solution that final concentration is 0.1mmol/L, and at being placed in 30 DEG C, 200rpm cultivates 8h, obtains the bacterium liquid after inducing, for subsequent use;
The alkaline lysis of step 2, thalline: the bacterium liquid after being induced by step one gained is placed in the centrifugal 1min of 8000g, after removing supernatant, thalline is resuspended in the phosphate buffered saline buffer of 100mmol/L, pH8.0 of 300 μ L, adding 1 μ L solubility is vortex oscillation after the Dnase aqueous solution of 1mg/mL, add the NaOH aqueous solution lysing cell of 50 μ L1mol/L, leave standstill 2min after vibration 30s mixing, then add 50 μ L1mol/LKH
2pO
4the aqueous solution neutralizes; Mixing is placed on the centrifugal 10min of 11000g, and collect supernatant liquor, the supernatant liquor collected is the thick liquid of CPC acetylase;
Step 3, the making of 7-ACA content standard curve: with 0.1mol/L, the phosphoric acid buffer preparation final concentration of pH8.0 is the 7-ACA of 1.5mg/mL, with the NaOH adjust pH to 8.0 of 1mol/L, get 0 respectively, 1, 2, 4, 6, 8, 12, the 7-ACA of 16 and 20 μ L is in centrifuge tube, use 0.1mol/L again, the phosphoric acid buffer of pH8.0 supplies 20 μ L, the CPC acetylase of 20 μ L13 DEG C preheating 3min is added in centrifuge tube, mixing, obtain mixture A, after mixture A being placed in 13 DEG C of water-bath 10min, add the stop buffer that volume is mixture A volume 5 times, the centrifugal 3min of 12000rpm after mixing, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, room temperature gets the light absorption value that 200 μ L survey 405nm after leaving standstill 10min, with No. 0 for blank, with the content of 7-ACA for ordinate zou, the light absorption value of 405nm is X-coordinate production standard curve, and obtain regression equation,
The high-throughout fast quantification of step 4,7-ACA: prepare the cephalosporin of 20g/L as substrate with the phosphoric acid buffer of 0.1mol/L, pH to 8.0 is regulated with the NaOH aqueous solution of 1mol/L, get 20 μ L substrates and 20 μ L dilute after the thick liquid of CPC acetylase mix in 96 orifice plates, hatch 10min for 13 DEG C, obtain mixture B; In mixture B, add volume is that the stop buffer of mixture B volume 5 times carries out termination reaction, gets the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, gets 200 μ L and utilize 96 orifice plates in microplate reader, survey the light absorption value of 405nm after room temperature reaction 10min;
The calculating of step 5, CPC acetylase activity: be defined as a Ge Meihuo unit (IU) with the per minute enzyme amount produced needed for 1 μm of ol7-ACA under condition determination; And then it is alive to calculate each mutant enzyme at low temperatures with EXCEL;
Described stop buffer, the ratio being the acetic acid aqueous solution of 20% and the NaOH aqueous solution 2:1 by volume of 0.05mol/L by mass percent concentration mixes;
Described developer is the dimethylaminobenzaldehyde of 0.5% with the mass percent concentration of methyl alcohol preparation.
Described polystyrene board is preferably Nunc 96DeepWell polystyrene board.
Described pipettor is preferably the pipettor of 8 passages.
Beneficial effect is:
The improvement that the method that the present invention is based on the low temperature CPC acetylase high flux screening of DAB colour developing has been done traditional p-DAB colorimetry, is beneficial to the mensuration of subsequent high pass amount.Utilize traditional expression and screening strategy from the mutated library of magnanimity, screen target low enzyme to be doomed to be a job of wasting time and energy, traditional CPC acetylase expression and the method for determination of activity can not meet the demand of rapid screening.Therefore, the method setting up low temperature CPC acetylase high flux screening is fast and accurately the important leverage obtaining cold-adapted enzyme from the mutated library of magnanimity, and tool is of great significance; The method has that screening is quick, easy and simple to handle, screening flux is large and accuracy advantages of higher, can accelerate the speed of CPC acetylase transformation, have huge application potential and economic worth.
