CN108642130B - High-throughput screening method for high-activity strain of tyrosine phenol lyase - Google Patents
High-throughput screening method for high-activity strain of tyrosine phenol lyase Download PDFInfo
- Publication number
- CN108642130B CN108642130B CN201810271360.3A CN201810271360A CN108642130B CN 108642130 B CN108642130 B CN 108642130B CN 201810271360 A CN201810271360 A CN 201810271360A CN 108642130 B CN108642130 B CN 108642130B
- Authority
- CN
- China
- Prior art keywords
- strain
- reaction
- sodium pyruvate
- activity
- lyase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- 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
- G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
Abstract
The invention discloses a high-throughput screening method of a high-activity strain of a tyrosine phenol lyase, which comprises the following steps: adding wet thalli obtained by fermentation culture of a strain to be detected into a substrate reaction solution as a strain sample to be detected, and centrifuging a shaker reaction solution to obtain a supernatant; adding the supernatant into the color reaction solution, standing at room temperature for color development, measuring a light absorption value at 465nm, obtaining the content of sodium pyruvate in the supernatant according to a sodium pyruvate standard curve, and further obtaining the activity of the tyrosine phenol lyase in a strain sample to be measured, thereby screening out a high-activity strain of the tyrosine phenol lyase; the method can directly screen and obtain the tyrosine phenol lyase with improved levodopa synthetic ability and the mutant thereof in high flux, the time is shortened from 20min for analyzing one sample to 1min for analyzing nearly 60 samples, and the error of the measured enzyme activity is controlled within 3%.
Description
(I) technical field
The invention relates to a method for high-throughput screening of tyrosine phenol lyase with improved levodopa synthesis capacity and mutants thereof.
(II) background of the invention
Tyrosine Phenol Lyase (TPL), also known as β -tyrosinase, is a class of pyridoxal-5' -phosphate (PLP) -dependent lyases. TPL can catalyze L-tyrosine to generate alpha, beta-elimination reaction to generate pyruvic acid, phenol and ammonia. The reaction is a reversible reaction, if catechol is used to replace phenol, the enzyme can catalyze the generation of levodopa, and the catalytic mechanism can be represented by the following formula:
levodopa is an important active substance in biological organisms and is the main drug for the treatment of parkinson's disease. The principle of treating Parkinson's disease is as follows: the derivative of levodopa, dopamine, is an important neurotransmitter for treating Parkinson's disease, but can not pass through the blood brain barrier, so that Parkinson's disease can not be treated by externally supplementing dopamine, and levodopa can pass through the blood brain barrier, reaches the central nervous system, and is converted into dopamine under the action of decarboxylase in vivo, so that the content of dopamine in brain tissues is increased, and the effect of treating Parkinson's syndrome is further exerted.
In view of unstable yield, complex steps, multiple chemical synthesis procedures, high cost and serious environmental pollution of the levodopa plant extraction method, the synthesis of levodopa by a microbial method is more and more concerned. The tyrosine phenol lyase catalyzes catechol, and the synthesis of levodopa from pyruvic acid and ammonia has the remarkable advantages of mild reaction conditions, high atom economy and the like.
With the rapid development of the genetic engineering technology, the efficiency of synthesizing levodopa by the catalysis of the tyrosine phenol lyase can be effectively improved through the technologies of enzyme high-throughput screening, molecular modification and the like. Due to the variety of the tyrosine phenol lyase existing in nature, the mutant library mutant obtained by the modification of enzyme molecules is huge in number (usually containing 10 percent of mutants)4～106Mutant) and the like, it is difficult to efficiently screen and obtain the tyrosine phenol lyase with improved catalytic performance by the traditional detection means such as chromatography and the like. Therefore, the establishment of a rapid and accurate high-throughput screening method for the tyrosine phenol lyase is of great significance.
Disclosure of the invention
The invention provides a method for screening high-throughput tyrosine phenol lyase based on micro culture of a deep-hole plate and micro detection of an enzyme-labeling instrument, aiming at overcoming the problems of long period, large workload and the like of screening high-activity tyrosine phenol lyase, and the mechanism is as follows: the substrate sodium pyruvate participating in the synthesis of levodopa and salicylaldehyde generate a chromogenic 1, 5-bis (2-hydroxyphenyl) -1, 4-pentadiene ketone product in a strong alkaline solution, and the product has an absorption value at the wavelength of 465 nm. As the concentration of sodium pyruvate decreased, the color changed from orange to light yellow. If the activity of the tyrosine phenol lyase is lower, the weaker the ability of the tyrosine phenol lyase to synthesize levodopa is, and the larger the residual concentration of the sodium pyruvate is, the darker the color of the color reaction liquid is, and if the activity of the tyrosine phenol lyase is higher, the stronger the ability of the tyrosine phenol lyase to synthesize levodopa is, and the smaller the residual concentration of the sodium pyruvate is, the lighter the color of the color reaction liquid is.
The technical scheme adopted by the invention is as follows:
the invention provides a high-throughput screening method of a high-activity strain of a tyrosine phenol lyase, which comprises the following steps: (1) and (3) conversion reaction: adding wet thallus obtained by fermentation culture of a strain to be detected (preferably the strain to be detected containing a tyrosine phenol lyase gene) into a substrate reaction solution as a strain sample to be detected, performing shake reaction at 10-30 ℃ and 100-300rpm for 20-120min (preferably 1M HCl or standing at 95 ℃ for 5-10min to terminate the reaction), and centrifuging the reaction solution to obtain a supernatant; the final concentration composition of the substrate reaction solution is as follows: 2-15g/L catechol, 2-20g/L sodium pyruvate, 5-50g/L ammonium acetate, 0.1-2g/L sodium sulfite, 0.1-2g/L EDTA-2Na, 0.2-2mM pyridoxal phosphate (PLP), pH 7.0-8.0, and solvent ultrapure water; (2) and (3) color development reaction: adding the supernatant obtained in the step (1) into a color reaction solution, standing at room temperature for color development, measuring a light absorption value at 465nm, obtaining the content of sodium pyruvate in the supernatant according to a sodium pyruvate standard curve, and further obtaining the activity of the tyrosine phenol lyase in a strain sample to be tested, so that a high-activity strain of the tyrosine phenol lyase with improved levodopa synthetic capacity is screened out; the color development reaction liquid consists of 10-250g/L sodium hydroxide aqueous solution, salicylaldehyde and ultrapure water in a volume ratio of 3: 0.1-3: 5-10; and the standard curve is formed by performing color reaction on a substrate reaction solution and a supernatant obtained after conversion reaction of recombinant escherichia coli containing a tyrosine phenol lyase gene under the same detection condition of a strain to be detected, and drawing by taking the concentration of sodium pyruvate as a horizontal coordinate and a light absorption value at 465nm as a vertical coordinate.
