CN112394050A - Detection method for high-throughput screening of ketone compounds and application of detection method in enzyme screening - Google Patents
Detection method for high-throughput screening of ketone compounds and application of detection method in enzyme screening Download PDFInfo
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- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
Abstract
The invention discloses a detection method for high-throughput screening of ketone compounds and application of the detection method in enzyme screening. The invention provides an application of 5-methoxy-2-aminobenzamide oxime, which is at least one of the following (a1) - (a 4): (a1) detecting the content of the ketone compound; (a2) detecting the activity of the enzyme; (a3) screening high-activity enzyme; (a4) directed evolution of the enzyme; the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds. The invention provides a high-throughput method for detecting ketone compounds, has wide substrate range, simple and convenient operation, sensitive reaction, accurate detection, low requirement on equipment and strong universality, and can quickly detect the content of ketone substances in an aqueous phase system and a non-aqueous phase system; and can be coupled with an enzyme which catalyzes the generation of ketone or consumes ketone to establish an enzyme activity high-throughput screening method. The invention has important significance for screening ketone compounds, efficiently excavating new enzymes or directionally evolving known enzymes.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection method for high-throughput screening of ketone compounds and application of the detection method in enzyme screening.
Background
The ketone compound is an important intermediate for medicine and fine chemical industry, and is widely applied to the fields of food, essence, spice additives and the like. Chemical synthesis of ketones requires expensive oxidizing agents or environmentally unfriendly halogenated hydrocarbons. The enzymatic synthesis of ketones such as alcohol dehydrogenase, alcohol oxidase, laccase, deaminase and the like has the advantages of mild reaction conditions, greenness and the like, and is an ideal substitute for chemical synthesis. However, natural enzymes have problems of low activity, selectivity, stability, and the like. Efficient mining of new enzymes or directed evolution of known enzymes requires the establishment of high throughput screening methods. However, high throughput screening methods for ketones have only been reported.
In 2016, Jiangnan university invented a high throughput screening method (CN 106047985A) for detecting alcohol dehydrogenase by using aliphatic or aryl substituted ketone and 2, 4-dinitrophenylhydrazine to perform color reaction under alkaline condition. However, 2, 4-dinitrophenylhydrazine can only carry out color reaction with ketone under alkaline condition, the pH range is narrow, and only aliphatic or aryl substituted ketone substrates can be measured.
Disclosure of Invention
The invention aims to provide a high-throughput screening method for ketone compounds, and simultaneously provides a high-throughput identification method for identifying the activity, substrate spectrum or directed evolution microorganisms and enzymes of enzymes or microorganisms for catalytically generating or consuming ketone compounds.
The invention firstly provides application of 5-methoxy-2-aminobenzamide oxime, which is at least one of the following (a1) - (a 4):
(a1) detecting the content of the ketone compound;
(a2) detecting the activity of the enzyme;
(a3) screening high-activity enzyme;
(a4) and (5) directed evolution screening of the enzyme.
The invention also protects the application of the 5-methoxy-2-aminobenzamide oxime in preparing products; the product has at least one of the following (a1) - (a 4):
(a1) detecting the content of the ketone compound;
(a2) detecting the activity of the enzyme;
(a3) screening high-activity enzyme;
(a4) and (5) directed evolution screening of the enzyme.
The invention also provides a kit comprising 5-methoxy-2-aminobenzamide oxime; the kit is used for at least one of the following (a1) - (a 4):
(a1) detecting the content of the ketone compound;
(a2) detecting the activity of the enzyme;
(a3) screening high-activity enzyme;
(a4) and (5) directed evolution screening of the enzyme.
The invention also provides a detection method of the content of the ketone compound, which comprises the following steps: adding 5-methoxy-2-aminobenzamide oxime and a sample to be detected into a buffer solution, mixing, placing at room temperature for reaction, and comparing the fluorescence values of the reaction system of the system to be detected and the reaction system of the control system in the excitation light of 360-400nm and the emission light of 500-540 nm; the fluorescence value of the system to be detected is higher than the corresponding fluorescence value of the comparison system, and the content of the ketone compound in the sample to be detected is higher; the contrast system is a reaction system without adding a sample to be detected.