Accompanying drawing explanation
Fig. 1 is the canonical plotting of 7-ACA content in the present invention;
Fig. 2 is the SDS-PAGE analysis chart of CPC acetylase and mutant thereof in the present invention;
In figure, mark is: 1, BL21/PET32a, 2, BL21/PET32a-HG6 (0mmol/LIPTG), 3, BL21/PET32a-HG6,4, P-254,5, P-166,6, P-835.
Embodiment
A kind of method of the low temperature CPC acetylase high flux screening based on DAB colour developing, the method is based on paradimethy laminobenzaldehyde (p-dimethylaminobenzaldehyde, p-DAB) principle of maximum absorption band is had at 405nm with the mixture of 7-ACA, 96 aseptic orifice plates are adopted to carry out low-temperature and low-concentration inductor abduction delivering to recombinant bacterium, SODIUM PHOSPHATE, MONOBASIC is utilized to neutralize after carrying out cellular lysate with dilute alkaline soln, and then the hydrolysis of substrate is carried out with 96 orifice plates, after p-DAB colour developing, utilize microplate reader to carry out the fast quantification of product, and then realize the high flux screening of CPC acetylase mutant.
Based on a method for the low temperature CPC acetylase high flux screening of DAB colour developing, working method more specifically, is preferably following scheme; Comprise the steps:
The high-throughput of step one, CPC acetylase is expressed: from the mutated library of spore rhzomorph C acetylase saturation mutation, the muton of picking individual colonies is seeded to that to be equipped with containing concentration be in the sterile polystyrene plate of 200 μ LLB substratum of 100 μ g/mL penbritins, at being placed in 37 DEG C, 200rpm overnight incubation; Be inoculated into by the product pipettor of incubated overnight in the LB liquid nutrient medium of 300 μ L, inoculum size is 4% of the LB liquid nutrient medium volume of 300 μ L; Then, at being placed in 37 DEG C, 200rpm cultivates 2.5h, finally adds the IPTG aqueous solution that final concentration is 0.1mmol/L, and at being placed in 30 DEG C, 200rpm cultivates 8h, obtains the bacterium liquid after inducing, for subsequent use;
The alkaline lysis of step 2, thalline: the bacterium liquid after being induced by step one gained is placed in the centrifugal 1min of 8000g, after removing supernatant, thalline is resuspended in the phosphate buffered saline buffer of 100mmol/L, pH8.0 of 300 μ L, adding 1 μ L solubility is vortex oscillation after the Dnase aqueous solution of 1mg/mL, add the NaOH aqueous solution lysing cell of 50 μ L1mol/L, leave standstill 2min after vibration 30s mixing, then add 50 μ L1mol/LKH
2pO
4the aqueous solution neutralizes; Mixing is placed on the centrifugal 10min of 11000g, and collect supernatant liquor, the supernatant liquor collected is the thick liquid of CPC acetylase;
Step 3, the making of 7-ACA content standard curve: with 0.1mol/L, the phosphoric acid buffer preparation final concentration of pH8.0 is the 7-ACA of 1.5mg/mL, with the NaOH adjust pH to 8.0 of 1mol/L, get 0 respectively, 1, 2, 4, 6, 8, 12, the 7-ACA of 16 and 20 μ L is in centrifuge tube, use 0.1mol/L again, the phosphoric acid buffer of pH8.0 supplies 20 μ L, the CPC acetylase of 20 μ L13 DEG C preheating 3min is added in centrifuge tube, mixing, obtain mixture A, after mixture A being placed in 13 DEG C of water-bath 10min, add the stop buffer that volume is mixture A volume 5 times, the centrifugal 3min of 12000rpm after mixing, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, room temperature gets the light absorption value that 200 μ L survey 405nm after leaving standstill 10min, with No. 0 for blank, with the content of 7-ACA for ordinate zou, the light absorption value of 405nm is X-coordinate production standard curve, and obtain regression equation,
The high-throughout fast quantification of step 4,7-ACA: prepare the cephalosporin of 20g/L as substrate with the phosphoric acid buffer of 0.1mol/L, pH to 8.0 is regulated with the NaOH aqueous solution of 1mol/L, get 20 μ L substrates and 20 μ L dilute after the thick liquid of CPC acetylase mix in 96 orifice plates, hatch 10min for 13 DEG C, obtain mixture B; In mixture B, add volume is that the stop buffer of mixture B volume 5 times carries out termination reaction, gets the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, gets 200 μ L and utilize 96 orifice plates in microplate reader, survey the light absorption value of 405nm after room temperature reaction 10min;
The calculating of step 5, CPC acetylase activity: be defined as a Ge Meihuo unit (IU) with the per minute enzyme amount produced needed for 1 μm of ol7-ACA under condition determination; And then it is alive to calculate each mutant enzyme at low temperatures with EXCEL;
Described stop buffer, the ratio being the acetic acid aqueous solution of 20% and the NaOH aqueous solution 2:1 by volume of 0.05mol/L by mass percent concentration mixes;
Described developer is the dimethylaminobenzaldehyde of 0.5% with the mass percent concentration of methyl alcohol preparation.
