CN117660595A - High-throughput screening method of alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio - Google Patents
High-throughput screening method of alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio Download PDFInfo
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- -1 alpha-amino acid ester Chemical class 0.000 title claims abstract description 68
- 108700016155 Acyl transferases Proteins 0.000 title claims abstract description 67
- 102000057234 Acyl transferases Human genes 0.000 title claims abstract description 67
- 235000008206 alpha-amino acids Nutrition 0.000 title claims abstract description 66
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 41
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000013537 high throughput screening Methods 0.000 title claims abstract description 31
- 230000000694 effects Effects 0.000 claims abstract description 24
- 239000000758 substrate Substances 0.000 claims abstract description 22
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 20
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 20
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 10
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims description 43
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 23
- 150000001413 amino acids Chemical class 0.000 claims description 22
- DWKPPFQULDPWHX-VKHMYHEASA-N l-alanyl ester Chemical compound COC(=O)[C@H](C)N DWKPPFQULDPWHX-VKHMYHEASA-N 0.000 claims description 21
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 20
- 235000001014 amino acid Nutrition 0.000 claims description 20
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 18
- 150000002148 esters Chemical class 0.000 claims description 12
- 230000008859 change Effects 0.000 claims description 10
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- 238000005070 sampling Methods 0.000 claims description 9
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- 239000006228 supernatant Substances 0.000 claims description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000002028 Biomass Substances 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 4
- 235000004279 alanine Nutrition 0.000 claims description 4
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- 238000004113 cell culture Methods 0.000 claims description 4
- 230000001276 controlling effect Effects 0.000 claims description 4
- JCXLZWMDXJFOOI-WCCKRBBISA-N ethyl (2s)-2-aminopropanoate;hydrochloride Chemical compound Cl.CCOC(=O)[C@H](C)N JCXLZWMDXJFOOI-WCCKRBBISA-N 0.000 claims description 4
- COQRGFWWJBEXRC-UHFFFAOYSA-N hydron;methyl 2-aminoacetate;chloride Chemical compound Cl.COC(=O)CN COQRGFWWJBEXRC-UHFFFAOYSA-N 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 2
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- CURNJKLCYZZBNJ-UHFFFAOYSA-M sodium;4-nitrophenolate Chemical compound [Na+].[O-]C1=CC=C([N+]([O-])=O)C=C1 CURNJKLCYZZBNJ-UHFFFAOYSA-M 0.000 claims description 2
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- 230000035484 reaction time Effects 0.000 description 6
- 239000003054 catalyst Substances 0.000 description 5
- 241000282326 Felis catus Species 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N Alanine Chemical compound CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
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- 230000002194 synthesizing effect Effects 0.000 description 2
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- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
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- 241001052560 Thallis Species 0.000 description 1
- 239000002696 acid base indicator Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
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- 238000000354 decomposition reaction Methods 0.000 description 1
- NJDNXYGOVLYJHP-UHFFFAOYSA-L disodium;2-(3-oxido-6-oxoxanthen-9-yl)benzoate Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=CC(=O)C=C2OC2=CC([O-])=CC=C21 NJDNXYGOVLYJHP-UHFFFAOYSA-L 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
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- 235000016236 parenteral nutrition Nutrition 0.000 description 1
- 229960003857 proglumide Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/91045—Acyltransferases (2.3)
- G01N2333/91051—Acyltransferases other than aminoacyltransferases (general) (2.3.1)
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Abstract
The invention discloses a high-throughput screening method of alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio, and belongs to the technical field of enzyme engineering. The invention utilizes the method to carry out high-efficiency screening on the mutant library through the detection of the synthesis activity and the hydrolysis activity of the alpha-amino acid ester acyltransferase, and obtains the mutant with high synthesis hydrolysis ratio. The high-throughput screening method provided by the invention has the characteristics of simplicity in operation, mature technology, intuitiveness, high efficiency, safety, environmental friendliness and the like, solves the problem of hydrolyzing substrates and products by wild alpha-amino acid ester acyltransferase, promotes the industrialized process of the prognostics dipeptide, and has wide application prospect and remarkable economic benefit.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for efficiently screening an alpha-amino acid ester acyltransferase mutant library.
