CN112662732A - High-throughput rapid screening method of pantothenate synthetase mutants - Google Patents

High-throughput rapid screening method of pantothenate synthetase mutants Download PDF

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CN112662732A
CN112662732A CN202011400326.5A CN202011400326A CN112662732A CN 112662732 A CN112662732 A CN 112662732A CN 202011400326 A CN202011400326 A CN 202011400326A CN 112662732 A CN112662732 A CN 112662732A
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alanine
beta
pantothenate synthetase
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柳志强
钟娜
张博
郑垦
郑裕国
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Zhejiang University of Technology ZJUT
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Abstract

The invention relates to a high-throughput rapid screening method of a pantothenate synthetase mutant. The invention takes pantothenate synthetase of Corynebacterium glutamicum as target protein, utilizes saturation mutation PCR technology to carry out directed evolution on pantothenate synthetase gene, the mutant gene is recombined in expression vector pET-28a (+), and is introduced into Escherichia coli BL21(DE3) to construct mutant library. And (3) putting beta-alanine and a derivatization reagent into a hole of an enzyme label plate, immediately putting the plate into an enzyme label instrument to detect the change of a fluorescence value with time under the conditions of an excitation wavelength of 355nm and an emission wavelength of 445nm, and correlating the concentration of the beta-alanine with the maximum value in a curve to obtain a beta-alanine standard curve. The concentration of beta-alanine in the reaction solution is obtained by calculation, and the pantothenate synthetase mutants are screened according to the concentration of the beta-alanine, so that the mutants with effective characters can be rapidly screened. The condensation reaction liquid and the pantothenate synthetase mutant whole-cell reaction liquid are fully and uniformly mixed, after the reaction is finished, cell precipitation is removed through centrifugation, supernate is taken to react with a derivatization reagent, and then the reaction liquid is placed in an enzyme labeling instrument to detect the change of a fluorescence value along with time.

Description

High-throughput rapid screening method of pantothenate synthetase mutants
(I) technical field
The invention relates to a high-throughput rapid screening method of a pantothenate synthetase mutant.
(II) background of the invention
Pantothenic acid, also known as vitamin B5, is a key precursor for the synthesis of coenzyme a in living cells and is a vitamin essential for life. It can be synthesized by microorganisms and plants, but not by humans and other animals. Therefore, it has been widely used as an active ingredient in feed and food additives, as well as in the cosmetic and pharmaceutical industries. Particularly, the growth and development of poultry, livestock and fish animals, and the synthesis and decomposition of fats are indispensable for calcium pantothenate. The deficiency of pantothenic acid results in growth retardation, reproductive dysfunction and reduced adaptability in poultry and livestock. Pantothenic acid is formed by enzymatic condensation of pantoic acid and beta-alanine, has optical activity, and only D type has biological activity. Pure free pantothenic acid is a yellow viscous oil, acidic, readily soluble in water and ethanol, and insoluble in benzene and chloroform. Pantothenic acid cannot exist stably under acid, alkali and light and heat conditions. Pantothenic acid is generally involved in the production of energy in the body and can control the metabolism of fat, and is an essential nutrient for the brain and nerves. At present, calcium pantothenate is added into many health care products at home and abroad, such as 'adult vitamins' and 'growth happiness' of health preserving rooms. Calcium pantothenate is in the fastest growing consumer demand in this area.
At present, the methods for producing D-pantothenic acid are: the physical induced crystallization method utilizes the characteristic that the solubility of the racemic calcium pantothenate is greater than that of D-type or L-type to carry out induced crystallization, and the method has mature process, but can only produce calcium pantothenate and cannot be used for producing other pantothenic acid derivatives. ② a chemical resolution method, chiral resolving agents such as chloramine and the like are used for resolution, but the resolving agents are expensive and difficult to separate, and the problems of toxicity and environmental pollution also exist. The microbiological method comprises a metabolic engineering method, a fermentation method and a biological enzyme method, Zhang and the like utilize the metabolic engineering method to produce the D-pantothenic acid, the method is complex, the yield is low, and the highest yield is only 28.45 g/L; the yield of the fermentation method is high, but the problems that the components of the fermentation product are complex and are not beneficial to downstream extraction and the like exist, and substances generated in the fermentation process also have certain influence on the quality of food; the biological enzyme method is to utilize Pantothenate Synthetase (PS) to catalyze D-pantoic acid and beta-alanine to carry out condensation reaction to generate D-pantothenic acid, the reaction condition is mild, no by-product is generated, and the product separation is facilitated.