Described polystyrene board is preferably Nunc 96DeepWell polystyrene board.
Described pipettor is preferably the pipettor of 8 passages.
Embodiment 1
Based on a method for the low temperature CPC acetylase high flux screening of DAB colour developing, comprise the steps:
Step one, the high-throughput of CPC acetylase is expressed: the penbritin using the muton of pipettor picking P-1 ~ No. P-1000 single bacterium colony and protoenzyme bacterial strain BL21/pET32a-HG6 to be seeded to be equipped with 200 μ LLB(100 μ g/mL from mutated library respectively) in the aseptic Nunc 96DeepWell polystyrene board of substratum, 37 DEG C, 200rpm overnight incubation, about 14h, by the product of incubated overnight with the pipettor of 8 passages with 4% inoculum size be forwarded in the fresh LB liquid nutrient medium of 300 μ L, 37 DEG C, 200rpm cultivates 2.5h, then the IPTG that final concentration is 0.1mmol/L is added, 30 DEG C, 200rpm induces 8h, obtain the bacterium liquid after inducing, for subsequent use,
The alkaline lysis of step 2, thalline: the bacterium liquid after being induced by step one gained is placed in the centrifugal 1min of 8000g, after removing supernatant, thalline is resuspended in the phosphate buffered saline buffer of 100mmol/L, pH8.0 of 300 μ L, adding 1 μ L solubility is vortex oscillation after the Dnase aqueous solution of 1mg/mL, add the NaOH aqueous solution lysing cell of 50 μ L1mol/L, leave standstill 2min after vibration 30s mixing, then add 50 μ L1mol/LKH
2pO
4the aqueous solution neutralizes; Mixing is placed on the centrifugal 10min of 11000g, and collect supernatant liquor, the supernatant liquor collected is the thick liquid of CPC acetylase;
The making of step 3,7-ACA content standard curve:
With 0.1mol/L, the phosphoric acid buffer preparation final concentration of pH8.0 is the 7-ACA of 1.5mg/mL, pH to 8.0 is adjusted with the NaOH of 1mol/L, get 0 respectively, 1, 2, 4, 6, 8, 12, 16, the 7-ACA of 20 μ L is in centrifuge tube, use 0.1mol/L again, the phosphoric acid buffer of pH8.0 supplies 20 μ L, Xiang Guanzhong adds the CPC of 20 μ L13 DEG C preheating 3min, mixing, the stop buffer of 5 times of volumes is added after 13 DEG C of water-bath 10min, the centrifugal 3min of 12000rpm after mixing, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, room temperature gets the light absorption value that 200 μ L survey 405nm after leaving standstill 10min, with No. 0 for blank, with the content of 7-ACA for ordinate zou, OD405 is X-coordinate production standard curve, as shown in Figure 1, and obtain regression equation.