In the method, when quantitative detection is carried out, the content of the ketone compound in the sample to be detected is determined as follows: after the reaction is finished, substituting the measured fluorescence value into a standard curve equation, thereby calculating the content of the ketone compound in the sample to be detected; the standard curve equation is obtained as follows: and detecting by using a series of standard solutions of the to-be-detected ketone compounds with known concentrations, and detecting the fluorescence value corresponding to the standard solutions with various concentrations, thereby obtaining a standard curve equation between the concentration of the ketone compounds and the fluorescence value.
In the method, the concentration of the 5-methoxy-2-aminobenzamide oxime in the reaction system is 1mM-50 mM. The 5-methoxy-2-aminobenzamide oxime can be added into a reaction system in a mother liquor form, and DMSO or other organic solvents can be adopted to dissolve the 5-methoxy-2-aminobenzamide oxime when the mother liquor is prepared.
In the method, the pH of the buffer solution is 3-14. The pH of the buffer is preferably 4.5 to 7.0. The buffer may specifically be a PBS buffer, more specifically a50 mM PBS buffer.
In the method, the reaction time is 0.5h-24 h.
In the method, the sample to be detected can be an aqueous phase solution or a non-aqueous phase solution.
The invention also protects the application of any one of the methods in enzyme activity detection or high-activity enzyme screening; the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds.
When the method is applied to enzyme activity detection or high-activity enzyme screening, if the enzyme to be detected is an enzyme capable of catalyzing and generating a ketone compound, the enzyme to be detected is adopted to carry out a catalytic reaction for synthesizing the ketone compound, the ketone compound content in a reaction system is detected by using the method, and if the generated ketone compound content is higher, the enzyme activity to be detected is higher.
When the method is applied to enzyme activity detection or high-activity enzyme screening, if the enzyme to be detected is an enzyme consuming a ketone compound, the enzyme to be detected is adopted to carry out a catalytic reaction of ketone compound consumption, the ketone compound content in the reaction system is detected by using the method, and if the generated ketone compound content is lower, the enzyme activity to be detected is higher.
The invention also protects the application of any one of the methods in enzyme directed evolution screening; the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds.
When the method is applied to enzyme directed evolution screening, after the natural enzyme is artificially modified, the modified enzyme activity is detected by adopting the method, and the modified enzyme with high enzyme activity is screened out.
Any of the ketone compounds described above include aliphatic ketones, aromatic ketones, hydroxyketones, and heterocyclic ketones. Preferably one of the following: raspberry ketone, p-fluorophenylalanine, phenyl butanone, 1-hydroxy-2-butanone, 2-hydroxybutanone, 1-hydroxyacetone, acetone, 1-hydroxy-3-butanone, 2-methylcyclohexanone, cyclopentanone, cyclohexanone, cycloheptanone, 3-methyl-cyclohexanone, pentanone, hexanone.
Any of the above enzymes is an enzyme that catalyzes the production of a ketone compound or the consumption of a ketone compound. More specifically, it may be alcohol dehydrogenase, transaminase or alcohol oxidase.
The invention has the positive effects that: (1) a high-throughput method is provided for the detection of ketone compounds, and the substrate range is wide; (2) the method is simple and convenient to operate, sensitive in reaction, accurate in detection, low in equipment requirement and strong in universality, and can be used for quickly detecting the content of ketone substances in an aqueous phase system and a non-aqueous phase system; (3) and can be coupled with an enzyme which catalyzes the generation of ketone or consumes ketone to establish an enzyme activity high-throughput screening method. The invention has important significance for screening ketone compounds, efficiently excavating new enzymes or directionally evolving known enzymes.
Drawings
FIG. 1 shows the reaction equation of PMA with 1-hydroxy-2-butanone.
FIG. 2 is a time plot of the reaction of PMA with 1-hydroxy-2-butanone.
FIG. 3 shows pH optimization of the reaction of PMA with 1-hydroxy-2-butanone.
FIG. 4 is a graph showing the reaction time of PMA with various concentrations of 1-hydroxy-2-butanone at pH 7.0.
FIG. 5 is a linear fit of fluorescence and GC for four ketone compounds.
FIG. 6 shows fluorescence intensity values of different ketone compounds reacted with PMA.
FIG. 7 is a schematic diagram of a high-throughput detection method for ketone compounds applied to alcohol dehydrogenase activity screening.
FIG. 8 shows fluorescence values of the alcohol dehydrogenase library.
FIG. 9 is a graph of the fit of the alcohol dehydrogenase screening fluorescence value to the GC peak area.