Further, the substrate reaction solution has a final concentration composition of: 4-8g/L of catechol, 4-10g/L of sodium pyruvate, 30-50g/L of ammonium acetate, 0.5-1g/L of sodium sulfite, 1-2g/L of EDTA-2Na, 0.5-1mM of pyridoxal phosphate, pH 7.0-8.0, ultrapure water as a solvent, and more preferably the substrate reaction solution has the following final concentration: 5g/L of catechol, 5g/L of sodium pyruvate, 50g/L of ammonium acetate, 1g/L, EDTA-2Na2g/L, PLP 1mM of sodium sulfite, and the solvent is ultrapure water.
Further, the color reaction liquid consists of 250g/L sodium hydroxide aqueous solution, salicylaldehyde and ultrapure water in a volume ratio of 3:0.1: 6.7.
Further, the color reaction in the step (2) is carried out according to the following steps: adding 1ml of 250g/L NaOH aqueous solution, 200 mu L of the supernatant obtained in the step (1), 6.7ml of ultrapure water, 100 mu L of salicylaldehyde and 2ml of 250g/L NaOH aqueous solution respectively, uniformly mixing to form a 10ml color reaction system, standing at room temperature for color development, and measuring the light absorption value at 465 nm.
Further, the sodium pyruvate standard curve is prepared as follows: (1) and (3) conversion reaction: adding recombinant escherichia coli wet thalli containing a tyrosol lyase gene into a substrate reaction solution, carrying out shaking table reaction at 30 ℃ and 150rpm for 5-5 h, sampling at regular time (sampling at 10,20,30,40,50,60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 300min respectively), adding 1M HCl to terminate the reaction, and centrifuging at 3000rpm for 10min to obtain supernatants with different reaction times; the final concentration composition of the substrate reaction solution is as follows: 5g/L catechol, 5g/L sodium pyruvate, 50g/L ammonium acetate, 1g/L, EDTA-2Na2g/L sodium sulfite, 1mM pyridoxal phosphate and ultrapure water as solvent, wherein the pH value is 7.0-8.0; the final concentration of the wet thalli is 4 g/L;
(2) and (3) color development reaction: and (2) sequentially adding 1mL of 250g/L NaOH aqueous solution, 200 μ L of the supernatant obtained in the step (1) at different reaction times, 6.7mL of ultrapure water, 100 μ L of salicylaldehyde and 2mL of 250g/L NaOH aqueous solution, uniformly mixing to form a chromogenic reaction system of 10mL, standing at room temperature for 2h, measuring absorbance at 465nm by using a microplate reader, and obtaining a sodium pyruvate standard curve by using the absorbance as a vertical coordinate and the conversion rate of sodium pyruvate as a horizontal coordinate.
Furthermore, the recombinant Escherichia coli containing the tyrosine phenol lyase gene is obtained by introducing a gene of a nucleotide sequence shown in SEQ ID NO.1 into Escherichia coli.
Further, the fermentation method of the strain to be detected comprises the following steps: inoculating the strain to be tested to a fermentation medium, and carrying out shake cultivation for 6-12h at 20-37 ℃ and 100-; the fermentation medium comprises the following components: 5-20g/L of peptone, 2-10g/L of yeast powder, 2-10g/L of sodium chloride, 10-30g/L of IPTG, water as solvent and natural pH value.
Further, before fermentation, the strain to be tested is subjected to seed activation culture, and then a seed solution is inoculated to a fermentation medium in an inoculum size of 2-5% in volume concentration, wherein the seed activation culture method comprises the following steps: inoculating the strain to be detected to a seed culture medium, and performing shake cultivation at 30-37 ℃ and 100-300rpm for 10-14h to obtain a seed solution; the seed culture medium comprises the following components: 5-20g/L of peptone, 2-10g/L of yeast powder, 2-10g/L of sodium chloride, 50-100 mu g/mL of kanamycin, water as a solvent and natural pH value.
Further, the method comprises the step of carrying out reaction in a 96-pore plate, wherein the 96-pore plate is a deep-pore plate, and the upper hole of a silica gel pore pad of the deep-pore plate corresponds to the deep hole of the deep-pore plate, so that each micropore can independently exchange air with the outside. The liquid loading amount of the culture medium is 30-50% of the pore volume of the deep hole plate, and the inoculation amount is 20-50% of the volume of the fermentation culture medium.