The high-throughout fast quantification of step 4,7-ACA: with the CPC substrate of the phosphoric acid buffer preparation 20g/L of 0.1mol/L, pH to 8.0 is regulated with the NaOH of 1mol/L, get the crude enzyme liquid that 20 μ L substrates and 20 μ L dilute suitable multiple to mix in 96 orifice plates, hatch 10min for 13 DEG C, obtain mixture B; In mixture B, add volume is that the stop buffer of mixture B volume 5 times carries out termination reaction, the centrifugal 3min of 12000rpm, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, the light absorption value that 200 μ L utilize 96 orifice plates Fast Measurement 405nm in microplate reader is got after room temperature reaction 10min, and then it is alive to calculate each mutant enzyme at low temperatures with EXCEL, from mutated library, successfully having screened two, catalytic activity is apparently higher than mutant P-166 and P-835 of protoenzyme at 13 DEG C, and having screened one does not have activated mutant P-254 yet simultaneously.Utilized the Enzyme activity assay method of improvement to the active replication of P-166, P-254 and P-835 6 times, it is better that result shows the result repeatability measured for 6 times, and the error often organizing test is all less than 0.5%.
Step 5, the product of BL21/pET32a, BL21/pET32a-HG6, P-166, P-254 and P-835 is carried out SDS-PAGE analysis in the lump, result as shown in Figure 2.Fig. 2 shows, and object band does not appear in recon BL21/pET32a and the BL21/pET32a-HG6 without induction; And all have solubility expression through the product of BL21/pET32a-HG6, P-166 and P-835 of induction, and define significantly large small subunit, the apparent molecular weight that QuantityOne software estimates large subunit is 60kDa, the apparent molecular weight of small subunit is 40kDa, also has part apparent molecular weight to be the precursor of 100kDa simultaneously; And the product of P-254 only has apparent molecular weight to be the precursor of 100kDa, this also further illustrates the accuracy of enzyme activity determination method.
Described stop buffer, the ratio being the acetic acid aqueous solution of 20% and the NaOH aqueous solution 2:1 by volume of 0.05mol/L by mass percent concentration mixes;
Described developer is the dimethylaminobenzaldehyde of 0.5% with the mass percent concentration of methyl alcohol preparation.
Embodiment 2
Based on a method for the low temperature CPC acetylase high flux screening of DAB colour developing, comprise the steps:
The high-throughput of step one, CPC acetylase is expressed: from the mutated library of spore rhzomorph C acetylase saturation mutation, the muton of picking individual colonies is seeded to that to be equipped with containing concentration be in the sterile polystyrene plate of 200 μ LLB substratum of 100 μ g/mL penbritins, at being placed in 37 DEG C, 200rpm overnight incubation; Be inoculated into by the product pipettor of incubated overnight in the LB liquid nutrient medium of 300 μ L, inoculum size is 4% of the LB liquid nutrient medium volume of 300 μ L; Then, at being placed in 37 DEG C, 200rpm cultivates 2.5h, finally adds the IPTG aqueous solution that final concentration is 0.1mmol/L, and at being placed in 30 DEG C, 200rpm cultivates 8h, obtains the bacterium liquid after inducing, for subsequent use;
The alkaline lysis of step 2, thalline: the bacterium liquid after being induced by step one gained is placed in the centrifugal 1min of 8000g, after removing supernatant, thalline is resuspended in the phosphate buffered saline buffer of 100mmol/L, pH8.0 of 300 μ L, adding 1 μ L solubility is vortex oscillation after the Dnase aqueous solution of 1mg/mL, add the NaOH aqueous solution lysing cell of 50 μ L1mol/L, leave standstill 2min after vibration 30s mixing, then add 50 μ L1mol/LKH
2pO
4the aqueous solution neutralizes; Mixing is placed on the centrifugal 10min of 11000g, and collect supernatant liquor, the supernatant liquor collected is the thick liquid of CPC acetylase;