FIG. 10 is a schematic diagram of directed evolution of a ketone high-throughput screening method applied to alcohol dehydrogenase.
FIG. 11 shows fluorescence values of TbSADH W110 site saturation mutation library screening.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The compound information used in the examples of the present invention is as follows:
5-methoxy-2-aminobenzamide oxime (para-methoxy-2-amino benzamidoxime, PMA): CAS No.1359828-78-6, which can be synthesized according to the literature (J.Am.chem.Soc.2014136238149-8152 and DOI: 10.1021/ja5023909), and has the following structural formula:
1-hydroxy-2-butanone: CAS No. 5077-67-8.
Benzyl acetone: CAS No. 2550-26-7.
2-pentanone: CAS No. 107-87-9.
2-methylcyclohexanone: CAS No. 583-60-8.
Racemic 1, 2-butanediol: CAS No. 26171-83-5.
Racemic rhododendrol: CAS No. 501-96-2.
Raspberry ketone: CAS No. 5471-51-2.
The invention provides a ketone high-throughput detection method, which is a screening method for generating detectable fluorescent substances based on the reaction of 5-methoxy-2-aminobenzamide oxime (PMA) and ketone compounds. The following detailed description is provided by way of illustration and explanation and is not intended to limit the scope of the invention.
Example 1 fluorescence detection of reaction of PMA with 1-hydroxy-2-butanone
This example illustrates a specific method for detecting ketone compounds using PMA, using 1-hydroxy-2-butanone as an example.
The reaction scheme of PMA with 1-hydroxy-2-butanone is shown in FIG. 1.
1. Dissolving 1-hydroxy-2-butanone in acetonitrile to obtain 1-hydroxy-2-butanone mother liquor. The concentration of 1-hydroxy-2-butanone in the mother liquor was 100 mM.
2. Dissolving PMA in DMSO to obtain PMA mother liquor. The concentration of PMA in the mother liquor was 1M.
3. mu.L of the PMA mother liquor prepared in step 2 was added to 89. mu.L of PBS (pH 4.5, 50mM) buffer, and 10. mu.L of the 1-hydroxy-2-butanone mother liquor prepared in step 1 was added to obtain a reaction system of the experimental group. Meanwhile, a control group reaction system without adding 1-hydroxy-2-butanone mother liquor is arranged.
Placing the experimental group reaction system and the control group reaction system in a microplate reader (the temperature is set to be 25 ℃) to start reaction, setting the exciting light (Ex) and the emitting light (Em) of the microplate reader to be 380nm and 520nm respectively, and detecting the change of the fluorescence value in the reaction process.
The results are shown in FIG. 2. The result shows that PMA reacts with 1-hydroxy-2-butanone for 2h to reach the equilibrium, and the fluorescence value is improved by about 20 times compared with PMA. The reaction of PMA and 1-hydroxy-2-butanone can form a detectable fluorescent substance in a short time, and can be used for establishing a ketone substrate detection method.
Example 2 pH optimization of the reaction of PMA with 1-hydroxy-2-butanone
1. Dissolving 1-hydroxy-2-butanone in acetonitrile to obtain 1-hydroxy-2-butanone mother liquor. The concentration of 1-hydroxy-2-butanone in the mother liquor was 100 mM.
2. Dissolving PMA in DMSO to obtain PMA mother liquor. The concentration of PMA in the mother liquor was 1M.
3. mu.L of the PMA mother liquor prepared in step 2 was added to 89. mu.L of 50mM PBS buffers having different pH values (3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 and 8), and 10. mu.L of the 1-hydroxy-2-butanone mother liquor prepared in step 1 was added to obtain a reaction system of the experimental group. Meanwhile, a control group reaction system without adding 1-hydroxy-2-butanone mother liquor is arranged.
Placing the experimental group reaction system and the control group reaction system in a microplate reader (the temperature is set to be 25 ℃) to start reaction, setting the exciting light (Ex) and the emitting light (Em) of the microplate reader to be 380nm and 520nm respectively, and detecting the change of the fluorescence value in the reaction process.
The results are shown in FIG. 3. The results show that PMA is capable of forming a fluorescent species with 1-hydroxy-2-butanone in the pH range of 3.5 to 8 of the assay, and the fluorescence value is 5-20 times that of the negative control. It is noted that PMA can react with ketones to form fluorescent substances at pH 3.5 to pH 8.0. Wherein the effect is best when the pH is 4.5-7.0.