Further, the method comprises the following steps: (1) inoculating a strain to be detected into a deep-hole plate I filled with a seed culture medium, and performing shake cultivation at 30-37 ℃ and 100-300rpm for 10-14h to obtain a seed solution; (2) correspondingly inoculating the seed liquid in the deep-well plate I into a deep-well plate II filled with a fermentation culture medium, and performing shake cultivation at 20-37 ℃ and 100-300rpm for 6-12 h; placing the deep-hole plate II in a hole plate centrifuge, centrifuging for 10-30min at 3000 Xg of 1000-; (3) adding 400 mu L of 100-plus substrate reaction solution into the deep-hole plate II, carrying out shaking table reaction at 10-30 ℃ and 300rpm for 20min-2h, and adding 400 mu L of 1M HCl with 100-plus to terminate the reaction; placing the deep-hole plate II in a hole plate centrifuge, and centrifuging for 10-30min at 3000 Xg of 1000-; (4) adding 1ml of 250g/L NaOH aqueous solution into a deep-hole plate III in sequence, respectively adding 200 mu L of supernatant, 6.7ml of ultrapure water, 100 mu L of salicylaldehyde and 2ml of 250g/L NaOH aqueous solution into a deep-hole plate III in the step (3), uniformly mixing to form a 10ml color reaction system, standing at room temperature for 20min-2h for color reaction, detecting the light absorption value at 465nm by using an enzyme labeling instrument, obtaining the content of sodium pyruvate in the supernatant according to a sodium pyruvate standard curve, further obtaining the conversion rate and activity of the recombinant bacteria to a substrate, and screening to obtain the strain containing the improved activity of the tyrosine phenol lyase when the activity is higher than 20% of that of a reference strain.
The higher the activity of the tyrosol lyase is, the lower the content of the residual sodium pyruvate in the reaction is, the content of the sodium pyruvate in the supernatant is obtained according to the corresponding relation between the concentration of the sodium pyruvate and the light absorption value, the capability of the recombinant tyrosol lyase for synthesizing levodopa is obtained, and when the activity is higher than 20% of that of the original strain, the recombinant tyrosol lyase is determined to be a strain with improved activity, and the high performance liquid chromatography analysis is further carried out for determination.
The strain to be detected comprises recombinant escherichia coli containing a tyrosine phenol lyase gene and a mutant gene thereof, and the source of the tyrosine phenol lyase gene sequence is not limited.
The invention provides a high-throughput method for screening a tyrosine phenol lyase-containing strain with improved levodopa synthesis capacity, which is carried out on the basis that a chromogenic 1, 5-bis (2-hydroxyphenyl) -1, 4-pentadiene ketone product is generated by substrate sodium pyruvate synthesized by levodopa and salicylaldehyde in a strong alkali solution, wherein the concentration of the sodium pyruvate is different, the degree of color development is different, and the tyrosine phenol lyase with improved levodopa synthesis capacity and a mutant thereof are rapidly screened and obtained according to the corresponding relation between the concentration of the sodium pyruvate and a light absorption value at 465 nm.
Compared with the prior art, the invention has the following beneficial effects: the screening method can directly screen and obtain the tyrosine phenol lyase with improved levodopa synthetic capacity and mutants thereof in high flux, compared with the traditional high performance liquid detection method, the time is shortened from 20min for analyzing a sample to only 1min for analyzing nearly 60 samples, the method comprehensively utilizes a porous plate to carry out rapid culture and fermentation of strains, utilizes an enzyme-labeling instrument to carry out rapid determination, has the advantages of simple and convenient operation, rapid detection, economy, practicability and the like, is easy to realize mechanical automatic operation, brings great convenience for rapid screening of the tyrosine phenol lyase for efficiently synthesizing levodopa, is sensitive and effective, compared with the high performance liquid method, the measured enzyme activity error is controlled within 3%, and compared with a control strain, the strain with the enzyme activity improved by 20% can be directly screened and obtained.
(IV) description of the drawings
FIG. 1 is a high throughput screening process of tyrosine phenol lyase and high-activity bacteria thereof.
FIG. 2 is a corresponding relationship between sodium pyruvate concentration and absorbance during high throughput screening.
FIG. 3 is a graph showing the variation of absorbance with time for different substrate concentrations in different development times during a high throughput screening process; concentration of sodium pyruvate: 0mM (■), 2.5mM (●), 5mM (. tangle-solidup.), 10mM (. diamond-solid.), 20mM (. XX), 40mM (□), 60mM (. smallcircle.), 80mM (. DELTA.), 100mM (. diamond-solid.).
FIG. 4 is the corresponding relationship between the concentration of sodium pyruvate and the light absorption value in the simulated reaction process without adding bacteria.
FIG. 5 is a graph showing the relationship between the absorbance and the conversion in the course of the reaction when cells were added.
(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:
the Ultrapure water (ultra water) in the embodiment of the invention is also called UP water, and is water with a resistivity of 18M Ω cm (25 ℃).
Example 1 correlation between sodium pyruvate concentration and light absorption value
A sodium pyruvate solution of 0 to 100mM (0mM, 2.5mM, 5mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM) was prepared with ultrapure water. 1mL of 250g/L NaOH aqueous solution, 200 μ L of sodium pyruvate solution with different concentrations, 6.7mL of ultrapure water, 100 μ L of salicylaldehyde and 2mL of 250g/L NaOH aqueous solution are respectively added into a reaction system of 10mL, and the mixture is shaken up after the salicylaldehyde is added so as to fully react with the sodium pyruvate for color development. Standing at room temperature for 2h, and measuring absorbance at 465nm by using a microplate reader. The absorbance was plotted on the ordinate and the concentration of sodium pyruvate was plotted on the abscissa, and the results are shown in FIG. 2. The higher the concentration of sodium pyruvate, the greater the absorbance, and the linear relationship between the two: y is 0.0434X +0.3046, R2＝0.9998。
EXAMPLE 2 determination of the color reaction time
A sodium pyruvate solution of 0 to 100mM (0mM, 2.5mM, 5mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM) was prepared with ultrapure water. 1mL of 250g/L NaOH aqueous solution, 200 μ L of sodium pyruvate solution with different concentrations, 6.7mL of ultrapure water, 100 μ L of salicylaldehyde and 2mL of 250g/L NaOH aqueous solution are respectively added into a reaction system of 10mL, and the mixture is uniformly mixed. Standing at room temperature, sampling every 10min, measuring absorbance at 465nm with a microplate reader, and reacting for 2 h. The absorbance was plotted as ordinate and the color reaction time as abscissa, and the light absorption curve was plotted, and the result is shown in FIG. 3. Indicating that the light absorption value basically tends to be stable in the color reaction for more than 1 h.