The biosynthesis of D-pantothenic acid in microorganisms is relatively complex, and its last synthesis is catalyzed by the pantothenate synthetase (panC): one molecule of pantoic acid and one molecule of beta-alanine consumes one molecule of ATP to form one molecule of pantothenic acid. Wherein the beta-alanine is derived from the catalytic action of aspartate decarboxylase (panD) and the pantoate is derived from alpha-ketoisovalerate, an intermediate of the valine synthetic pathway, alpha-ketoisovalerate being first reduced by hydroxymethyltransferase (panB) to ketopantoate, which is reduced by ketopantoate reductase (panE) or ketoreductoisomerase (ilvC) to pantoate. Virtually all of these pathways use Pantothenate Synthetase (PS) to condense D-pantoate and beta-alanine to produce D-pantothenic acid.
However, very little research has focused on the enzyme Pantothenate Synthetase (PS), a key enzyme for the biological production of D-pantothenic acid. Pantothenate Synthetase (Panthenate synthase) encoded by the PanC gene. The enzyme is best activated in the presence of magnesium or manganese ions by catalyzing the condensation of D-pantoate and beta-alanine to form pantothenic acid, vitamin B5, under ATP dependence. Up to now, 70 pantothenate synthases including 1 empty (apo) structure and 69 crystal complex structures were searched in the PDB library website. The full length of the PS protein sequence contains 287 amino acids, which consists of A, B two chains, and the two subunits of the dimer have the same structure.
The prior art screens the mutant PS mainly by taking the screening object, namely the mutant PS, as a catalyst for synthesizing D-pantothenic acid and screening the PS mutant by detecting the content of the D-pantothenic acid; the method for detecting D-pantothenic acid mainly adopts a High Performance Liquid Chromatography (HPLC), is time-consuming and labor-consuming in operation, low in efficiency and not suitable for high-throughput rapid screening of the PS mutant, so that a high-throughput rapid screening method of the pantothenate synthetase mutant needs to be developed.
Disclosure of the invention
The invention aims to provide a high-throughput rapid screening method of a pantothenate synthetase mutant.
The technical scheme adopted by the invention is as follows:
a method for high-throughput rapid screening of pantothenate synthetase mutants, said method comprising:
(1) preparing a reagent: selecting a single clone of a pantothenate synthetase mutant to be screened, and preparing a whole-cell reaction solution of the pantothenate synthetase mutant; preparing a derivatization reaction reagent of beta-alanine; preparing pantoate condensation reaction liquid; the pantothenate synthetase mutants to be screened can be randomly selected from a pantothenate synthetase mutation library; the pantoate condensation reaction liquid is prepared from pantoate, beta-alanine, ATP, magnesium chloride, potassium chloride and the like, wherein the pantoate is prepared by hydrolyzing D-pantolactone and sodium hydroxide; the derivatization reaction reagent is prepared from sodium borate buffer solution, 1, 2-diacetylbenzene, beta-mercaptoethanol, methanol, ethanol and the like;
(2) drawing a beta-alanine standard curve: taking beta-alanine and a derivatization reagent, immediately placing the beta-alanine and the derivatization reagent in an enzyme labeling instrument to detect the change condition of a fluorescence value along with time, drawing a scatter diagram by taking the concentration of the beta-alanine as an independent variable x and the fluorescence value as a dependent variable y, and obtaining a regression equation y ═ ax + b, wherein a is the obtained slope;
(3) determination of enzymatic Activity of pantothenate synthetase mutants: taking a pantoate condensation reaction solution and a pantothenate synthetase mutant whole-cell reaction solution, fully and uniformly mixing, centrifuging to remove cell precipitates after the reaction is finished, collecting reaction supernatant, and diluting by n times until the concentration of beta-alanine is 0.01-10 mM;
taking the diluted supernatant to react with a derivatization reagent, immediately placing the reaction product in an enzyme-labeling instrument to detect the change condition of a fluorescence value along with time, and determining the fluorescence value of the beta-alanine;
(4) screening: according to the fluorescence value of beta-alanine in the supernatant of the sample to be detected, the concentration C of beta-alanine in the reaction solution is calculated by contrasting the beta-alanine standard curve(beta-alanine)When the concentration of beta-alanine is higher than the value of (fluorescence value + b)/a × n, the mutants of the pantothenate synthetase with the corresponding higher activity are screened, and the mutants with the consumption of beta-alanine in the reaction solution being more than 10% of that of the original bacterium are high-activity mutants, and the further re-screening can be continued.