Example 3 reaction time curves of PMA with different concentrations of 1-hydroxy-2-butanone at pH 7.0
1. Dissolving 1-hydroxy-2-butanone in acetonitrile to obtain 1-hydroxy-2-butanone mother liquor. The concentration of 1-hydroxy-2-butanone in the mother liquor was 100 mM.
2. Dissolving PMA in DMSO to obtain PMA mother liquor. The concentration of PMA in the mother liquor was 1M.
3. mu.L of the PMA mother liquor prepared in step 2 was taken and added to 89. mu.L of 50mM PBS buffer solution having pH 7, and then different volumes of the 1-hydroxy-2-butanone mother liquor prepared in step 1 were added (so that the concentration of 1-hydroxy-2-butanone in the reaction system was 0mM, 2mM, 4mM, 6mM, 8mM, and 10mM) to obtain experimental group reaction systems.
The reaction system of the experimental group is placed in a microplate reader (the temperature is set to be 25 ℃) to start reaction, the exciting light (Ex) and the emitting light (Em) of the microplate reader are respectively set to be 380nm and 520nm, and the change of the fluorescence value is detected in the reaction process.
The results are shown in FIG. 4. The results show that PMA can form fluorescent substances with 1-hydroxy-2-butanone in 0.5h-24 h. It shows that PMA can react with ketone substance within 0.5-24 h to produce fluorescent substance, which can be used to build high-throughput screening method.
Example 4 comparison of the consistency of the fluorescence detection method with the gas phase detection method
The ketone compound to be tested: 1-hydroxy-2-butanone (for hydroxyketone), benzylacetone (for aromatic ketone), 2-pentanone (for aliphatic ketone) and 2-methylcyclohexanone (for heterocyclic ketone).
The fluorescence detection method and the gas phase detection method are respectively adopted to detect the ketone compound to be detected, and the consistency of the fluorescence detection values of the four ketones and the GC detection peak area is evaluated.
The fluorescence detection method comprises the following steps:
1. and dissolving the ketone compound to be detected in acetonitrile to obtain a ketone compound mother solution. The concentration of the ketone compound in the mother liquor was 100 mM.
2. Dissolving PMA in DMSO to obtain PMA mother liquor. The concentration of PMA in the mother liquor was 1M.
3. mu.L of PMA mother liquor prepared in step 2 was added to 89. mu.L of 50mM PBS buffer solution having pH 4.5, and 10. mu.L of ketone compound mother liquor prepared in step 1 was added to obtain a reaction system of the experimental group. Meanwhile, a control group reaction system without adding the ketone compound mother liquor to be detected is arranged.
Placing the experimental group reaction system and the control group reaction system in a microplate reader (the temperature is set to be 25 ℃) to start reaction, setting the exciting light (Ex) and the emitting light (Em) of the microplate reader to be 380nm and 520nm respectively, and detecting the change of the fluorescence value in the reaction process.
The gas phase detection method comprises the following steps:
the shimadzu GC2030 was equipped with a hydrogen ion flame detector, high purity nitrogen as carrier gas, injection port temperature: 250 ℃, chromatographic column flow rate of 1mL/min, detection port temperature: 280 ℃.
The chromatographic columns, temperature program and retention time of the four ketones to be tested are shown in Table 1.
TABLE 1 GC conditions and retention times for four ketone assays
a the retention times of the four ketones were determined with standards.
1-hydroxy-2-butanone standard product: shanghai Bide pharmaceutical science and technology Limited, Cat number: BD 59017.
Benzyl acetone standard: shanghai Michelin Biochemical technology, Inc., cat #: p815872.
Pentanone standard: shanghai Michelin Biochemical technology, Inc., cat #: m812745.
And (3) methyl cyclohexanone standard product: shanghai Michelin Biochemical technology, Inc., cat #: p815619.
A comparison of the consistency of the two methods is shown in figure 5. The result shows that the correlation coefficient R is obtained after the fluorescence detection values of four ketone and PMA reactions are fitted with the GC detection peak area2>0.996. The method has high detection accuracy.
Example 5 substrate spectra of high throughput screening assay for Ketone Compounds
Substrate to be tested: PMA and the compound shown in FIG. 6B.
1. And dissolving the substrate to be detected in acetonitrile to obtain a substrate solution. The concentration of the substrate in the substrate solution was 100 mM.