Example 3 correspondence between sodium pyruvate concentration and light absorption value without adding a bacteria reaction system
The final concentration of the substrate reaction solution (pH 7.0-8.0) was composed of: 5g/L of catechol, 0g/L of sodium pyruvate (0, 1, 2.5, 5, 7.5, 10, 15, 20,30 and 40g/L of sodium pyruvate), 50g/L of ammonium acetate, 1g/L, EDTA-2Na2g/L of sodium sulfite, 1mM of pyridoxal phosphate (PLP) and ultrapure water as a solvent. Sequentially reacting sodium pyruvate with different concentrations for 30min at 30 ℃ by a shaking table at 150rpm, and respectively adding 400 mu L of 1M HCl to terminate the reaction to obtain reaction solutions of the sodium pyruvate with different concentrations.
The color development reaction comprises the following steps: 1mL of 250g/L NaOH aqueous solution, 200 μ L of reaction solution with different sodium pyruvate concentrations, 6.7mL of ultrapure water, 100 μ L of salicylaldehyde and 2mL of 250g/L NaOH aqueous solution are respectively added into a color reaction system of 10mL, and the mixture is uniformly mixed. Standing at room temperature for 2h, and measuring absorbance at 465nm by using a microplate reader. The absorbance was plotted on the ordinate and the sodium pyruvate concentration was plotted on the abscissa, and the results are shown in FIG. 4. The light absorption values and the sodium pyruvate concentrations under different sodium pyruvate concentrations are shown to be in a linear relationship under the condition of no bacteria: y ═ 0.0282X +0.1630, R2＝0.9973。
Example 4 correlation between sodium pyruvate concentration and light absorption value in a cell reaction System
(1) Constructing recombinant Escherichia coli containing a tyrosine phenol lyase gene derived from Fusobacterium nucleatum: the recombinant escherichia coli containing the tyrosol lyase gene derived from fusobacterium nucleatum is obtained by introducing a coding gene (shown in SEQ ID NO. 1) of the tyrosol lyase into escherichia coli for construction, and a specific construction method reference (Enzyme Microb Tech,2018,266: 20-26).
The recombinant escherichia coli containing the fusobacterium nucleatum-derived tyrosine phenol lyase is obtained by culturing in the following way: culturing the recombinant strain in LB culture medium at 37 ℃ to OD600After 0.6-0.8, 1mmol/L IPTG is added, and induction is carried out for 10-12h at 28 ℃. After completion of the culture, the wet cells were collected by centrifugation and washed 2 times with 0.85% physiological saline.
(2) The final concentration of the substrate reaction solution (pH 7.0-8.0) was composed of: 5g/L of catechol, 5g/L of sodium pyruvate, 50g/L of ammonium acetate, 1g/L, EDTA-2Na2g/L, PLP 1mM of sodium sulfite, and the solvent is ultrapure water.
The specific conversion reaction steps are as follows: sucking 400 μ L of substrate reaction solution, adding recombinant large intestine wet thallus containing tyrosol lyase derived from fusobacterium nucleatum (the final concentration of the added wet thallus is 4g/L), mixing thallus and substrate reaction solution uniformly, shaking at 30 ℃ and 150rpm for 5min-5h (respectively 10,20,30,40,50,60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 300min), adding 400 μ L of 1M HCl to terminate the reaction, centrifuging at 3000rpm for 10min, and taking supernatant of different reaction time for color reaction.
(4) Color reaction
10mL of the color reaction system is added with 1mL of 250g/L NaOH aqueous solution, 200 mu L of the supernatant obtained by different reaction times, 6.7mL of ultrapure water, 100 mu L of salicylaldehyde and 2mL of 250g/L NaOH aqueous solution respectively, and the mixture is mixed uniformly. Standing at room temperature for 2h, measuring absorbance at 465nm with an enzyme-labeling instrument, and measuring conversion rate of sodium pyruvate with high performance liquid chromatography. The absorbance was plotted on the ordinate and the conversion of sodium pyruvate was plotted on the abscissa, and the results are shown in FIG. 5. The linear relationship between the conversion rate of sodium pyruvate and the light absorption value is shown under the condition of adding thalli: -0.01076X +1.2423, R2＝1。
The liquid phase detection conditions were as follows: the liquid chromatogram is Shimadzu LC-16 (Japan), and the chromatographic columns are: c18 column (Welch,5 μm. times.250X 4.6 mm); column temperature: 34 ℃; flow rate: 1 mL/min; sample introduction amount: 10 mL; detection wavelength: UV210 nm; mobile phase: 20mM KH2PO4(pH adjusted to 2.6 with HCl): methanol 9: 1.
Example 5 comparison of the high throughput screening method of the present invention with conventional HPLC analysis
(1) Taking the recombinant escherichia coli containing the tyrosol lyase gene obtained by the method in the embodiment 4 as a strain to be detected, coating the recombinant escherichia coli as an LB agar culture medium containing 100ug/ml kanamycin resistance, culturing at 37 ℃ for 12h, and taking an obtained single colony as a high-activity strain of the tyrosol lyase to be screened;
(2) under the aseptic condition, taking a single colony of a high-activity strain of the tyrosol lyase to be screened, inoculating the single colony into a 96-well plate containing a seed culture medium, and activating for later use;
(3) inoculating the activated bacterial liquid into a 96-well plate containing a fermentation culture medium according to the volume concentration of 2%, culturing at 28 ℃ for 10h, and centrifuging at 1000-;
(4) and (3) adding 400 mu L of substrate reaction liquid into a 96-well plate of the thallus obtained in the step (3), carrying out shaking table reaction at 30 ℃ and 150rpm, respectively sampling after 10,20,30,40,50 and 60min of reaction, placing the sampled sample at 95 ℃ for 5-10min to terminate the reaction, and centrifuging at 3000 Xg for 10 min. The final concentration composition of the substrate reaction solution (pH 8.0) was: 5g/L of catechol, 8g/L of sodium pyruvate, 50g/L of ammonium acetate, 2g/L, EDTA-2Na 1g/L, PLP 1mM of sodium sulfite, and the solvent is ultrapure water;
(5) and (3) performing color development reaction on the centrifuged supernatant in the step (4) for 1h, measuring the absorbance at 465nm by using an enzyme-linked immunosorbent assay, adding 1ml of 250g/L NaOH aqueous solution into 10ml of the color development reaction system, sequentially and respectively adding 200 mu L of centrifuged supernatant of samples obtained from the step (4) at different reaction times, 6.7ml of ultrapure water, 100 mu L of salicylaldehyde and 2ml of 250g/L NaOH aqueous solution, and uniformly mixing. According to the curve Y of the corresponding relation between the concentration of the sodium pyruvate and the light absorption value obtained in the embodiment 4, the content of the sodium pyruvate in the supernatant is calculated, the conversion rate of the recombinant bacteria to the substrate is further obtained, and the strains with high activity of the tyrosine phenol lyase are obtained by screening.