The invention rapidly screens the catalytic activity of the pantothenate synthetase mutants by judging the concentration of beta-alanine, namely screening the pantothenate synthetase mutants with high and low corresponding activities according to the concentration of the beta-alanine. The pantothenate synthetase mutants to be screened can be from a pantothenate synthetase mutant library.
Specifically, the whole-cell reaction solution of the pantothenate synthetase mutant is obtained by the following method: selecting a single clone of the pantothenate synthetase mutant to be screened, inoculating the single clone into an LB liquid culture medium containing 50 mu g/mL kanamycin, and culturing at 37 ℃ and 180rpm overnight; on the next day, the cell culture solution is sucked and then transferred into LB liquid culture medium containing 50 mug/mL kanamycin, IPTG is added to the final concentration of 0.1mM, and the culture is stopped after 10 hours of induction at 28 ℃; after OD600 measurement, centrifugation is carried out, the thalli are collected and added with HEPES buffer solution for heavy suspension, and the thalli are preserved at the temperature of minus 80 ℃.
The derivatization reaction reagent of the beta-alanine is prepared from the following components: 0.2M, pH9.5 sodium borate buffer, 210mg/mL1, methanol solution of 2-diacetylbenzene and 5.7mg/mL ethanol solution of beta-mercaptoethanol were mixed in a volume ratio of 1:1: 1.
The pantoate condensation reaction liquid is prepared from the following components: after stirring 25mM D-pantolactone and 25mM NaOH at room temperature for 3h, 25mM beta-alanine, 4.5mM ATP, 10mM MgCl were added215mM KCl, diluting to a constant volume of 200mL, adjusting the pH value to 8.0, and storing at 2-4 ℃ for use.
The method for drawing the beta-alanine standard curve in the step (2) is as follows: dissolving 0.089g of beta-alanine in 10mL of sterile water, and diluting to prepare 0, 1,2, 4, 6, 8 and 10mM beta-alanine working solution respectively; adding 2.5 mu L0.2M, pH9.5 sodium borate buffer solution +2.5 mu L of 10mg/mL1, 2-diacetylbenzene methanol solution +2.5 mu L of 5.7mg/mL beta-mercaptoethanol ethanol solution into a 96-well plate, immediately placing the plate in a microplate reader to detect the change of fluorescence values along with time, reading once every 4min and 31 times, and recording the concentration of the beta-alanine related to the maximum value of the fluorescence values; and (3) drawing a scatter diagram by taking the concentration of beta-alanine as an independent variable x and the fluorescence value as a dependent variable y to obtain a regression equation y as ax + b.
The principle of the High-Throughput Screening method (HTS for short) of the present invention is shown in FIG. 1. Pantothenate synthetase catalyzes the production of D-pantothenic acid from D-pantoic acid and β -alanine, as well as ATP. The high throughput screening method developed by the present invention is based on the selective fluorescent response of primary amines with o-diacetylbenzene and mercaptoethanol. Beta alanine contains primary amine, and can generate fluorescence with o-diacetylbenzene and mercaptoethanol, thereby relating the concentration of beta alanine (R represents any functional group, such as methyl, ethyl, etc., except carboxyl).