2. Dissolving PMA in DMSO to obtain PMA mother liquor. The concentration of PMA in the mother liquor was 1M.
3. mu.L of the PMA stock solution prepared in step 2 was added to 89. mu.L of 50mM PBS buffer pH 4.5, and 10. mu.L of the substrate solution prepared in step 1 was added to obtain a reaction system of the experimental group. Meanwhile, a control reaction system without adding a substrate solution was set.
Placing the experimental group reaction system and the control group reaction system in a microplate reader (the temperature is set to be 25 ℃) to start reaction, setting the exciting light (Ex) and the emitting light (Em) of the microplate reader to be 380nm and 520nm respectively, and detecting the change of the fluorescence value in the reaction process.
The results are shown in FIG. 6. The result shows that the high-throughput screening detection method for ketone compounds established by the invention has a wide substrate spectrum and can act on hydroxyketone, aromatic ketone, aliphatic ketone, heterocyclic ketone and the like.
Example 6 application of high throughput detection method for ketone compounds to alcohol dehydrogenase activity screening
A schematic diagram of the ketone high-throughput detection method applied to the alcohol dehydrogenase activity screening method is shown in FIG. 7.
1. BL21(DE3) strains containing alcohol dehydrogenases, which were constructed in the laboratory, all of which were synthesized from the whole gene of Biotech, Inc., of Beijing engine, and inserted into the corresponding vectors through restriction enzyme sites, are described in Table 1. Streaks were made on LB plates containing kanamycin to a final concentration of 50. mu.g/mL, and incubated overnight at 37 ℃.
TABLE 1
2. After completion of step 1, the single colonies were picked and transferred to a 96-well deep-well plate containing 300. mu.L of LB medium, kanamycin was added to a final concentration of 50. mu.g/mL (LB medium and kanamycin were premixed and dispensed), and after sealing (the front and back sides of the sealing film could not be reversed during the screening process to prevent the splashed bacterial solution from contaminating other mutants) the culture was carried out at 37 ℃ for 12 hours with shaking at 800 rpm.
3. After completion of step 3, 800. mu. LTB medium containing 0.2mM IPTG and 50. mu.g/mL kanamycin was added to the 96-well deep-well plate, and cultured with shaking at 30 ℃ and 800rpm for 12 hours for protein expression.
4. After completion of step 3, the expressed cells were collected by centrifugation at 4000rpm for 10min at 4 ℃ and washed with 400. mu.L of 50mM potassium phosphate buffer pH 7.4. 400 μ L of 50mM pH 7.0 potassium phosphate buffer was added to the washed cells to resuspend the cells, and 6U/mL of LDNase I (Beijing ancient China Changsheng Biotechnology Co., Ltd., product No.: E1127h) and 1mg/mL of lysozyme (purchased from Beijing Solebao technology Co., Ltd., product No.: L8120) were added thereto and subjected to shaking culture at 30 ℃ and 800rpm for 1 hour to disrupt the cells, and then the supernatant was centrifuged at 4000rpm for 30min and 4 ℃ and collected for enzyme activity measurement.
The enzyme activity determination method comprises the following steps:
the racemic solution 1, 2-butanediol acetonitrile solution (50. mu.L dissolved in acetonitrile, rac 1,the concentration of 2-butanediol in the enzyme activity reaction system is 10mM), NADP+Solution 50. mu.L (dissolved in ultrapure water, NADP)+The concentration of the LreNox enzyme powder in the enzyme activity reaction system is 1mM), and an LreNox (NADH oxidase from Lactobacillus reuteri) enzyme powder solution (dissolved in PBS with pH 7.0 and 50mM, and the concentration of the LreNox enzyme powder in the enzyme activity reaction system is 2mg/mL) are supplemented to the total volume of 500 mu L with pH 7.0 and 50mM, so as to obtain the reaction system.
5. After completing step 4, 1. mu.l of PMA mother liquor (PMA is dissolved in DMSO to obtain PMA mother liquor, the concentration of PMA in the mother liquor is 1M) is added into 99. mu.L of the reaction system in step 4, the mixture is placed at room temperature for 4h, excitation light (Ex) and emission light (Em) of an microplate reader are respectively set to be 380nm and 520nm, and fluorescence values are detected.