Background
The proglumide (Ala-Gln) has the advantages of strong thermal stability, high solubility, high decomposition speed and small water burden on human bodies, is an important parenteral nutrition supplement, is widely applied to the fields of foods, cosmetics, medical treatment, health care products and the like, and has wide application prospect and remarkable social and economic benefits. Compared with other chemical/enzymatic synthesis methods, the biological synthesis method for directly synthesizing the proglutide by catalyzing alanine methyl ester hydrochloride (AlaOMe) and glutamine (Gln) with alpha-amino acid ester acyltransferase (protein_ID in NCBI is QKI 29181.1) has the advantages of low raw material cost, simple reaction steps, less byproducts, less environmental pollution and the like, and is suitable for being used as a new path for green biological production of the proglutide.
The acyl-enzyme complex formed by the alpha-amino acid ester acyltransferase and the acyl donor (AlaOMe) can be combined with the nucleophile glutamine to form the proglutide, and can be combined with the competitive nucleophile (H 2 O) is hydrolyzed into alanine, and the generated proglutide is hydrolyzed, so that the problem that the accumulation amount of the product is reduced with the time, and the industrialization process of the proglutide is seriously hindered. Therefore, in order to increase the accumulation of the glutamine dipeptide, a screening method is needed to obtain an alpha-amino acid ester acyltransferase mutant with a high synthetic hydrolysis ratio, and on the basis of the known basic structural information of the alpha-amino acid ester acyltransferase, a high throughput screening method for rapidly and accurately detecting the synthetic activity and the hydrolytic activity of the mutant is needed to be established at present.
Disclosure of Invention
In view of the above, the present invention aims to provide a high throughput screening method for alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio, based on the high throughput screening method for detecting synthetic activity of alanine methyl ester hydrochloride and glutamine as substrates (according to the principle that the alanin dipeptide has strong absorption at near ultraviolet wavelength and the absorption value of the alanine methyl ester hydrochloride and the glutamine is smaller in the wavelength range, the change of the absorption value can reflect the amount of the alanin dipeptide produced) and detecting hydrolytic activity of alanine methyl ester hydrochloride as substrates (according to the principle that the hydrolysis of the alanine methyl ester hydrochloride releases hydrogen ions, so that the sodium fluorescein is protonated to be colorless, and the change of the color and the absorption value of an indicator can reflect the degree of the alanine methyl ester hydrochloride, etc., the invention has the characteristics of simple operation, mature technology, intuitiveness, high efficiency, safety, environmental protection, etc.
The invention aims at realizing the following steps:
the invention provides a high-throughput screening method of alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio, which comprises the steps of detecting the synthetic activity of the alpha-amino acid ester acyltransferase and detecting the hydrolysis activity, and screening to obtain the alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio.
Based on the technical scheme, further, the detection of the synthesis activity of the alpha-amino acid ester acyltransferase mainly comprises the following steps:
(1) Dissolving substrate amino acid ester hydrochloride and amino acid in a reaction solution, and regulating the pH value to 7.0-10.0;
(2) Adding an alpha-amino acid ester acyltransferase or a cell expressing an alpha-amino acid ester acyltransferase to the reaction solution in step (1);
(3) Controlling the reaction temperature at 15-35 ℃, carrying out oscillation reaction, sampling in a certain time range, transferring the supernatant into a micro-pore plate, detecting the change of light absorption value under the ultraviolet wavelength of 200-380nm, and screening to obtain the alpha-amino acid ester acyltransferase or the cell expressing the alpha-amino acid ester acyltransferase with high synthesis activity.
Based on the above technical scheme, further, the amino acid ester hydrochloride in the step (1) comprises alanine methyl ester hydrochloride, alanine ethyl ester hydrochloride and glycine methyl ester hydrochloride; the amino acids include glutamine, glycine and alanine; the concentration of the amino acid ester hydrochloride and the concentration of the amino acid are 50-100mM, preferably 90mM; the reaction solution is any one of water, buffer solution and cell culture solution, and the pH value is controlled at 8.5.
Based on the technical scheme, further, alpha in the step (2)The addition of the amino acid ester acyltransferase or the cell expressing the alpha-amino acid ester acyltransferase is carried out in a manner comprising volume percent, mass percent and biomass OD 600 Values.
Based on the technical scheme, the reaction temperature in the step (3) is controlled at 25 ℃, the sampling time is 6, 12 and 18min, and the ultraviolet wavelength is 237nm.