The invention has the following beneficial effects: (1) the method can rapidly screen 96 PS mutants at high flux for 2h, is greatly superior to the traditional HPLC detection (3 samples/hour), and can rapidly screen PS mutants with high D-pantothenic acid yield; (2) the method is not interfered by impurities in the reaction liquid, and has the advantages of high sensitivity and accuracy, good repeatability, simple operation and convenient popularization and application.
(IV) description of the drawings
FIG. 1 is a schematic diagram of the high throughput screening of this patent.
FIG. 2 is a schematic diagram of SDS-PAGE gel electrophoresis of recombinant bacteria expression protein.
FIG. 3 is a standard curve for detecting beta-alanine by the high throughput screening method of this patent.
FIG. 4 is a standard curve of ninhydrin detection for β -alanine coloration.
FIG. 5 is a standard curve for the detection of beta-alanine by OPA and NAC derivatizing agents.
FIG. 6 is a HPLC chromatogram of the production of D-pantothenic acid catalyzed by the PS mutant.
FIG. 7 is a standard curve for liquid phase detection of D-pantothenic acid.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: construction of recombinant plasmid for pantothenate synthetase
Corynebacterium glutamicum ATCC 13032 purchased from the website of American Type Culture Collection (ATCC) was inoculated into a 5mL LB tube, cultured at 37 ℃ with shaking for 12 hours, centrifuged at 12000rpm to obtain cells for genome extraction (FastDNA)
Figure BDA0002816740220000041
Kit).
1.1mL of CLS-TC was added to a 1.5mL centrifuge tube containing the cells to suspend and lyse the cells, transferred to a disruption tube, and disrupted in a cell disrupter.
2. The disruption tube was placed in a high speed centrifuge at 12000rpm for 5-10 minutes.
3. Absorbing 700-800 mu L of supernatant into a new 2mL centrifuge tube, adding Binding Matrix with the same volume (placing at 37 ℃ for preheating before use, mixing evenly), and washing, blowing and mixing evenly.
4. Gently invert upside down for 5 minutes.
5. Centrifuge at 12000rpm for 1min and discard the supernatant.
6. 500. mu.L of SEWS-M was added to resuspend the cells.
7. Centrifuge at 12000rpm for 1min and discard the supernatant.
8. After 1 minute of idling at 12000rpm, excess supernatant was aspirated.
9. Add 100. mu.L of DES and heat in a 55 ℃ water bath for 5 minutes.
10. Centrifuging at 12000rpm for 2 min, and obtaining the supernatant, namely the extracted genome.
The desired pantothenate synthetase gene panC of interest was isolated from the genome P of Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) by means of PCR, the nucleotide sequence of which is shown in SEQ ID No. 1.
The PCR amplification reaction system is as follows: 25. mu.L of 2 XPPhanta Max Buffer, 0.5. mu.L of Phanta Max Super-Fidelity DNA Polymerase, 2. mu.L of dNTP, two primers panC-F, panC-R (sequences shown in Table 1), 1. mu.L each, 0.5. mu.L of template (Corynebacterium glutamicum ATCC 13032 genome), 20. mu.L of ddH2O。
PCR program Table for construction of pET-28a-panC
Figure BDA0002816740220000051
Expression vector pET-28a (+) was linearized: 25 μ L of 2 XPhanta Max Buffer, 0.5 μ L of Phanta Max Super-Fidelity DNA Polymerase, 2 μ L of dNTP, two primers pET-28a-F, pET-28a-Each of R (sequence shown in Table 1) was 1. mu.L, 0.5. mu.L of template (pET-28a (+) plasmid, available from Okagaceae), and 20. mu.L of ddH2O。
Construction of PCR schedule for linearized pET-28a +
Figure BDA0002816740220000052
After the linearized plasmid and the PCR product were subjected to clean up (using clean up kit from Shanghai bioengineering Co., Ltd.), the PCR product and the linearized expression vector pET-28a (+) were ligated to construct a recombinant plasmid pET-28 a-panC. The connecting system is as follows: 2 μ L of Lexnase II (C112-02-AB), 4 μ L of 5 XBuffer, 0.5 μ L of panC fragment, 0.5 μ L of pET-28a (+) linearized plasmid, ddH2O make up to 20. mu.L. The reaction conditions are as follows: 30min at 37 ℃.