The results are shown in FIG. 8. The result shows that the method can detect the alcohol dehydrogenase which catalyzes the catalytic oxidation of 1, 2-butanediol to generate 1-hydroxy-2-butanone. Further, the fluorescence detection value was linearly fitted to the peak area of 1-hydroxy-2-butanone by GC (the GC detection method is described in example 4), and the results are shown in fig. 9. The result shows that the fluorescence detection values of 18 alcohol dehydrogenase screens are fitted with a correlation coefficient R with GC detection peak area20.8562, the method can be applied to enzyme activity screening.
Example 7 application of the high-throughput detection method for ketones to directed evolution of alcohol dehydrogenase
A schematic diagram of the directed evolution of the ketone high-throughput detection method applied to alcohol dehydrogenase is shown in FIG. 10.
1. Construction of an alcohol dehydrogenase TbSADH (alcohol dehydrogenase from Thermoanaerobacter brockii) W110 single-site saturated mutant library: TbSADH W110 site two rounds of PCR construction were performed using PrimesSTAR DNA polymerase to design primers according to Tang method (Biotechniques,2012,52(3): 149-.
One round of PCR reaction system: 50 μ L of reaction system, PrimesSTAR DNA polymerase (Takara Shuzo Co., Ltd., product No. R045Q)25 μ L, forward and reverse primers each 1 μ L (see Table 2, wherein the forward four primers contain NDT, VMA, ATG and TTG, and respectively encode 12, 6, 1 and 1 amino acids, and the four primers are sequentially addedMixing the two in a volume ratio of 12:6:1:1 to form a W110 site saturation mutation library, constructing an upstream primer for the first round of PCR amplification), and constructing a pRSFDuet-TbSADH plasmid template with a final concentration of 0.2. mu.M (the template is constructed according to the Journal of the American Chemical Society,2012,135(5):1665-1668.), and a final concentration of 50-200 ng and 22. mu.L of sterilized ddH2And O. And (3) PCR reaction conditions: 98 ℃ for 2min, (98 ℃ for 10s,55 ℃ for 15s,72 ℃ for 15s) x 25 cycles,72 ℃ for 5 min.
Second round PCR reaction System: 50 mu.L of reaction system, 25 mu.L of PrimesSTAR DNApolymerase, 2 mu.L of one-round product, 1 mu.L of pRSFDuet-TbSADH plasmid template, 50-200 ng of final concentration, and 22 mu.L of sterilized ddH2And O. Two rounds of PCR reaction conditions: 98 ℃ for 2min, (98 ℃ for 10s,55 ℃ for 15s,72 ℃ for 2min) x 30 cycles,72 ℃ for 5 min.
The PCR products of both rounds were analyzed by electrophoresis using a nucleic acid gel, and after the size of the band was correct (303 bp for the first round PCR product and 4888bp for the second round PCR product), the products of the two rounds of PCR were digested with Dpn I enzyme, and 0.5. mu.L of Dpn I enzyme was added to 10. mu.L of the second round PCR product, and digested at 37 ℃ for 3 hours or more. mu.L of the digested two-round PCR product was added to 50. mu.L of E.coli BL21(DE3) for electrotransformation. More than 300 single clones were collected after electrotransformation, and plasmids were submitted to sequencing for peak overlap assessment (saturated mutant library plasmid assessment as described in Advanced synthesis & catalysis, 2009,351(18): 3287-.
TABLE 2 primers required for the construction of the W110G library
2. The TbSADH W110G single-site saturation mutant pool strain was treated according to the procedures 1-4 of example 5 to obtain a supernatant for enzyme activity assay.
The enzyme activity determination method comprises the following steps:
and taking 300 mu L of supernatant, and adding 50 mu L of rhododendron racemic alcohol solution (dissolved in acetonitrile, the concentration of the rhododendron racemic alcohol in an enzyme activity reaction system is 10mM), 50 mu L of NADP + solution (dissolved in ultrapure water, the concentration of the NADP + in the enzyme activity reaction system is 1mM) and LreNox enzyme powder solution (dissolved in PBS with the pH value of 7.0 and 50mM, and the concentration of the LreNox enzyme powder in the enzyme activity reaction system is 2mg/mL) to obtain a reaction system. Samples were taken at 37 ℃ and 24 hours at 1000rpm for testing. BL21(DE3)/pET28a was used as a blank control and a wild type TbSADH negative control (wild type strain was constructed in accordance with the Journal of the American Chemical Society,2012,135(5): 1665-1668.).