Based on the technical scheme, further, the detection of the hydrolysis activity of the alpha-amino acid ester acyltransferase mainly comprises the following steps:
1) Dissolving a substrate amino acid ester hydrochloride in a reaction solution, adding a pH indicator and adjusting the pH value to 7.0-10.0;
2) Adding an alpha-amino acid ester acyltransferase or a cell expressing an alpha-amino acid ester acyltransferase to the reaction solution of step 1);
3) Controlling the reaction temperature at 15-35 ℃, carrying out oscillation reaction, sampling in a certain time range, transferring the supernatant into a micro-pore plate, detecting the change of light absorption value at the visible light wavelength of 400-700nm, and screening to obtain the alpha-amino acid ester acyltransferase with low hydrolytic activity or the cell expressing the alpha-amino acid ester acyltransferase.
Based on the above technical scheme, further, the amino acid ester hydrochloride in the step 1) comprises alanine methyl ester hydrochloride, alanine ethyl ester hydrochloride and glycine methyl ester hydrochloride; the concentration of the amino acid ester hydrochloride is 20-200mM, preferably 100mM; the reaction solution is any one of water, buffer solution and cell culture solution; the pH indicator comprises disodium fluoroflavum, sodium paranitrophenolate and methyl red, the concentration is 4-8mg/L, and the pH value is controlled at 8.0.
Based on the technical scheme, further, the addition modes of the alpha-amino acid ester acyltransferase or the cell expressing the alpha-amino acid ester acyltransferase in the step 2) comprise volume percent, mass percent and biomass OD 600 Values.
Based on the technical scheme, the reaction temperature in the step 3) is controlled at 25 ℃, the sampling time is 10, 20, 18 and 30min, and the ultraviolet wavelength is 492nm.
In another aspect, the invention provides a high throughput screening method as described above to obtain an alpha-amino acid ester acyltransferase of high synthetic hydrolysis ratio or a cell expressing an alpha-amino acid ester acyltransferase of high synthetic hydrolysis ratio.
Based on the technical scheme, further, the amino acid sequence of the alpha-amino acid ester acyltransferase is shown as SEQ ID NO: 2.
The invention also provides the application of the alpha-amino acid ester acyltransferase with high synthesis hydrolysis ratio or the cell expressing the alpha-amino acid ester acyltransferase with high synthesis hydrolysis ratio in dipeptide synthesis.
Compared with the prior art, the invention has the following beneficial effects:
1. the high-throughput screening method can reflect the synthesis/hydrolysis activity of the mutant according to the change of the light absorption value, and has the characteristics of simplicity in operation, mature technology, intuitiveness, high efficiency, safety, environment friendliness and the like.
2. The high throughput screening method of the present invention was used at about 10 4 The beneficial mutant of alpha-amino acid ester acyltransferase with improved synthetic hydrolysis ratio can be screened from the library capacity of the grade mutant, and the high efficiency and the effectiveness of the method are proved.
3. The high-throughput screening method of the invention is used to obtain the alpha-amino acid ester acyltransferase mutant of the high-yield proglutamic dipeptide, and the synthetic hydrolysis ratio is 1.56 times of that of the wild type; catalytic efficiency k cat /k m The value is improved by 1.93 times, the yield of the glutamine dipeptide is improved by 30 percent, the production economic benefit is increased, and a solid and scientific basis is laid for promoting the industrialization process of the glutamine dipeptide.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
FIG. 1 shows changes in absorbance of alanine methyl ester hydrochloride (A), glutamine (B) and proglutidine (C) at different wavelengths.
FIG. 2 shows the conversion versus time for different reaction conditions, wherein A: substrate concentration 50-50mM, B: substrate concentration 70-70mM, C: the substrate concentration is 90-90mM.
FIG. 3 shows the change in absorbance of fluorescence Huang Erna at different wavelengths at different concentrations.
FIG. 4 shows the absorbance of fluorescence Huang Erna at pH 8.5 at various concentrations of alanine methyl ester hydrochloride.
FIG. 5 shows the effect of different pH values on the absorbance of fluorescence Huang Erna, wherein A: ph=7.5, b: ph=8.0.
FIG. 6 is an optimized graph of optimal fluorescent disodium salt concentration.
Fig. 7 is a high throughput screening method accuracy test chart, wherein a: synthesizing the proglutide, and B: alanine methyl ester hydrochloride hydrolysis.
FIG. 8 is a graph of color of disodium fluorescent yellow over time.
FIG. 9 shows the catalytic performance of wild-type and whole cell catalyst G9-2, wherein A: comparison of the synthetic activity of alpha-amino acid ester acyltransferase, B: hydrolytic activity of alpha-amino acid ester acyltransferase, C: comparison of wild-type to whole cell catalyst G9-2 malodipeptide synthesis concentration.
Detailed Description
The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.