Step 3, the making of 7-ACA content standard curve: with 0.1mol/L, the phosphoric acid buffer preparation final concentration of pH8.0 is the 7-ACA of 1.5mg/mL, with the NaOH aqueous solution adjust pH to 8.0 of 1mol/L, get 0 respectively, 1, 2, 4, 6, 8, 12, the 7-ACA of 16 and 20 μ L is in centrifuge tube, use 0.1mol/L again, the phosphoric acid buffer of pH8.0 supplies 20 μ L, the CPC acetylase of 20 μ L13 DEG C preheating 3min is added in centrifuge tube, mixing, obtain mixture A, after mixture A being placed in 13 DEG C of water-bath 10min, add the stop buffer that volume is mixture A volume 5 times, the centrifugal 3min of 12000rpm after mixing, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, room temperature gets the light absorption value that 200 μ L survey 405nm after leaving standstill 10min, with No. 0 for blank, with the content of 7-ACA for ordinate zou, the light absorption value of 405nm is X-coordinate production standard curve, and obtain regression equation,
The high-throughout fast quantification of step 4,7-ACA: prepare the cephalosporin of 20g/L as substrate with the phosphoric acid buffer of 0.1mol/L, pH to 8.0 is regulated with the NaOH aqueous solution of 1mol/L, get 20 μ L substrates and 20 μ L dilute after the thick liquid of CPC acetylase mix in 96 orifice plates, hatch 10min for 13 DEG C, obtain mixture B; In mixture B, add volume is that the stop buffer of mixture B volume 5 times carries out termination reaction, the centrifugal 3min of 12000rpm, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, get 200 μ L after room temperature reaction 10min and utilize 96 orifice plates in microplate reader, survey the light absorption value of 405nm;
The calculating of step 5, CPC acetylase activity: be defined as a Ge Meihuo unit (IU) with the per minute enzyme amount produced needed for 1 μm of ol7-ACA under condition determination; And then it is alive to calculate each mutant enzyme at low temperatures with EXCEL;
Described stop buffer, the ratio being the acetic acid aqueous solution of 20% and the NaOH aqueous solution 2:1 by volume of 0.05mol/L by mass percent concentration mixes;
Described developer is the dimethylaminobenzaldehyde of 0.5% with the mass percent concentration of methyl alcohol preparation.
Described pipettor is the pipettor of 16 passages.
Described polystyrene board is 96 hole transparent polystyrene microwell plates.
Claims (4)
1. the method based on the low temperature CPC acetylase high flux screening of DAB colour developing; it is characterized in that: the principle having maximum absorption band based on the mixture of paradimethy laminobenzaldehyde and 7-ACA at 405nm; 96 aseptic orifice plates are adopted to carry out low-temperature and low-concentration inductor abduction delivering to recombinant bacterium; SODIUM PHOSPHATE, MONOBASIC is utilized to neutralize after carrying out cellular lysate with dilute alkaline soln; and then the hydrolysis of substrate is carried out with 96 orifice plates; after dimethylaminobenzaldehyde colour developing, utilize microplate reader to carry out the fast quantification of product, and then realize the high flux screening of CPC acetylase mutant.
2. the method for the low temperature CPC acetylase high flux screening based on DAB colour developing according to claim 1, is characterized in that: comprise the following steps:
The high-throughput of step one, CPC acetylase is expressed: from the mutated library of spore rhzomorph C acetylase saturation mutation, the muton of picking individual colonies is seeded to that to be equipped with containing concentration be in the sterile polystyrene plate of 200 μ LLB substratum of 100 μ g/mL penbritins, at being placed in 37 DEG C, 200rpm overnight incubation; Be inoculated into by the product pipettor of incubated overnight in the LB liquid nutrient medium of 300 μ L, inoculum size is 4% of the LB liquid nutrient medium volume of 300 μ L; Then, at being placed in 37 DEG C, 200rpm cultivates 2.5h, finally adds the IPTG aqueous solution that final concentration is 0.1mmol/L, and at being placed in 30 DEG C, 200rpm cultivates 8h, obtains the bacterium liquid after inducing, for subsequent use;
The alkaline lysis of step 2, thalline: the bacterium liquid after being induced by step one gained is placed in the centrifugal 1min of 8000g, after removing supernatant, thalline is resuspended in the phosphate buffered saline buffer of 100mmol/L, pH8.0 of 300 μ L, adding 1 μ L solubility is vortex oscillation after the Dnase aqueous solution of 1mg/mL, add the NaOH aqueous solution lysing cell of 50 μ L1mol/L, leave standstill 2min after vibration 30s mixing, then add 50 μ L1mol/LKH
2pO