Table 1: primer sequences
Figure BDA0002816740220000061
Example 2: construction of recombinant Escherichia coli
The recombinant plasmid pET-28a-panC obtained in example 1 was transformed into the expression host E.coli BL21(DE3) by a chemical transformation method, which comprises the following specific steps:
(1) 10. mu.L of the homologous recombination product was introduced into 100. mu.L of E.coli BL21(DE3) competent cells;
(2) performing ice bath for 20-30 min;
(3) performing water bath heat shock at 42 ℃ for 90s, taking out, rapidly placing into ice, standing, and performing ice bath for 3-5 min;
(4) adding 650 mu L of non-resistant LB culture medium, mixing uniformly, and culturing at 37 ℃ and 200rpm for 1 h;
(5) centrifuging at 5000rpm for 1min to collect bacteria;
(6) the supernatant was removed and the remaining 100. mu.L of the solution was applied by pipetting and spreading onto kanamycin-resistant plates (kanamycin concentration 0.05 mg/L).
The transformed bacterial liquid is coated on an LB plate containing 0.1mmol/L kanamycin resistance, the bacterial liquid is cultured for 12h at 37 ℃, a single colony growing on the LB kanamycin resistance plate (kanamycin concentration is 0.05mg/L) is selected, colony PCR verification is carried out by using a T7 universal primer, then a positive transformant is selected and inoculated in a liquid LB culture medium containing kanamycin resistance (kanamycin concentration is 0.05mg/L), the plasmid is extracted after the bacterial liquid is cultured for 12h at 37 ℃, the sequencing is carried out, and the corresponding glycerol tube is stored with correct sequence.
Example 3: recombinant escherichia coli inducible expression pantothenate synthetase
The recombinant Escherichia coli E.coli BL21(DE3) of example 2 was streaked and isolated into single colonies, the single colonies were inoculated into LB seed medium containing 0.1mM kanamycin, cultured overnight at 37 ℃ and 200rpm, transferred to 100mL of LB medium containing 0.1mmol/L kanamycin resistance in an inoculum size of 2% (v/v), and cultured at 37 ℃ and 200rpm to OD6000.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mM for induction, collecting thalli for ultrasonic disruption after induction for 12h at 28 ℃, and verifying through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel electrophoresis that the recombinant bacteria correctly express the target protein. The expression is shown in FIG. 2.
Example 4: high throughput screening method beta-alanine standard curve drawing
Dissolving 0.089g of beta-alanine in 10mL of sterile water, and diluting to prepare 0, 1,2, 4, 6, 8 and 10mM beta-alanine working solution respectively; adding 2.5 mu L of beta-alanine working solution, 150 mu L of 0.2M sodium borate buffer solution, 2.5 mu L of 10mg/mL1, 2-diacetylbenzene (dissolved in methanol), 2.5 mu L of 5.7mg/mL beta-mercaptoethanol (dissolved in ethanol) into a 96-well plate, immediately placing the plate in a microplate reader to detect the change of a fluorescence value along with time, reading the plate once every 4min, reading the plate 31 times, and recording the concentration of the beta-alanine related to the maximum value of the fluorescence value; and (3) drawing a scatter diagram by taking the concentration of beta-alanine as an independent variable x and the fluorescence value as a dependent variable y to obtain a regression equation y as ax + b. The standard curve is shown in fig. 3.