The mutant library of the alpha-amino acid ester acyltransferase is obtained by randomly mutating the pocket region gene sequence (namely the front 911bp base) of the alpha-amino acid ester acyltransferase by using an error-prone PCR technology, firstly, changing the mutation rate of amino acids by adjusting the concentration of a template and the circulation times in a PCR reaction system, then amplifying the rest gene fragments (6283 bp) by using high-fidelity polymerase, and completing the construction of the gene mutation library by in vitro fusion.
EXAMPLE 1 establishment and optimization of a high throughput screening method for the Synthesis of the alpha-amino acid ester acyltransferase mutant library of proglutide
(1) Optimization of working wavelength: scanning light absorption values of alanine methyl ester hydrochloride, glutamine and proglutin with different concentrations at 200-240 nm;
(2) Optimization of reaction conditions: the substrate alanine methyl ester hydrochloride and glutamine concentrations were selected at 50/50, 70/70 and 90/90mM; the reaction time is selected to be 8, 16, 24 and 32 minutes; the amount of the whole cell catalyst BPA (host BL21, protein_ID QKI 29181.1) used in the reaction OD 600 Values were chosen to be 0.5, 0.75, 1.0.
The result of the selection of the working wavelength is shown in fig. 1, and the result of the full wavelength scanning shows that the alanyl dipeptide has higher absorbance in the wavelength range of 200-240nm in the near ultraviolet region compared with other components in the reaction system. In particular, the light absorption coefficient of the substrate is the lowest when the wavelength is controlled within the range of 230-240nm, wherein the interference of the substrate on the light absorption value of the glutamine dipeptide is the smallest when the wavelength is 237nm, and the light absorption value of the glutamine dipeptide has good linear relation with the concentration. Therefore, 237nm is selected as the working wavelength of the high-throughput screening method for detecting the synthetic activity.
As shown in FIG. 2, the reaction conditions were optimized such that the synthesis rate was almost zero at 8min and the synthesis was almost completely stopped at 16min at a substrate concentration of 50/50mM, and the cell usage (OD 600 Value) and reaction time can not be adjusted to sample at the initial stage of the reaction, and too high a reaction speed can lead to lower detection sensitivity in the subsequent high-throughput screening of synthetic activity. At a substrate concentration of 70/70mM, although there was formation of glutamine, the concentration was low, the difference in absorbance was small, and at 8min the substrate conversion rate exceeded 40%, and the accuracy of the experiment was not ensured. In addition, OD 600 When the values of 0.75 and 1.0 are selected, the reaction speed is too high regardless of the change of the substrate concentration and the reaction time, and sampling in the initial stage of the reaction is not facilitated, so that a smaller OD should be selected 600 Values.
In summary, the high throughput screening conditions for synthetic activity are: the detection wavelength is 237nm; the substrate alanine methyl ester hydrochloride and glutamine concentrations were 90/90mM; OD (optical density) 600 A value of 0.5; the reaction time was adjusted to 6, 12, 18min.
EXAMPLE 2 establishment and optimization of a high throughput screening method for alanine methyl ester hydrochloride hydrolysis of alpha-amino acid ester acyltransferase mutant library
(1) Optimization of working wavelength: scanning the absorbance of fluorescence Huang Erna with different concentrations at 400-700 nm;
(2) Optimization of reaction conditions: the concentration of the substrate alanine methyl ester hydrochloride is selected to be 50mM, 80 mM and 100mM; the reaction time is selected to be 10, 30 and 60 minutes; OD (optical density) 600 Value selection 1.25; the pH is selected to be 7.5, 8.0 and 8.5; the concentration of the acid-base indicator is selected to be 4-10mg/L.
The result of the operation wavelength selection is shown in fig. 3, and it is found by full wavelength scanning that the disodium fluorescent yellow has a high absorbance in the wavelength range of 482-502nm in the visible light region, and particularly the absorbance is maximum at a wavelength of 492nm. As shown in FIGS. 4 and 5, when the concentration of the substrate is 50mM, the peak area of the hydrolysis product alanine is smaller under the condition of liquid chromatography, which is unfavorable for improving the experimental sensitivity, and when the concentration of the substrate is 80 mM and 100mM, the reaction degree is small within 30min, the change of the absorbance value is small, and the detection method of long-time reaction is unfavorable for improving the flux in the subsequent high-flux screening, so that the reduction of the pH value is considered. At OD 600 At a value of 1.25 and a pH indicator concentration of 10mg/L, the absorbance of the solution after 10min of the reaction at pH=7.5 is reduced to be very small, and the solution is basically unchanged after 30min, so that the color of the pH indicator cannot accurately indicate the progress of the reaction. In comparison, the optimal reaction pH should be chosen to be 8.0. The optimum concentration of the indicator is shown in FIG. 6, and the reaction degree can be well indicated when the working concentration of the disodium fluorescent yellow is controlled within the range of 4-8mg/L, so that the optimum concentration of the pH indicator selected by the invention is 8mg/L.