(6) And (4) simultaneously taking the centrifuged supernatant of the samples obtained in the step (4) at different reaction times and simultaneously carrying out high performance liquid chromatography analysis. The liquid phase detection conditions of sodium pyruvate were as follows: the liquid chromatogram is Shimadzu LC-16 (Japan), and the chromatographic columns are: c18 column (Welch,5 μm. times.250X 4.6 mm); column temperature: 34 ℃; flow rate: 1 mL/min; sample introduction amount: 10 mL; detection wavelength: UV210 nm; mobile phase: 20mM KH2PO4(pH adjusted to 2.6 with HCl): methanol 9: 1. Under the chromatographic conditions, the peak time of sodium pyruvate was 3.37 min. And (3) taking the supernatant to be detected to dilute by 20 times, analyzing by using a high performance liquid chromatography, and calculating the conversion rate of the recombinant bacteria to the substrate.
The result of comparison between the high-throughput screening method and the traditional high performance liquid chromatography analysis method is shown in table 1, the conversion rate of catechol is improved along with the prolonging of the reaction time, the concentration of sodium pyruvate is reduced, the absorbance is reduced, the conversion rate measured by the color reaction is positively correlated with the conversion rate measured by the high performance liquid chromatography, the testing method is accurate, the error range is within 3 percent, and the feasibility of the 96-pore plate enzyme-linked immunosorbent assay is proved.
TABLE 1 comparison of the conversion of sodium pyruvate measured in different ways at the same reaction time
Comparison of the detection times by 2 methods (see table 2) the difference in the time of pretreatment of the samples was negligible.
TABLE 2 comparison of detection times of the method and HPLC
| Method for producing a composite material | High performance liquid chromatography | |
| Detection time (57 samples)/ |
1 | 1140 |
The high-throughput screening method (a 96-well plate is selected), each sample is paralleled for three times, 57 samples are detected by one 96-well plate, and the detection time is about 1 min. The procedure of high performance liquid chromatography was 20min per sample, and detection of 57 samples took about 1140 min. Therefore, the high-throughput screening method established by the invention greatly reduces the screening time and improves the screening efficiency.
Example 6 validation of screening of multiple unknown strains
(1) Under the aseptic condition, inoculating unknown single colony (containing tyrosine phenol lyase gold) to be screened into a 96-well plate containing a seed culture medium, and activating for later use;
(2) inoculating the activated bacterial liquid into a 96-well plate containing a fermentation culture medium according to the volume concentration of 2%, culturing at 28 ℃ for 10h, and centrifuging at 1000-;
(3) adding 400 μ L substrate reaction solution into 96-well plate of thallus obtained in step (2), shaking at 30 deg.C and 150rpm for reaction, respectively sampling after 30min reaction, standing the sample at 95 deg.C for 5-10min to terminate the reaction, and centrifuging at 3000 Xg for 10 min. The final concentration composition of the substrate reaction solution (pH 8.0) was: 5g/L of catechol, 8g/L of sodium pyruvate, 50g/L of ammonium acetate, 1g/L, EDTA-2Na2g/L, PLP 1mM of sodium sulfite, and the solvent is ultrapure water;
(4) and (3) performing color development reaction on the centrifuged supernatant in the step (3) for 1h, measuring the absorbance at 465nm by using an enzyme-linked immunosorbent assay, adding 100 mu L of 250g/L NaOH aqueous solution into 1ml of the color development reaction system, adding 20 mu L of centrifuged supernatant of samples obtained from the step (3) at different reaction times, 670 mu L of ultrapure water, 10 mu L of salicylaldehyde and 200 mu L of 250g/L NaOH aqueous solution, and uniformly mixing. According to the curve Y of the corresponding relation between the concentration of the sodium pyruvate and the light absorption value obtained in the embodiment 4, the concentration of the sodium pyruvate in the supernatant is obtained through calculation, the conversion rate of the recombinant bacteria to the substrate is further obtained, and the strain with high activity of the tyrosol lyase is obtained.
(5) And (4) taking the centrifuged supernatant of the sample obtained in the step (3) and simultaneously carrying out high performance liquid chromatography analysis. The sodium pyruvate liquid phase detection conditions were as follows: the liquid chromatogram is Shimadzu LC-16 (Japan), and the chromatographic columns are: c18 column (Welch,5 μm. times.250X 4.6 mm); column temperature: 34 ℃; flow rate: 1 mL/min; sample introduction amount: 10 mL; detection wavelength: UV210 nm; mobile phase: 20mM KH2PO4(pH adjusted to 2.6 with HCl): methanol 9: 1. Under the chromatographic conditions, the peak time of sodium pyruvate was 3.37 min. And (3) taking the supernatant to be detected, diluting by 20 times, analyzing by using a high performance liquid chromatography, and calculating the conversion rate of the unknown bacteria to the substrate.
Comparison of sodium pyruvate conversion rates of 12 unknown strains in the first row of a well plate in Table 396 by different methods
The high-throughput screening is carried out on the strains to be tested, and the result shows that the conversion rate measured by the chromogenic reaction established by the invention is positively correlated with the conversion rate measured by the high performance liquid chromatography, and the testing method is accurate.