0.15g of recombinant strain BL21/pET-28a (+) -panC was weighed, and 3mL of HEPES buffer (100mM HEPES, 20mM magnesium chloride, 1mM EDTA, pH8.0) was added to the mixture to conduct resuspension. To 1.1mL of a reaction substrate system (25mM D-pantoate, 25 mM. beta. -alanine, 4.5mM ATP, 10mM magnesium chloride, 15mM potassium chloride) was added 20. mu.L of the whole cell suspension, and the reaction was stirred at 37 ℃ for 8min and stopped by adding hydrochloric acid. The reaction solution was diluted 5 times after centrifugation at 12000rpm for 10min, 2.5. mu.L of the diluted solution was added to the high-throughput screening system of this example, and the system was quickly placed on a microplate reader for detection, and the fluorescence value was 3290, which corresponds to a β -alanine concentration of 17.14mM, thus 7.86mM of β -alanine was consumed during the reaction. And judging whether the enzyme activity is improved compared with the original bacteria or not according to the content of the beta-alanine consumed in each hole during subsequent 96-hole plate screening, wherein mutants with the consumption of the beta-alanine in the reaction liquid being more than 10% of that of the original bacteria are high-activity mutants, and the next step of re-screening can be continuously carried out.
Example 5: indantrione method for detecting beta-alanine
Preparation of acidic ninhydrin solution: dissolving 250mg ninhydrin in 6mL acetic acid and 4mL concentrated hydrochloric acid, standing at room temperature for 30min, and ultrasonic dissolving if dissolution is incomplete.
And (3) color development reaction: mixing 500 mu L of acid ninhydrin, 500 mu L of acetic acid and 500 mu L of beta-alanine standard substance (0.1-1mol/L), boiling in boiling water bath for 10min, taking out and rapidly cooling, then taking 200 mu L into an enzyme label plate, and measuring the yield of beta-alanine at the ultraviolet wavelength of 560 nm. The linear relationship of beta-alanine with absorbance at 560nm is shown in FIG. 4. R of the process20.9987, the linear relationship is not as good as the method provided by the present invention. In addition, the method has more influence factors, the color development condition is influenced by heating time, heating temperature, heating condition and the like, and a compound formed by the method and beta-alanine is easy to oxidize in the air to cause color fading, so that the method is not suitable for high-throughput screening.
Example 6: detection of beta-alanine by OPA and NAC derivatization method
Referring to a high-throughput detection method of glufosinate-ammonium, derivatization reagent OPA and NAC is used for derivatization of beta-alanine. Wherein the molar concentrations of OPA and NAC in the derivatization reagent are respectively 27.6mM and 13.8mM, the mixture is dissolved by 10mL of absolute ethyl alcohol, then the volume is determined to be 50mL by 140mM of boric acid buffer solution (pH9.8), the mixture is uniformly mixed and stored in ice bath for three days, when sample detection is carried out, beta-alanine is diluted to a certain concentration range, the beta-alanine reacts with the derivatization reagent according to the volume ratio of 2:1, the mixture is placed at 30 ℃ for 40min, and detection is carried out under the conditions that lambda ex is 340nm and lambda ex is 455 nm. The standard curve is shown in fig. 5. OPA and NAC reagents are short in storage time and poor in linear relation when detecting beta-alanine, so that the OPA and NAC reagents are not suitable for high-throughput screening.
Example 7: HPLC determination standard curve drawing of D-pantothenic acid content
The detection method comprises the following steps:
chromatographic conditions are as follows: c18Column (250X 4.6mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, Calif., USA), detection wavelength: 200nm, column temperature: 30 ℃;
sample treatment: diluting the sample with ultrapure water to maintain the content of D-pantothenic acid between 0.05g/L and 0.40 g/L;
mobile phase: acetonitrile/water/phosphoric acid: (50/949/1);
data acquisition time: and (4) 18 min.