In summary, the conditions for high throughput screening of hydrolytic activity are: the detection wavelength is 492nm; the substrate AlaOMe concentration was 100mM; OD (optical density) 600 A value of 1.25; the pH value is 8.0; the reaction time is selected to be 10, 20 and 30 minutes.
Example 3: high throughput screening method accuracy test
The invention uses three strains of BPA (BL 21 as host), OPA (Origami 2 as host) and SPA (Shuffle T7 as host) with known synthesis/hydrolysis activities stored in a laboratory to evaluate the established high-throughput screening method, wherein the three strains contain pET29a vector and express the same alpha-amino acid ester acyltransferase (protein_ID is QKI 29181.1), and high performance liquid chromatography is used to prove that the synthesis and hydrolysis activities of the three strains are OPA > SPA > BPA respectively.
(1) BPA, OPA, SPA inoculating to 4mL LB medium containing Cana resistance, and culturing at 37deg.C and 180rpm for 12-16 hr;
(2) Inoculating 1% -5% of the strain into 100mL LB culture medium containing kana resistance, and culturing at 37deg.C and 180rpm to OD 600 Adding 60-100 μm of IPTG at final concentration between 0.6-0.8, inducing at 16deg.C and 180rpm for 12-16 hr, and measuring OD 600 ；
(3) Centrifugally collecting thalli at 4 ℃ and 8000rpm, and discarding the supernatant;
(4) Preparing alanine methyl ester hydrochloride and glutamine solution with concentration of 90-90mM, pH of 8-10, adding final OD 600 The bacterial solution was 0.5, reacted at 25℃and 180rpm for 6, 12 and 18 minutes, sampled and centrifuged at 8000rpm for 1 to 5 minutes, and the supernatant was transferred to a microplate and absorbance was measured at 237nm.
(5) Preparing alanine methyl ester hydrochloride solution with concentration of 100mM, adding fluorescence Huang Erna with final concentration of 8mg/L, adjusting pH to 8.0-9.0, adding final OD 600 The bacterial solution was 1.25, reacted at 25℃and 180rpm for 10, 20 and 30 minutes, sampled and centrifuged at 8000rpm for 1 to 5 minutes, and the supernatant was transferred to a microplate and absorbance was measured at 492nm.
The reaction results are shown in figures 7 and 8, and the high-throughput screening method established by the invention can rapidly and accurately reflect the difference of synthesis/hydrolysis activities among different strains (which is the same as the synthesis and hydrolysis activities OPA > SPA > BPA of the prior art), and is consistent with the trend of HPLC rechecking results (broken lines), thereby proving the high efficiency and accuracy of the method. Particularly in the hydrolysis reaction, the pH indicator has weak fluorescence signal and small light absorption value when the hydrolysis activity is strong, and the degree of hydrolysis can be rapidly distinguished by comparing the fluorescence brightness among different strains intuitively.
Example 4: synthesis and hydrolysis of mutant G9-2
The same method as in example 3 was used to perform the synthesis reaction of glutamine and the hydrolysis reaction of alanine methyl ester hydrochloride in this example, except that the whole cell catalyst used in this example was G9-2 (an enzyme mutant library constructed by error-prone PCR, transferred into BL21 host, and obtained by high throughput screening, the amino acid sequence of the encoded alpha-amino acid ester acyltransferase was shown as SEQ ID NO: 2) and BPA (protein_ID was QKI 29181.1).
The G9-2 mutant has a high synthetic hydrolysis ratio compared with BPA expressing wild-type alpha-amino acid ester acyltransferase, and can synthesize proglutide with high efficiency, and the results are shown in Table 1, specifically, G9-2 has a synthetic hydrolysis ratio 1.56 times that of wild-type; catalytic efficiency k cat /k m The value is improved by 1.93 times, and the yield of the proglutin is improved by 30 percent.