4the aqueous solution neutralizes; Mixing is placed on the centrifugal 10min of 11000g, and collect supernatant liquor, the supernatant liquor collected is the thick liquid of CPC acetylase;
Step 3, the making of 7-ACA content standard curve: with 0.1mol/L, the phosphoric acid buffer preparation final concentration of pH8.0 is the 7-ACA of 1.5mg/mL, with the NaOH adjust pH to 8.0 of 1mol/L, get 0 respectively, 1, 2, 4, 6, 8, 12, the 7-ACA of 16 and 20 μ L is in centrifuge tube, use 0.1mol/L again, the phosphoric acid buffer of pH8.0 supplies 20 μ L, the CPC acetylase of 20 μ L13 DEG C preheating 3min is added in centrifuge tube, mixing, obtain mixture A, after mixture A being placed in 13 DEG C of water-bath 10min, add the stop buffer that volume is mixture A volume 5 times, the centrifugal 3min of 12000rpm after mixing, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, room temperature gets the light absorption value that 200 μ L survey 405nm after leaving standstill 10min, with No. 0 for blank, with the content of 7-ACA for ordinate zou, the light absorption value of 405nm is X-coordinate production standard curve, and obtain regression equation,
The high-throughout fast quantification of step 4,7-ACA: prepare the cephalosporin of 20g/L as substrate with the phosphoric acid buffer of 0.1mol/L, pH to 8.0 is regulated with the NaOH aqueous solution of 1mol/L, get 20 μ L substrates and 20 μ L dilute after the thick liquid of CPC acetylase mix in 96 orifice plates, hatch 10min for 13 DEG C, obtain mixture B; In mixture B, add volume is that the stop buffer of mixture B volume 5 times carries out termination reaction, the centrifugal 3min of 12000rpm, get the supernatant of 200 μ L and the chromogenic reagent of 40 μ L, get 200 μ L after room temperature reaction 10min, utilize 96 orifice plates in microplate reader, survey the light absorption value of 405nm;
The calculating of step 5, CPC acetylase activity: be defined as a Ge Meihuo unit with the per minute enzyme amount produced needed for 1 μm of ol7-ACA under condition determination; And then it is alive to calculate each mutant enzyme at low temperatures;
Described stop buffer, the ratio being the acetic acid aqueous solution of 20% and the NaOH aqueous solution 2:1 by volume of 0.05mol/L by mass percent concentration mixes;
Described developer is the dimethylaminobenzaldehyde of 0.5% with the mass percent concentration of methyl alcohol preparation.
3., as claimed in claim 2 based on the method for the low temperature CPC acetylase high flux screening of DAB colour developing, it is characterized in that: described polystyrene board is Nunc 96DeepWell polystyrene board.
4., as claimed in claim 2 based on the method for the low temperature CPC acetylase high flux screening of DAB colour developing, it is characterized in that: described pipettor is the pipettor of 8 passages.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610002076.7A CN105543332A (en) | 2016-01-06 | 2016-01-06 | DAB color development-based high-throughput screening method for CPC histone deacetylases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610002076.7A CN105543332A (en) | 2016-01-06 | 2016-01-06 | DAB color development-based high-throughput screening method for CPC histone deacetylases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105543332A true CN105543332A (en) | 2016-05-04 |
Family
ID=55822890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610002076.7A Pending CN105543332A (en) | 2016-01-06 | 2016-01-06 | DAB color development-based high-throughput screening method for CPC histone deacetylases |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105543332A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108642130A (en) * | 2018-03-29 | 2018-10-12 | 浙江工业大学 | A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain |
| CN110904188A (en) * | 2019-12-23 | 2020-03-24 | 福州大学 | High-throughput rapid screening method of L-threonine transaldolase mutant |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0496993A1 (en) * | 1990-12-21 | 1992-08-05 | ANTIBIOTICOS, S.p.A. | Enzymatic process for preparing 7-aminocephalosporanic acid and derivatives |
| CN101240285A (en) * | 2008-03-19 | 2008-08-13 | 清华大学 | Cephalosporin C acrylase and its vector and application |
| CN102925423A (en) * | 2012-11-16 | 2013-02-13 | 清华大学 | Mutated cephalosporin C acylase |
-
2016