2. After completing step 2, 1. mu.l of PMA mother liquor (PMA is dissolved in DMSO to obtain PMA mother liquor, the concentration of PMA in the mother liquor is 1M) is added into 99. mu.L of the reaction system in step 2, the mixture is placed at room temperature for 4h, excitation light (Ex) and emission light (Em) of an enzyme reader are respectively set to be 380nm and 520nm, and fluorescence values are detected. And selecting a mutant with a fluorescence value higher than that of the wild TbSADH for GC detection, and performing parallel detection twice.
GC detection conditions are as follows: HP-5 achiral column, the injection port temperature is 300 ℃, and the flow rate of the chromatographic column is 1 mL/min; temperature rising procedure: the initial temperature of the chromatographic column is 100 ℃, the retention time is 1min, the heating rate is 15 ℃/min, the temperature is raised to 170 ℃, the retention time is 1min, the heating rate is 3 ℃/min, the temperature is raised to 180 ℃, and the retention time is 1 min; the detection port temperature was 320 ℃. Raspberry ketone (Beijing Hanlongda science and technology development Co., Ltd., product number: PS2151-0050), and rhododendrol (Beijing Hanlongda science and technology development Co., Ltd., product number: R134727).
The fluorescence values of TbSADH W110 site saturation mutation library screening are shown in figure 11, a strain with a fluorescence value higher than that of a wild control and a strain with a fluorescence value lower than that of BL21(DE3)/pET28a blank control are selected and sent to Beijing Optimalaceae biotechnology limited company for sequencing, and the sequencing results are shown in table 3.
TABLE 3 TbSADH mutants and corresponding transformation rates
a) Number of screening acquisitions in 96-well plates. b) And calculating the conversion rate according to the generated amount of the raspberry ketone of the product and the raspberry ketone standard product.
The result shows that 13 strains are selected for sequencing through fluorescence value screening, all the strains to be sent are mutants, and the GC detection conversion rate and the fluorescence value have better consistency, which indicates that the ketone high-throughput detection method can be applied to directed evolution screening of enzymes.
Claims (10)
- The application of 5-methoxy-2-aminobenzamide oxime is at least one of the following (a1) - (a 4):(a1) detecting the content of the ketone compound;(a2) detecting the activity of the enzyme;(a3) screening high-activity enzyme;(a4) directed evolution screening of enzymes;the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds.
- 2.5-methoxy-2-aminobenzamide oxime in preparing products; the product has at least one of the following (a1) - (a 4):(a1) detecting the content of the ketone compound;(a2) detecting the activity of the enzyme;(a3) screening high-activity enzyme;(a4) directed evolution screening of enzymes;the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds.
- 3. A kit comprising 5-methoxy-2-aminobenzamide oxime; the kit is used for at least one of the following (a1) - (a 4):(a1) detecting the content of the ketone compound;(a2) detecting the activity of the enzyme;(a3) screening high-activity enzyme;(a4) directed evolution screening of enzymes;the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds.
- 4. The detection method of the content of the ketone compound comprises the following steps: adding 5-methoxy-2-aminobenzamide oxime and a sample to be detected into a buffer solution, mixing, placing at room temperature for reaction, and comparing the fluorescence values of the reaction system of the system to be detected and the reaction system of the control system in the excitation light of 360-400nm and the emission light of 500-540 nm; the fluorescence value of the system to be detected is higher than the corresponding fluorescence value of the comparison system, and the content of the ketone compound in the sample to be detected is higher; the contrast system is a reaction system without adding a sample to be detected.
- 5. The method of claim 4, wherein: the concentration of the 5-methoxy-2-aminobenzamide oxime in the reaction system is 1mM-50 mM.
- 6. The method of claim 4 or 5, wherein: the pH value of the buffer solution is 3-14.
- 7. The method of any of claims 4 to 6, wherein: the reaction time is 0.5h-24 h.
- 8. Use of the method of any one of claims 4 to 7 for the detection of enzymatic activity or for the screening of highly active enzymes; the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds.
- 9. Use of the method of any one of claims 4 to 7 in directed evolution screening of enzymes; the enzyme is an enzyme that can catalyze the production of ketone compounds or the consumption of ketone compounds.
- 10. The use or method or kit as claimed in any one of claims 1 to 9, wherein: the ketone compounds include aliphatic ketones, aromatic ketones, hydroxyketones, and heterocyclic ketones.
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