The source of the tyrosine phenol lyase related to the invention is not limited to the Fusobacterium nucleatum source enzyme mentioned in the examples, but is generally applicable to tyrosine phenol lyase from other sources and mutants thereof.
The invention is not limited by the specific text described above. The invention can be varied within the scope outlined by the claims and these variations are within the scope of the invention.
Sequence listing
<110> Zhejiang industrial university
<120> high-throughput screening method of high-activity bacterial strain of tyrosine phenol lyase
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1383
<212> DNA
<213> Unknown (Unknown)
<400> 1
atgagatttg aagattatcc agcagagcca tttagaatta aaagtgtaga aactgttaaa 60
atgattgata aggcagcaag agaagaagta attaaaaaag caggatataa tactttctta 120
attaactctg aagatgttta cattgattta ttaactgata gtggaactaa tgctatgagt 180
gataaacaat ggggtggatt aatgcaaggt gatgaagctt atgcaggaag tagaaatttc 240
ttccacttag aagaaactgt aaaagaaata tttgggttta aacatatagt tcctactcac 300
caaggaagag gagcagaaaa tattttatct caaatagcta taaaacctgg acaatatgtt 360
cctggaaata tgtattttac aactactaga tatcaccaag aaagaaatgg tggaatattt 420
aaagatatta tcagagatga ggcacatgat gctactctta atgttccttt caaaggagat 480
attgacttaa ataaattaca aaaattaata gatgaagttg gagcagaaaa cattgcttat 540
gtttgtttag ctgtaactgt aaaccttgct ggtggacaac cagtttctat gaaaaatatg 600
aaagcagtta gagaactaac taaaaaacat ggaataaaag ttttctatga tgcaactaga 660
tgtgttgaaa atgcttactt cattaaagaa caagaagaag gatatcaaga taaaactata 720
aaggaaatag tgcatgaaat gtttagctat gctgatggat gtactatgag tggtaaaaaa 780
gattgtcttg ttaatatagg tggattttta tgtatgaatg atgaagattt attcttagct 840
gcaaaagaaa tagttgttgt ttatgaaggt atgccatctt atggtggact tgctggtaga 900
gatatggaag ctatggcaat agggttaaga gaatctttac aatatgaata cattagacat 960
agaattttac aagttagata cttaggagaa aaattaaaag aagctggtgt acctatactt 1020
gaaccagttg gaggacatgc tgtattccta gatgctagaa gattctgtcc tcatatccca 1080
caagaagaat tcccagctca agctcttgca gcagctatct atgttgaatg tggtgtaaga 1140
actatggaaa gaggaataat ttctgctggt agagatgtaa aaactggtga aaaccataaa 1200
cctaaactag aaactgttag agttactatt ccaagaagag tttatactta taaacatatg 1260
gatgtagtag cagaaggtat aatcaaatta tataaacata aagaagatat aaaaccatta 1320
gaatttgtat atgaaccaaa acaattaaga ttctttacag ctagatttgg aataaaaaaa 1380
taa 1383
Claims (8)
1. A high-throughput screening method of a high-activity strain of a tyrosol lyase is characterized in that the method comprises the following steps: (1) and (3) conversion reaction: adding wet thalli obtained by fermentation culture of a strain to be detected into a substrate reaction solution as a strain sample to be detected, carrying out shaking table reaction at 10-30 ℃ and 100-300rpm for 20-120min, and centrifuging the reaction solution to obtain a supernatant; the final concentration composition of the substrate reaction solution is as follows: 2-15g/L of catechol, 2-20g/L of sodium pyruvate, 5-50g/L of ammonium acetate, 0.1-2g/L of sodium sulfite, 0.1-2g/L of EDTA-2Na, 0.2-2mM of pyridoxal phosphate, 7.0-8.0 of pH and ultrapure water as a solvent; (2) and (3) color development reaction: sequentially and respectively adding 1ml of 250g/L NaOH aqueous solution, 200 mu L of the supernatant obtained in the step (1), 6.7ml of ultrapure water, 100 mu L of salicylaldehyde and 2ml of 250g/L NaOH aqueous solution, uniformly mixing to form a 10ml color reaction system, standing at room temperature for color development, measuring an absorbance at 465nm, obtaining the content of sodium pyruvate in the supernatant according to a sodium pyruvate standard curve, and further obtaining the activity of the tyrosine phenol lyase in a strain sample to be detected, thereby screening out the high-activity strain of the tyrosine phenol lyase; and the standard curve is formed by performing color reaction on a substrate reaction solution and a supernatant obtained after conversion reaction of recombinant escherichia coli containing a tyrosine phenol lyase gene under the same detection condition of a strain to be detected, and drawing by taking the concentration of sodium pyruvate as a horizontal coordinate and a light absorption value at 465nm as a vertical coordinate.
2. The method for high throughput screening of the highly active tyrosol lyase strain according to claim 1, wherein the final concentration of the substrate reaction solution is: 4-8g/L of catechol, 4-10g/L of sodium pyruvate, 30-50g/L of ammonium acetate, 0.5-1g/L of sodium sulfite, 1-2g/L of EDTA-2Na, 0.5-1mM of pyridoxal phosphate, pH 7.0-8.0 and ultrapure water as a solvent.