TABLE 1 comparison of catalytic Properties of wild type and Whole cell catalyst G9-2
| Mutant | k cat (min -1 ) | k m (mmol/L) | k cat /k m (L/(mmol·min)) |
| WT | 236±24.87 | 0.7±0.075 | 337.14 |
| G9-2 | 3737.14±51.36 | 3.78±0.0055 | 988.62 |
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The high-throughput screening method of the alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio is characterized by comprising the steps of detecting the synthetic activity of the alpha-amino acid ester acyltransferase and detecting the hydrolysis activity, and screening to obtain the alpha-amino acid ester acyltransferase with high synthetic hydrolysis ratio.
2. The high throughput screening method of claim 1, wherein said detecting of the synthetic activity of α -amino acid ester acyltransferase comprises the steps of:
(1) Dissolving substrate amino acid ester hydrochloride and amino acid in a reaction solution, and regulating the pH value to 7.0-10.0;
(2) Adding an alpha-amino acid ester acyltransferase or a cell expressing an alpha-amino acid ester acyltransferase to the reaction solution in step (1);
(3) Controlling the reaction temperature at 15-35 ℃, carrying out oscillation reaction, sampling in a certain time range, transferring the supernatant into a micro-pore plate, detecting the change of light absorption value under the ultraviolet wavelength of 200-380nm, and screening to obtain the alpha-amino acid ester acyltransferase or the cell expressing the alpha-amino acid ester acyltransferase with high synthesis activity.
3. The high throughput screening method of claim 2, wherein said amino acid ester hydrochloride in step (1) comprises alanine methyl ester hydrochloride, alanine ethyl ester hydrochloride, and glycine methyl ester hydrochloride; the amino acids include glutamine, glycine and alanine; the concentration of the amino acid ester hydrochloride and the concentration of the amino acid are 50-100mM, preferably 90mM; the reaction solution is any one of water, buffer solution and cell culture solution, and the pH value is controlled at 8.5.
4. The high throughput screening method of claim 2, wherein the addition of the α -amino acid ester acyltransferase or the α -amino acid ester acyltransferase-expressing cells in step (2) comprises volume percent, mass percent and biomass OD 600 A value; in the step (3), the reaction temperature is controlled at 25 ℃, the sampling time is 6, 12 and 18min, and the ultraviolet wavelength is 237nm.
5. The high throughput screening method of claim 1, wherein said detecting of the hydrolytic activity of α -amino acid ester acyltransferase comprises the steps of:
1) Dissolving a substrate amino acid ester hydrochloride in a reaction solution, adding a pH indicator and adjusting the pH value to 7.0-10.0;
2) Adding an alpha-amino acid ester acyltransferase or a cell expressing an alpha-amino acid ester acyltransferase to the reaction solution of step 1);
3) Controlling the reaction temperature at 15-35 ℃, carrying out oscillation reaction, sampling in a certain time range, transferring the supernatant into a micro-pore plate, detecting the change of light absorption value at the visible light wavelength of 400-700nm, and screening to obtain the alpha-amino acid ester acyltransferase with low hydrolytic activity or the cell expressing the alpha-amino acid ester acyltransferase.
6. The high throughput screening method of claim 5, wherein said amino acid ester hydrochloride of step 1) comprises alanine methyl ester hydrochloride, alanine ethyl ester hydrochloride, and glycine methyl ester hydrochloride; the concentration of the amino acid ester hydrochloride is 20-200mM, preferably 100mM; the reaction solution is any one of water, buffer solution and cell culture solution; the pH indicator comprises disodium fluoroflavum, sodium paranitrophenolate and methyl red, the concentration is 4-8mg/L, and the pH value is controlled at 8.0.
7. The high throughput screening method according to claim 5, wherein the addition of the alpha-amino acid ester acyltransferase or the cells expressing the alpha-amino acid ester acyltransferase in step 2) comprises volume percent, mass percent and biomass OD 600 A value; in the step 3), the reaction temperature is controlled at 25 ℃, the sampling time is 10, 20, 18 and 30min, and the ultraviolet wavelength is 492nm.
8. The high throughput screening method of any one of claims 1-7 resulting in a high synthetic hydrolysis ratio of an alpha-amino acid ester acyltransferase or a cell expressing a high synthetic hydrolysis ratio of an alpha-amino acid ester acyltransferase.
9. The high synthetic hydrolysis ratio α -amino acid ester acyltransferase or cell expressing the same according to claim 8, wherein the α -amino acid ester acyltransferase has an amino acid sequence as set forth in SEQ ID NO: 2.
10. Use of the high synthetic hydrolysis ratio α -amino acid ester acyltransferase or the cell expressing the high synthetic hydrolysis ratio α -amino acid ester acyltransferase of claim 8 or 9 in dipeptide synthesis.
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