- 2016-01-06 CN CN201610002076.7A patent/CN105543332A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0496993A1 (en) * | 1990-12-21 | 1992-08-05 | ANTIBIOTICOS, S.p.A. | Enzymatic process for preparing 7-aminocephalosporanic acid and derivatives |
| CN101240285A (en) * | 2008-03-19 | 2008-08-13 | 清华大学 | Cephalosporin C acrylase and its vector and application |
| CN102925423A (en) * | 2012-11-16 | 2013-02-13 | 清华大学 | Mutated cephalosporin C acylase |
Non-Patent Citations (4)
| Title |
|---|
| OLAJIRE A. ADEGOKE等: "Novel spectrophotometric determinations of some", 《ARABIAN JOURNAL OF CHEMISTRY》 * |
| POLLEGIONI等: "Evolution of an acylase active on cephalosporin C", 《PROTEIN SCIENCE》 * |
| 章莫桦 等: "国产固定化GL-7-ACA酰化酶制备7-ACA的工艺优化", 《中南林学院学报》 * |
| 马晨露等: "头孢菌素C乙酰化酶的半理性改造及7-ACA的生物合成", 《中国生物工程杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108486209B (en) * | 2018-03-22 | 2022-03-18 | 浙江工业大学 | Method for rapidly screening (R) -2- (4-hydroxyphenoxy) propionic acid producing strains in high flux |
| CN108642130A (en) * | 2018-03-29 | 2018-10-12 | 浙江工业大学 | A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain |
| CN108642130B (en) * | 2018-03-29 | 2021-10-15 | 浙江工业大学 | High-throughput screening method for high-activity strain of tyrosine phenol lyase |
| CN110904188A (en) * | 2019-12-23 | 2020-03-24 | 福州大学 | High-throughput rapid screening method of L-threonine transaldolase mutant |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | A simplified diphenylamine colorimetric method for growth quantification | |
| Chen et al. | A sensitive impedance biosensor based on immunomagnetic separation and urease catalysis for rapid detection of Listeria monocytogenes using an immobilization-free interdigitated array microelectrode | |
| CN105543332A (en) | DAB color development-based high-throughput screening method for CPC histone deacetylases | |
| Du et al. | An efficient intergeneric conjugation of DNA from Escherichia coli to mycelia of the lincomycin-producer Streptomyces lincolnensis | |
| CN101676719A (en) | Method for measuring diastatic enzyme activity using a spectrophotometer | |
| CN102520168B (en) | Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof | |
| Burdock et al. | Effect of assay conditions on the measurement of dehydrogenase activity of Streptomyces venezuelae using triphenyl tetrazolium chloride | |
| CN108300758A (en) | A kind of Hemin hybridized nanometers flower and its preparation method and application | |
| CN111272717B (en) | One-step hydrothermal synthesis based on novel fluorescent carbon dots of ionic liquid and detection application of novel fluorescent carbon dots to sulfathiazole | |
| Cao et al. | Types and applications of nicking enzyme-combined isothermal amplification | |
| CN104232734A (en) | High-throughput screening method of high-yield microbial strains for adenosine | |
| WO2009135214A3 (en) | Rna polyphosphatase compositions, kits, and uses thereof | |
| Chen et al. | A galactosidase-activatable fluorescent probe for detection of bacteria based on BODIPY | |
| Shen et al. | Synthesis of N-substituted dimethylmaleimides and their antifungal activities against Sclerotinia sclerotiorum | |
| Fluxá et al. | Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test | |
| WO2020006940A1 (en) | Method and kit thereof for quantitatively detecting lactulose in liquid milk by using microplate reader enzymatic method | |
| AU1162801A (en) | Assay of micro-organisms in cell cultures | |
| Dai et al. | Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples | |
| Yang et al. | A rapid fluorescence assay for hSMUG1 activity based on modified molecular beacon | |
| CN112538518B (en) | Method for detecting histone acetyl transferase based on nano enzyme | |
| CN103409389B (en) | Culture medium and method for producing trehalase by fermentation of Lecanicillium lecanii | |
| Zhou et al. | The construction of CRISPR/Cas9-mediated FRET 16S rDNA sensor for detection of Mycobacterium tuberculosis | |
| Haq et al. | Influence of dissolved oxygen concentration on intracellular pH for regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor | |
| Liu et al. | Soft sensor modeling for key parameters of marine alkaline protease MP fermentation process | |
| Hu et al. | Sensitive and direct analysis of pseudomonas aeruginosa through self-primer-assisted chain extension and CRISPR-Cas12a-based color reaction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160504 |