The pantothenate standard curve is shown in FIG. 7.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (6)

1. A method for high-throughput rapid screening of pantothenate synthetase mutants, said method comprising:
(1) preparing a reagent: selecting a single clone of a pantothenate synthetase mutant to be screened, and preparing a whole-cell reaction solution of the pantothenate synthetase mutant; preparing a derivatization reaction reagent of beta-alanine; preparing pantoate condensation reaction liquid;
(2) drawing a beta-alanine standard curve: taking beta-alanine and a derivatization reagent, immediately placing the beta-alanine and the derivatization reagent in an enzyme labeling instrument to detect the change condition of a fluorescence value along with time, drawing a scatter diagram by taking the concentration of the beta-alanine as an independent variable x and the fluorescence value as a dependent variable y, and obtaining a regression equation y ═ ax + b, wherein a is the obtained slope;
(3) determination of enzymatic Activity of pantothenate synthetase mutants: taking a pantoate condensation reaction solution and a pantothenate synthetase mutant whole-cell reaction solution, fully and uniformly mixing, centrifuging to remove cell precipitates after the reaction is finished, collecting reaction supernatant, and diluting by n times until the concentration of beta-alanine is 0.01-10 mM;
taking the diluted supernatant to react with a derivatization reagent, immediately placing the reaction product in an enzyme-labeling instrument to detect the change condition of a fluorescence value along with time, and determining the fluorescence value of the beta-alanine;
(4) screening: according to the fluorescence value of beta-alanine in the supernatant of the sample to be detected, comparing with a beta-alanine standard curve, calculating to obtain the concentration of beta-alanine in the reaction solutionDegree C(beta-alanine)When the pantothenate synthetase mutants having high and low activities were selected based on the concentration of β -alanine (fluorescence value + b)/a × n, the mutants having a β -alanine consumption in the reaction mixture of 10% or more higher than that of the original strain were high-activity mutants.
2. The method as claimed in claim 1, wherein the pantothenate synthetase mutants to be screened are from the pantothenate synthetase mutant library.
3. The method according to claim 1, wherein the whole-cell reaction solution of the pantothenate synthetase mutant is obtained by: selecting a single clone of the pantothenate synthetase mutant to be screened, inoculating the single clone into an LB liquid culture medium containing 50 mu g/mL kanamycin, and culturing at 37 ℃ and 180rpm overnight; on the next day, the cell culture solution is sucked and then transferred into LB liquid culture medium containing 50 mug/mL kanamycin, IPTG is added to the final concentration of 0.1mM, and the culture is stopped after 10 hours of induction at 28 ℃; after OD600 measurement, centrifugation is carried out, the thalli are collected and added with HEPES buffer solution for heavy suspension, and the thalli are preserved at the temperature of minus 80 ℃.
4. The method of claim 1, wherein said derivatized reactant of β -alanine is formulated as follows: 0.2M, pH9.5 sodium borate buffer, 210mg/mL1, methanol solution of 2-diacetylbenzene and 5.7mg/mL ethanol solution of beta-mercaptoethanol were mixed in a volume ratio of 1:1: 1.
5. The method according to claim 1, wherein the pantoate condensation reaction solution is prepared with the following composition: after stirring 25mM D-pantolactone and 25mM NaOH at room temperature for 3h, 25mM beta-alanine, 4.5mM ATP, 10mM MgCl were added215mM KCl, diluting to a constant volume of 200mL, adjusting the pH value to 8.0, and storing at 2-4 ℃ for use.
6. The method according to claim 1, wherein the standard curve of β -alanine in step (2) is prepared as follows: dissolving 0.089g of beta-alanine in 10mL of sterile water, and diluting to prepare 0, 1,2, 4, 6, 8 and 10mM beta-alanine working solution respectively; adding 2.5 muL of 0.2M, pH9.5 sodium borate buffer solution, 2.5 muL of 10mg/mL1, 2-diacetylbenzene methanol solution and 2.5 muL of 5.7mg/mL beta-mercaptoethanol ethanol solution into a 96-well plate, immediately placing the plate in a microplate reader to detect the change of fluorescence values along with time, reading once every 4min and 31 times, and recording the maximum value of the fluorescence values to correlate the concentration of the beta-alanine; and (3) drawing a scatter diagram by taking the concentration of beta-alanine as an independent variable x and the fluorescence value as a dependent variable y to obtain a regression equation y as ax + b.
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