3. The method for high throughput screening of the high-activity tyrosol lyase strain according to claim 1, wherein the standard curve of sodium pyruvate is prepared as follows: (1) and (3) conversion reaction: adding the recombinant escherichia coli wet thalli containing the tyrosol lyase gene into a substrate reaction solution, carrying out shake reaction at 30 ℃ and 150rpm for 5min-5h, regularly sampling, adding 1M HCl to terminate the reaction, and then centrifuging at 3000rpm for 10min to obtain supernatants with different reaction times; the final concentration composition of the substrate reaction solution is as follows: 5g/L catechol, 5g/L sodium pyruvate, 50g/L ammonium acetate, 1g/L, EDTA-2Na2g/L sodium sulfite, 1mM pyridoxal phosphate and ultrapure water as solvent, wherein the pH value is 7.0-8.0; the final concentration of the wet thalli is 4 g/L;
(2) and (3) color development reaction: and (2) sequentially adding 1mL of 250g/L NaOH aqueous solution, 200 μ L of the supernatant obtained in the step (1) at different reaction times, 6.7mL of ultrapure water, 100 μ L of salicylaldehyde and 2mL of 250g/L NaOH aqueous solution, uniformly mixing to form a chromogenic reaction system of 10mL, standing at room temperature for 2h, measuring absorbance at 465nm by using a microplate reader, and obtaining a sodium pyruvate standard curve by using the absorbance as ordinate and the concentration of sodium pyruvate as abscissa.
4. The high-throughput screening method of the highly active tyrosine phenol lyase strain according to claim 3, wherein the recombinant Escherichia coli containing the tyrosine phenol lyase gene is obtained by introducing a gene having a nucleotide sequence represented by SEQ ID NO.1 into Escherichia coli.
5. The method for screening high-throughput strains with high activity of the tyrosine phenol lyase according to claim 1, wherein the fermentation method of the strain to be tested comprises the following steps: inoculating the strain to be tested to a fermentation medium, and carrying out shake cultivation for 6-12h at 20-37 ℃ and 100-; the fermentation medium comprises the following components: 5-20g/L of peptone, 2-10g/L of yeast powder, 2-10g/L of sodium chloride, 10-30g/L of IPTG, water as solvent and natural pH value.
6. The high-throughput screening method of the high-activity tyrosine phenol lyase strain according to claim 5, wherein the strain to be tested is subjected to seed activation culture before fermentation, and then the seed solution is inoculated to the fermentation medium in an inoculum size of 2-5% by volume concentration, and the seed activation culture method comprises: inoculating the strain to be detected to a seed culture medium, and performing shake cultivation at 30-37 ℃ and 100-300rpm for 10-14h to obtain a seed solution; the seed culture medium comprises the following components: 5-20g/L of peptone, 2-10g/L of yeast powder, 2-10g/L of sodium chloride, 50-100 mu g/mL of kanamycin, water as a solvent and natural pH value.
7. The method for high throughput screening of the high-activity strain of the tyrosol lyase according to claim 1, wherein the method comprises performing the reaction in a 96-well plate.
8. The method for high throughput screening of the high-activity tyrosol lyase strain according to claim 7, wherein the method comprises: (1) inoculating a strain to be detected into a deep-hole plate I filled with a seed culture medium, and performing shake cultivation at 30-37 ℃ and 100-300rpm for 10-14h to obtain a seed solution; (2) correspondingly inoculating the seed liquid in the deep-well plate I into a deep-well plate II filled with a fermentation culture medium, and performing shake cultivation at 20-37 ℃ and 100-300rpm for 6-12 h; placing the deep pore plate II in a pore plate centrifuge, centrifuging for 10-30min at 3000 Xg of 1000-; (3) adding 100-; placing the deep pore plate II in a pore plate centrifuge, and centrifuging for 10-30min at 3000 Xg for 1000-; (4) and (3) sequentially and respectively adding 1ml of 250g/L NaOH aqueous solution into a deep-hole plate III, uniformly mixing 200 mu L of supernatant in the step (3), 6.7ml of ultrapure water, 100 mu L of salicylaldehyde and 2ml of 250g/L NaOH aqueous solution to form a 10ml color reaction system, standing at room temperature for 20min-2h for color reaction, detecting the light absorption value at 465nm by using an enzyme labeling instrument, obtaining the content of sodium pyruvate in the supernatant according to a sodium pyruvate standard curve, and screening to obtain the strain with improved activity of the tyrosine phenol lyase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810271360.3A CN108642130B (en) | 2018-03-29 | 2018-03-29 | High-throughput screening method for high-activity strain of tyrosine phenol lyase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810271360.3A CN108642130B (en) | 2018-03-29 | 2018-03-29 | High-throughput screening method for high-activity strain of tyrosine phenol lyase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108642130A CN108642130A (en) | 2018-10-12 |
| CN108642130B true CN108642130B (en) | 2021-10-15 |
Family
ID=63744826
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810271360.3A Active CN108642130B (en) | 2018-03-29 | 2018-03-29 | High-throughput screening method for high-activity strain of tyrosine phenol lyase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108642130B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580604A (en) * | 2018-11-28 | 2019-04-05 | 江南大学 | A kind of method of high-throughput detection naringenin |
| CN109929889B (en) * | 2019-03-29 | 2022-10-21 | 安徽华恒生物科技股份有限公司 | Preparation method of L-tyrosine |
| CN110331153B (en) * | 2019-06-24 | 2021-04-30 | 浙江工业大学 | Kluyveromyces tyrosol lyase mutant and application thereof |
| CN112662732A (en) * | 2020-12-04 | 2021-04-16 | 浙江工业大学 | High-throughput rapid screening method of pantothenate synthetase mutants |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995019169A2 (en) * | 1994-01-07 | 1995-07-20 | Sugen, Inc. | Treatment of platelet derived growth factor related disorders such as cancer using inhibitors of platelet derived growth factor receptor |
| WO2001064842A1 (en) * | 2000-03-02 | 2001-09-07 | Korea Research Institute Of Bioscience And Biotechnology | A novel obligately symbiotic thermophile symbiobacterium toebii sc-1 producing thermostable l-tyrosine phenol-lyase and l-tryptophan indole-lyase and a method for screening its relative bacteria |
| CN102994387A (en) * | 2012-09-17 | 2013-03-27 | 天津工业生物技术研究所 | High throughput screening method of aminopeptidase and high-yield strain thereof |
| CN104232734A (en) * | 2014-09-18 | 2014-12-24 | 中国科学院天津工业生物技术研究所 | High-throughput screening method of high-yield microbial strains for adenosine |
| CN105543332A (en) * | 2016-01-06 | 2016-05-04 | 南阳师范学院 | DAB color development-based high-throughput screening method for CPC histone deacetylases |
| CN105886450A (en) * | 2016-05-04 | 2016-08-24 | 浙江绿创生物科技有限公司 | Trosine phenol lyase engineering bacteria as well as construction method thereof and application thereof |
| CN106318989A (en) * | 2016-11-17 | 2017-01-11 | 山东鲁抗医药股份有限公司 | Fermentation method for preparing levodopa |
| CN106754846A (en) * | 2016-12-02 | 2017-05-31 | 浙江工业大学 | A kind of Fusobacterium nucleatum tyrosine phenol lyase mutant, gene, carrier, engineering bacteria and its application |
| CN107586797A (en) * | 2017-09-25 | 2018-01-16 | 长兴制药股份有限公司 | The method that one pot of enzyme process prepares levodopa |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4513377B2 (en) * | 2004-03-29 | 2010-07-28 | 味の素株式会社 | Mutant tyrosine repressor gene and its use |
-
2018
- 2018-03-29 CN CN201810271360.3A patent/CN108642130B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995019169A2 (en) * | 1994-01-07 | 1995-07-20 | Sugen, Inc. | Treatment of platelet derived growth factor related disorders such as cancer using inhibitors of platelet derived growth factor receptor |
| WO2001064842A1 (en) * | 2000-03-02 | 2001-09-07 | Korea Research Institute Of Bioscience And Biotechnology | A novel obligately symbiotic thermophile symbiobacterium toebii sc-1 producing thermostable l-tyrosine phenol-lyase and l-tryptophan indole-lyase and a method for screening its relative bacteria |
| CN102994387A (en) * | 2012-09-17 | 2013-03-27 | 天津工业生物技术研究所 | High throughput screening method of aminopeptidase and high-yield strain thereof |
| CN104232734A (en) * | 2014-09-18 | 2014-12-24 | 中国科学院天津工业生物技术研究所 | High-throughput screening method of high-yield microbial strains for adenosine |
| CN105543332A (en) * | 2016-01-06 | 2016-05-04 | 南阳师范学院 | DAB color development-based high-throughput screening method for CPC histone deacetylases |
| CN105886450A (en) * | 2016-05-04 | 2016-08-24 | 浙江绿创生物科技有限公司 | Trosine phenol lyase engineering bacteria as well as construction method thereof and application thereof |
| CN106318989A (en) * | 2016-11-17 | 2017-01-11 | 山东鲁抗医药股份有限公司 | Fermentation method for preparing levodopa |
| CN106754846A (en) * | 2016-12-02 | 2017-05-31 | 浙江工业大学 | A kind of Fusobacterium nucleatum tyrosine phenol lyase mutant, gene, carrier, engineering bacteria and its application |
| CN107586797A (en) * | 2017-09-25 | 2018-01-16 | 长兴制药股份有限公司 | The method that one pot of enzyme process prepares levodopa |
Non-Patent Citations (7)
Also Published As
| Publication number | Publication date |
|---|---|
| CN108642130A (en) | 2018-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108642130B (en) | High-throughput screening method for high-activity strain of tyrosine phenol lyase | |
| Zhu et al. | Generation of high rapamycin producing strain via rational metabolic pathway‐based mutagenesis and further titer improvement with fed‐batch bioprocess optimization | |
| CN107475281B (en) | Bioconversion methanol metabolic pathway | |
| Graham et al. | Identification of the 7, 8-didemethyl-8-hydroxy-5-deazariboflavin synthase required for coenzyme F 420 biosynthesis | |
| CN111154704B (en) | Serratia marcescens mutant strain and method for producing histidine by fermentation | |
| US20220282293A1 (en) | Enterobacter chengduensis for producing nicotinamide mononucleotide and application thereof | |
| CN111057729A (en) | (R) -2- (2, 5-difluorophenyl) pyrrolidine and preparation method and application thereof | |
| CN112626100A (en) | Method for high-throughput screening of alpha-keto acid high-yield strains | |
| CN109370998B (en) | Omega-transaminase mutant I215F with improved catalytic efficiency | |
| Liu et al. | Breeding of Gluconobacter oxydans with high PQQ-dependent D-sorbitol dehydrogenase for improvement of 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose production | |
| Taran et al. | Enzymatic transglycosylation of natural and modified nucleosides by immobilized thermostable nucleoside phosphorylases from Geobacillus stearothermophilus | |
| CN112592958B (en) | Method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin | |
| CN109022282A (en) | A kind of screening technique producing marine low temperature urate oxidase bacterial strain | |
| Trentin et al. | Fixing N2 into cyanophycin: continuous cultivation of Nostoc sp. PCC 7120 | |
| CN109486780B (en) | Omega-transaminase mutant with improved catalytic efficiency | |
| WO2023103947A1 (en) | ANCESTRAL SEQUENCE RECONSTRUCTION-BASED ω-TRANSAMINASE MUTANT | |
| Xu et al. | Non-photosynthetic chemoautotrophic CO2 assimilation microorganisms carbon fixation efficiency and control factors in deep-sea hydrothermal vent | |
| CN108277215A (en) | A kind of high activity S- cyanalcohols lyases and its application | |
| CN104535511A (en) | Single enzyme reaction based L-glutamine colorimetric assay method and assay kit | |
| CN116396882A (en) | NRK producing strain, method for producing NRK and application | |
| CN105602913B (en) | Recombinate carbonyl reduction enzyme mutant ReCR-Mut, encoding gene, engineering bacteria and application | |
| Cañizares et al. | Enzymatic interference-free assay for oxalate in urine | |
| CN105505844B (en) | A kind of high-throughput screening method of cytidine high yield microbial strains | |
| CN103487571B (en) | Detection method of acidophilic thermophilic bacterium in fruit juice, and inducer and color development agent of detection method | |
| CN110951706A (en) | Recombinant R-omega-transaminase, mutant and application in asymmetric synthesis of sitagliptin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |