CN110938579A - Recombinant tyrosine ammonia lyase strain, tyrosine ammonia lyase and preparation method and application thereof - Google Patents
Recombinant tyrosine ammonia lyase strain, tyrosine ammonia lyase and preparation method and application thereof Download PDFInfo
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- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01023—Tyrosine ammonia-lyase (4.3.1.23)
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Abstract
The invention provides a tyrosine ammonia lyase recombinant bacterium, a tyrosine ammonia lyase, a preparation method and an application thereof, wherein the construction method of the tyrosine ammonia lyase recombinant bacterium comprises the following steps: extracting a target gene with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2; connecting a target gene to a plasmid to obtain a recombinant plasmid; and introducing the recombinant plasmid into the bacteria to obtain the tyrosine ammonia lyase recombinant bacteria. Aims to provide a tyrosine ammonia lyase recombinant strain, a tyrosine ammonia lyase, a preparation method and application thereof, and the tyrosine ammonia lyase has high conversion efficiency and good stability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a tyrosine ammonia lyase recombinant strain, a tyrosine ammonia lyase, a preparation method and an application thereof.
Background
TAL (Tyrosine ammonia lyase) is an important phenylpropanoid compound precursor conversion key enzyme, can efficiently convert Tyrosine into cinnamic acid, and realizes further product application. TAL is found in monocots such as sorghum, barley, wheat, oats, rice, corn, sweet sugarcane, parsley, and the like. Among the microorganisms, basidiomycetes, rhodobacter capsulatus, actinomycetes, rhodotorula glutinis and hyphomycete have been found. Due to the limitation of cinnamic acid content in nature, the biotransformation of the phenylpropane is limited in metabolism, and the application and development work of the phenylpropanoid active compounds is further limited. Therefore, it is required to develop an enzyme which promotes the metabolism of phenylpropane and thus improves the yield of the product.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the tyrosine ammonia lyase recombinant strain, the tyrosine ammonia lyase, the preparation method and the application thereof, and the tyrosine ammonia lyase has high conversion efficiency and good stability.
The purpose of the invention is realized by adopting the following technical scheme:
the invention provides a tyrosine ammonia lyase recombinant strain, which is a recombinant plasmid with a nucleotide sequence shown as SEQ ID NO.1 and/or SEQ ID NO. 2.
In a specific embodiment, the construction method of the tyrosine ammonia lyase recombinant bacteria comprises the following steps:
extracting a target gene with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2; connecting a target gene to a plasmid to obtain a recombinant plasmid; and introducing the recombinant plasmid into the bacteria to obtain the tyrosine ammonia lyase recombinant bacteria.
In one embodiment, the target gene is ligated to a plasmid to obtain a recombinant plasmid, comprising the steps of: amplifying a target gene and connecting the amplified target gene to a first plasmid to obtain a recombinant vector; carrying out enzyme digestion treatment on the recombinant vector to obtain a target gene fragment; and connecting the target gene fragment to a second plasmid to obtain a recombinant plasmid.
The invention also provides a preparation method of the tyrosine ammonia lyase, which comprises the steps of culturing the recombinant tyrosine ammonia lyase described in any embodiment to obtain thalli; crushing the thalli, performing centrifugal separation, and collecting supernatant; and separating and purifying the supernatant to obtain the tyrosine ammonia lyase.
In one embodiment, the method for culturing the tyrosine ammonia lyase recombinant bacteria to obtain the thallus comprises the following steps: in LB medium containing 5-70. mu.g/mL kanamycin, the culture is expanded to OD at 100-500 rpm and 25-45 ℃600When the concentration reaches 0.4-0.6, adding 80-320 mu M isopropyl- β -D-1-thiogalactopyranoside into the culture medium, and continuously culturing for 8-16 hours to obtain the thalli.
The invention also provides tyrosine ammonia lyase prepared by the preparation method of tyrosine ammonia lyase in any embodiment.
The invention also provides application of the tyrosine ammonia lyase in conversion of phenylpropanoid compound precursors.
In one embodiment, 0.01-0.05 mol volume of first tyrosine ammonia lyase and/or second tyrosine ammonia lyase is added into a phosphate buffer solution with the pH value of 7.0-12.0, 40-60 mol volume of glycine alkaline buffer solution and 10-20 mol volume of tyrosine or phenylalanine are added, the mixture is uniformly mixed and cultured for not less than 1 hour at the temperature of 30-70 ℃, then the mixture is treated in a boiling water bath for 8-15 min, and then the centrifugal treatment is carried out to obtain supernatant fluid, so as to obtain the 3- (4-hydroxyphenyl) -2-acrylic acid or 3-phenyl-2-acrylic acid.
The invention also provides application of the tyrosine ammonia lyase recombinant bacteria in conversion of phenylpropanoid compound precursors.
In a specific embodiment, the first tyrosine ammonia lyase recombinant bacterium and/or the second tyrosine ammonia lyase recombinant bacterium are cultured in an LB culture medium containing 5-70 mu g/mL kanamycin at 100-500 rpm and 25-45 ℃ until OD is achieved600When reaching 0.4-0.6, parallel toAdding 80-320 mu M isopropyl- β -D-1-thiogalactopyranoside into a culture medium, continuously culturing for 8-16 hours, separating and cleaning, collecting recombinant bacteria, transferring the recombinant bacteria into a phosphate buffer solution with the pH of 7-12, adding 0.01-0.05 molar volume of tyrosine or phenylalanine, uniformly mixing, reacting for 4-8 hours at the temperature of 28-37 ℃ at 100-500 rpm, adding 100-300 molar volume of hydrochloric acid into the reaction solution, centrifuging, and taking supernatant to obtain 3- (4-hydroxyphenyl) -2-acrylic acid or 3-phenyl-2-acrylic acid.
Compared with the prior art, the invention has the beneficial effects that:
the invention firstly extracts the target gene of the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2, obtains recombinant plasmid by PCR amplification and connecting to plasmid, and introduces the recombinant plasmid into escherichia coli to obtain the tyrosine ammonia lyase recombinant bacteria. And carrying out amplification culture, separation and purification on the obtained tyrosine ammonia lyase recombinant bacteria to obtain the tyrosine ammonia lyase.
In addition, both the tyrosine ammonia lyase recombinant bacteria and the tyrosine ammonia lyase have high-efficiency catalytic action on the phenylpropanoid compounds, and the production is easy to operate, good in stability and suitable for large-scale industrial production.
Drawings
FIG. 1 is an electrophoretogram of tyrosine ammonia lyase and a recombinant plasmid of the present invention;
FIG. 2 is a pH adaptation curve of the tyrosine ammonia lyase of the present invention;
FIG. 3 is a temperature adaptation curve of the tyrosine ammonia lyase of the invention;
FIG. 4 shows the liquid phase detection of the conversion of tyrosine to 3- (4-hydroxyphenyl) -2-propenoic acid product according to the present invention;
FIG. 5 shows the liquid phase detection of the product of the present invention for the conversion of phenylalanine to 3-phenyl-2-propenoic acid.
Detailed Description
The present invention will now be described in more detail with reference to the accompanying drawings, in which the description of the invention is given by way of illustration and not of limitation. The various embodiments may be combined with each other to form other embodiments not shown in the following description.
The invention also provides a recombinant plasmid which contains one or two tyrosine ammonia lyase expression genes with sequences shown as SEQ ID NO.1 or SEQ ID NO. 2.
The invention provides a tyrosine ammonia lyase recombinant bacterium, which is provided with a recombinant plasmid with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2, namely, the tyrosine recombinant bacterium comprises one or two recombinant expression plasmids for expressing one or two tyrosine ammonia resolvases.
Specifically, TAL (Tyrosine ammonia lyase) is an important phenylpropanoid compound precursor conversion key enzyme, and can efficiently convert Tyrosine into cinnamic acid, thereby realizing further product application. The invention analyzes and searches approximate genome of the gene of known TAL source strain, screens and confirms the approximate gene sequence in the gene library, and clones, copies and expands the strain sequence confirmed to have the corresponding gene sequence. Experimental research shows that the gene sequences with potential TAL approximate functions in the whole genome of Saccharothrix sp.NRRL B-16348 and Streptomyces sp.NRRL F-4489, and the suspected sequences are subjected to clone amplification and expression (engineering bacteria) by designing primer sequences and are tested by using substrates, so that the mutant strains are determined to have corresponding substrate conversion functions (tyrosine and phenylalanine conversion) and have better efficiency.
The invention provides a construction method of a tyrosine ammonia lyase recombinant strain, which comprises the following steps: extracting a target gene with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2; connecting a target gene to a plasmid to obtain a recombinant plasmid; and introducing the recombinant plasmid into the bacteria to obtain the tyrosine ammonia lyase recombinant bacteria. Wherein, the target gene is connected to the plasmid to obtain the recombinant plasmid, and the method comprises the following steps: amplifying a target gene and connecting the amplified target gene to a first plasmid to obtain a recombinant vector; carrying out enzyme digestion treatment on the recombinant vector to obtain a target gene fragment; and connecting the target gene fragment to a second plasmid to obtain a recombinant plasmid.
Specifically, the construction method of the tyrosine ammonia lyase recombinant bacteria comprises the following steps: (1) extracting genes: using a Quick-DNA complete sequence extraction kit (Zymo Research, USA) to extract a complete gene sequence from the saccharothrix strain and obtain a first target gene Sas-TAL, wherein the nucleotide sequence is shown as SEQ ID NO. 1; or using a Quick-DNA complete sequence extraction kit (Zymo Research, USA) to extract a complete gene sequence from the streptomyces strain and obtain a second target gene Sts-TAL, wherein the nucleotide sequence is shown as SEQ ID NO. 2.
(2) Constructing a plasmid: performing PCR amplification on the first target gene Sas-TAL, wherein the primer sequence for performing PCR amplification on the first target gene with the nucleotide sequence shown as SEQ ID NO.1 is as follows:
an upstream primer: AACATATGACCGACGCCCCCGTGGACCT the flow of the air in the air conditioner,
a downstream primer: TAAAGCTTCTCGAGTCAGTACCGGCCCCCGGTGACCG, wherein the amplification system is front end primer 1 μ L, whole gene sequence 0.2 μ L, dNTP mixed liquor 200 μ M, 5 × concentrated buffer 4 μ L, dimethyl sulfoxide 0.4 μ L, high fidelity DNA ligase 0.2 μ L (2U/μ L), adding ultrapure water to constant volume to 20 μ L amplification system; the amplification reaction condition is that the pre-denaturation treatment is carried out for 5min at the temperature of 98 ℃; performing denaturation treatment at 98 deg.C for 40s, at 56 deg.C for 40s, at 72 deg.C for 3.5min, and circulating for 30 groups; then the extension treatment is carried out for 10min at the temperature of 72 ℃. The method comprises the steps of mixing an amplified gene sequence with 0.8% agar Gel by using a first plasmid pJET1.2 and a GeneJET Gel extraction kit (Samefei) to obtain a first recombinant vector pJET1.2-Sas-TAL, carrying out enzyme digestion treatment on the first recombinant vector by using restriction enzymes aiming at enzyme digestion sites NdeI and HindIII to obtain a first target gene fragment, and connecting the first gene fragment to a corresponding site of a second plasmid pET28a of a receptor plasmid to form a target first recombinant plasmid pET28 a-Sas-TAL.
Performing PCR amplification on the second target gene Sts-TAL, wherein the primer sequence for performing PCR amplification on the second target gene with the nucleotide sequence shown as SEQ ID NO.2 is as follows: an upstream primer: AACATATGCCGAGTCTGGACAGCAT, downstream primer: TAAAGCTTCTCGAGTCAGGCGGCGCCGGTCAGCT are provided. Wherein the amplification system is 1 muL of front-end primer, 0.2 muL of whole gene sequence, 200 muM of dNTP mixed solution, 4 muL of 5 Xconcentrated buffer solution, 0.4 muL of dimethyl sulfoxide, 0.2 muL (2U/muL) of high-fidelity DNA ligase, and the amplification system is fixed to the volume of 20 muL by adding ultrapure water; the amplification reaction condition is that the pre-denaturation treatment is carried out for 5min at the temperature of 98 ℃; performing denaturation treatment at 98 deg.C for 40s, at 56 deg.C for 40s, at 72 deg.C for 3.5min, and circulating for 30 groups; then the extension treatment is carried out for 10min at the temperature of 72 ℃. Mixing the amplified gene sequence with 0.8% agar Gel by using a first plasmid pJET1.2 and a GeneJET Gel extraction kit (Samerfei) to obtain a second recombinant vector pJET1.2-Sts-TAL, carrying out enzyme digestion treatment on the second recombinant vector by using restriction enzymes aiming at enzyme digestion sites NdeI and HindIII to obtain a second target gene fragment, and connecting the second gene fragment to a corresponding site of a second plasmid pET28a of a receptor plasmid to form a target second recombinant plasmid pET28 a-Sts-TAL.
(3) Constructing a recombinant bacterium: introducing the first recombinant plasmid into receptor cells such as an escherichia coli vector DE3 by using a polydiethanol-lithium acetate mediated method to obtain a first tyrosine ammonia lyase recombinant bacterium DE 3-Sas-TAL; introducing the second recombinant plasmid into escherichia coli DE3 by using a polydiethanol-lithium acetate mediated method to obtain a second tyrosine ammonia lyase recombinant bacterium DE 3-Sts-TAL;
the invention also provides a preparation method of the tyrosine ammonia lyase, namely a separation and acquisition method of the Tyrosine Ammonia Lyase (TAL) active enzyme, which comprises the following steps: culturing the tyrosine ammonia lyase recombinant bacteria to obtain thalli, crushing the thalli, performing centrifugal separation, and collecting supernatant; and separating and purifying the supernatant to obtain the tyrosine ammonia lyase. Specifically, the first tyrosine ammonia lyase recombinant bacterium and/or the second tyrosine ammonia lyase recombinant bacterium are subjected to amplification culture in an LB culture medium containing 5-70 mu g/mL kanamycin at 100-500 rpm and 25-45 ℃ until OD is achieved600Adding 80-320 mu M isopropyl- β -D-1-thiogalactopyranoside into the culture medium when the concentration reaches 0.4-0.6, continuously culturing for 8-16 hours to obtain thalli, crushing the thalli, performing centrifugal separation, collecting supernatant, and performing Ni affinity on the supernatantAnd purifying to obtain a first tyrosine ammonia lyase and a second tyrosine ammonia lyase.
Specifically, the first tyrosine ammonia lyase recombinant bacterium and/or the second tyrosine ammonia lyase recombinant bacterium are cultured in LB broth medium added with kanamycin (50 mu g/mL), and are subjected to amplification culture at the conditions of 250rpm and 37 ℃ until OD is reached600Adjusting the culture temperature to 28 deg.C when the temperature reaches 0.4-0.6, adding 200 μ M isopropyl- β -D-1-thiogalactopyranoside into the culture medium, continuously culturing for 12 hr to obtain thallus, crushing thallus obtained by the above culture, centrifuging at 3000 Xg and 4 deg.C for 10min, washing the obtained solid with distilled water for 2 times, re-dissolving in 20mM tris (hydroxymethyl) aminomethane hydrochloric acid solution (pH 7.9, adding 0.5M sodium chloride), subjecting the re-dissolved solution to ultrasonic lysis in ice bath, centrifuging the lysate again, centrifuging at 10000 Xg and 4 deg.C for 10min, collecting supernatant, and subjecting the supernatant to sHiurTMThe Ni-NTA affinity separation column (Thermo Scientific, Rockford, USA) is used for separation and purification, the specific operation can be carried out according to the instruction of the equipment, and purified active proteases Sas-TAL and Sts-TAL are obtained respectively. Carrying out electrophoretic analysis on the obtained tyrosine ammonia lyase Sas-TAL and Sts-TAL, wherein the result is shown in figure 1, and A is the electrophoretic identification of the sequence of the tyrosine ammonia lyase Sas-TAL; b is electrophoretic identification of a Sts-TAL sequence of tyrosine ammonia lyase; in the C, 3 is pET28a-Sas-TAL recombinant plasmid sequence identification, and 4 is pET28a sequence identification; in D, 5 is pET28a-Sts-TAL recombinant plasmid sequence identification, and 6 is pET28a sequence identification. Testing the temperature adaptation range and the pH adaptation range of the tyrosine ammonia lyase Sas-TAL and the Sts-TAL, wherein the temperature adaptation range of the tyrosine ammonia lyase Sas-TAL and the Sts-TAL in vitro is 30-70 ℃ as shown in figure 2 and figure 3 respectively; the pH adaptation range of the tyrosine ammonia lyase Sas-TAL and the Sts-TAL in vitro is 7-12.
The invention also provides tyrosine ammonia lyase prepared by the preparation method of the tyrosine ammonia lyase. The invention also provides an application of the tyrosine ammonia lyase in the conversion of the phenylpropanoid compound precursor, which adopts a purification protease in-vitro conversion method and utilizes one or two of the tyrosine ammonia lyase Sas-TAL and Sts-TAL to prepare corresponding products. Wherein the phenylpropanoid compounds can be tyrosine, phenylalanine, etc.
In one embodiment, the preparation of the corresponding product is carried out in vitro with the tyrosine ammonia lyase active enzyme: adding 0.01-0.05 mol volume of first tyrosine ammonia lyase and/or second tyrosine ammonia lyase into a phosphate buffer solution with the pH value of 7.0-12.0, adding 40-60 mol volume of glycine alkaline buffer solution and 10-20 mol volume of tyrosine, uniformly mixing, culturing for not less than 1 hour at the temperature of 30-70 ℃, treating for 8-15 min in a boiling water bath, and then carrying out centrifugal treatment to obtain a supernatant to obtain 3- (4-hydroxyphenyl) -2-acrylic acid;
or adding 0.01-0.05 mol volume of first tyrosine ammonia lyase and/or second tyrosine ammonia lyase into a phosphate buffer solution with the pH value of 7.0-12.0, adding 40-60 mol volume of glycine alkaline buffer solution and 10-20 mol volume of phenylalanine, uniformly mixing, culturing for not less than 1 hour at the temperature of 30-70 ℃, treating in a boiling water bath for 8-15 min, and then carrying out centrifugal treatment to obtain a supernatant, thus obtaining the 3-phenyl-2-acrylic acid.
Specifically, 6 mu M of first tyrosine ammonia lyase and/or 3 mu M of second tyrosine ammonia lyase are put in a phosphate buffer solution system with 200 mu L, pH of 7.0-12.0, and the pH is preferably 11. Then, 10mM glycine basic buffer solution or potassium chloride basic buffer solution and 3mM tyrosine were added to the system, and the mixture was mixed well and incubated at 30 ℃ to 70 ℃ for 1 hour, preferably 50 ℃. Then, the mixture was treated in a boiling water bath for 10min, and the mixture was centrifuged at 130,000 Xg for 15min to obtain a supernatant. Performing High Performance Liquid Chromatography (HPLC) on the supernatant to detect and identify, and confirming to obtain 3- (4-hydroxyphenyl) -2-acrylic acid, wherein the HPLC detection result is shown in FIG. 4, wherein (i) is tyrosine liquid phase detection; (iii) liquid phase detection is carried out on the product of the conversion outside the active enzyme; (iv) is a liquid phase detection of a 3- (4-hydroxyphenyl) -2-acrylic acid standard substance.
Or, 6 μ M of the first tyrosine ammonia lyase and/or 3 μ M of the second tyrosine ammonia lyase are placed in 200 μ L, pH phosphate buffer solution of 7.0-12.0, preferably pH 11, 10mM glycine alkaline buffer solution or potassium chloride alkaline buffer solution and 3mM phenylalanine are added and mixed uniformly, the mixture is incubated for 1 hour at 30-70 ℃, preferably 50 ℃, then treated in boiling water bath for 10min, and then centrifuged to obtain supernatant. Detecting and identifying the supernatant by High Performance Liquid Chromatography (HPLC) to obtain the 3-phenyl-2-acrylic acid. The HPLC detection results are shown in FIG. 5, in which (i) phenylalanine liquid phase detection; (ii) liquid phase detection of the product of the conversion in the active enzyme; (iii) liquid phase detection of the active enzyme external conversion product; (iv) and (3) detecting the liquid phase of the 3-phenyl-2-acrylic acid standard substance.
The invention also provides an application of the tyrosine ammonia lyase recombinant bacteria in the conversion of phenylpropanoid compound precursors, a recombinant cell in-vivo conversion method, and the preparation of corresponding products is carried out by using one or two of the tyrosine ammonia lyase recombinant bacteria DE3-Sas-TAL and DE 3-Sts-TAL; wherein the phenylpropanoid compounds can be tyrosine, phenylalanine, etc. Culturing the first tyrosine ammonia lyase recombinant bacteria and/or the second tyrosine ammonia lyase recombinant bacteria in an LB culture medium containing 5-70 mu g/mL kanamycin at 100-500 rpm and 25-45 ℃ until OD is reached600And when the concentration reaches 0.4-0.6, adding 80-320 mu M isopropyl- β -D-1-thiogalactopyranoside into a culture medium, continuously culturing for 8-16 hours, separating and cleaning, collecting recombinant bacteria, transferring the recombinant bacteria into a phosphate buffer solution with the pH of 7-12, adding 0.01-0.05 molar of tyrosine, uniformly mixing, reacting for 4-8 hours at the temperature of 28-37 ℃ at 100-500 rpm, adding 100-300 molar of hydrochloric acid into a reaction solution, centrifuging, and taking a supernatant to obtain the 3- (4-hydroxyphenyl) -2-acrylic acid.
Or culturing the first tyrosine ammonia lyase recombinant bacteria and/or the second tyrosine ammonia lyase recombinant bacteria in an LB culture medium containing 5-70 mu g/mL kanamycin at the rpm of 100-500 and the temperature of 25-45 ℃ until OD is reached600When the pH value reaches 0.4-0.6, adding 80-320 mu M isopropyl- β -D-1-thiogalactopyranoside into the culture medium, continuously culturing for 8-16 hours, separating and cleaning, collecting recombinant bacteria, and transferring the recombinant bacteria into phosphate with the pH value of 7-12Adding phenylalanine with the molar volume of 0.01-0.05 into the buffer solution, uniformly mixing, and reacting for 4-8 hours at the temperature of 28-37 ℃ at the rpm of 100-500; and adding 100-300 mol (such as 300 mol) of hydrochloric acid into the reaction solution, centrifuging, and taking supernatant to obtain the 3-phenyl-2-acrylic acid.
Specifically, the first tyrosine ammonia lyase recombinant bacteria and/or the second tyrosine ammonia lyase recombinant bacteria are cultured in LB culture medium containing 50 mu g/mL kanamycin at 250rpm and 37 ℃ until OD is reached600Adjusting the culture temperature to 28 ℃ when the temperature reaches 0.4-0.6 ℃, adding 200 mu M isopropyl- β -D-1-thiogalactopyranoside into the culture medium, continuously culturing for 12 hours, then centrifuging the culture at 3,000 Xg and 4 ℃ for 10min to obtain a solid, washing the solid with distilled water for 2 times, transferring the product into a phosphate buffer solution system containing 10mM and 10mL and having a pH of 7-12, preferably having a pH of 8, adding 3mM tyrosine into the system, uniformly mixing, reacting at 250rpm and 28 ℃ for 6 hours, adding 3M hydrochloric acid into the reaction solution, centrifuging the mixture at 130,000 Xg for 15min to obtain a supernatant, carrying out detection and identification on the supernatant by High Performance Liquid Chromatography (HPLC) to obtain 3- (4-hydroxyphenyl) -2-acrylic acid, wherein the detection result of the HPLC liquid phase is shown in figure 4, wherein (ii) is the detection of the in vivo conversion product of the active enzyme;
or culturing the first tyrosine ammonia lyase recombinant bacteria and/or the second tyrosine ammonia lyase recombinant bacteria in LB culture medium containing 50 mug/mL kanamycin respectively at 250rpm and 37 ℃ until OD is reached600Adjusting the culture temperature to 28 ℃ when the temperature reaches 0.4-0.6, adding 200 mu M isopropyl- β -D-1-thiogalactopyranoside into the culture medium, continuously culturing for 12 hours, then centrifugally separating the culture at 3,000 Xg and 4 ℃ for 10min to obtain a solid, washing the solid with distilled water for 2 times, transferring the product into a phosphate buffer solution system containing 10mM and 10mL and having a pH of 7-12, preferably having a pH of 8, adding 3mM phenylalanine into the system, uniformly mixing, reacting for 6 hours at 250rpm and 28 ℃, adding 3M hydrochloric acid into the reaction solution, and then adding 3M hydrochloric acid into the reaction solutionThe mixture was centrifuged at 130,000 Xg for 15min to obtain a supernatant. Detecting and identifying the supernatant by High Performance Liquid Chromatography (HPLC) to obtain the 3-phenyl-2-acrylic acid. The HPLC assay results are shown in FIG. 5, in which (ii) the liquid phase assay of the product of the active enzymatic conversion is performed.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Sequence listing
<110> Hangzhou platinum only Biotechnology Ltd
<120> tyrosine ammonia lyase recombinant strain, tyrosine ammonia lyase and preparation method and application thereof
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>1485
<212>DNA
<213> Saccharothrix (Saccharothrix)
<400>1
gtgaccgacg cccccgtgga cctcggcgca ccgctgagga gcgaccacct ccaccgtgcc 60
gccgcgccgc taccggtgct cgtcagcccc gtcgtccggg aacgggtcga ccggggccgc 120
gagtacctgc gcacggtgct ggccgacgac gatcggcccg tctacggcgc caagaccggg 180
ttcggcgccc tgatcggctt cgcgggccgt gaggacgaag ccgaccaatg cgacaacacc 240
ctcgcacacc tcggcgcggg ccaagggccc gacctgcccg ccgacatcgc ccgcgccgcg 300
ctgctcgtgc gggcgtggtc gctcgcccag ggcgtctcgg gcgtgtccgc ggacgtgttg 360
gacgggctcg cggcgatgtt cgcgaccacg ttcgtgccgg cgatcccgcg ttacggctcg 420
gtcggcgcga gcggcgacct catcccgctc gcctacgcga cccaggcgtt gcgcggccgc 480
ggtcacgcct acctggacgg cgagcggatg cccgcggacg acgccctgcg ccgtgccggc 540
ctcacgccgc tcaccctcga cgggcgtgac gcgctggcgc tcgtcaacgg cacgtcggtc 600
acgaccgccg ccgcggctct cgcgctggac agcgtgcgcg cctcgcaccg tgcgatccag 660
gacttgacct gcctgctcgt ggacgtcgtc ggcggtgacc ccggtttcct cgacccacgg 720
ctgatctccg cgtacgggca ccccggcgcg gccaccgtcg ccgagcgcat gcgcgccacc 780
ctcgcgggca cggtgccgac cggtgagcgt gcgctccagg agccctacag catccggtgc 840
tcgccgcagc tgctcggcgc ggccgaggac gcgttgcgct acgtcgacgg cgtggtggcg 900
gccgacctgg gcggtgtcag cgacaacccg ctgttcttcc ccggtgacga cctcgtcgcg 960
cacggcggca acttcttcgg ccagccggcc gcgttcgccg ccgacgtgct ggccatggtc 1020
acggcgtcgt tgggcaacct cgccgaacgg cagctggacc tgctggtcga cccggtgcgc 1080
aacaccggcc tgccgccgat gctggcggcc ggacccggac gtcagcacgg gttgcagggc 1140
gtgcagctgg ccacgaccgc gttcgtcgtg gagatccgcc gcgcggcact gcccgcgagc 1200
acccagagcc tgccgacgaa cctgcacaac caggacgtcg tgccgttcgg cacgcaggcc 1260
gcgctgcgcg cctacgacgt cgcagaactg ctccggctgc tgtgcggctc gctggcgctc 1320
ggcctgcggc aggcggtcca cgtgggaggc cgccgaccga ccgcgccccg ctgcgccgct 1380
ctgctcgacg ccctcgccga cgcgatcgcc ccggtcgacc cggaccgccc gctcgacgcg 1440
gacgtgcggg cggccgccga cctggtcacc gggggccggt actga 1485
<210>2
<211>1479
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<213> Streptomyces (Streptomyces)
<400>2
atgccgagtc tggacagcat cgtcgaggcc gcctcctgga ccgccaagct cggcccgctc 60
accgacgccg acgtggcgcg gatggaccgc agcggcgcca ccgtcgacgc gtacctcgcc 120
gagggccggc ccgtctacgg gctcacccag ggcttcggcc cgctggtgac gtactccgcc 180
acctccgaga tggagcaggg cgcctcgctc atctcccacc tcggcaccgc ccagggccgc 240
ccgatcgacc cggacgcctc ccggctggtg ttctggctgc ggctgaacag catgcgcaag 300
ggcttctccg cggtctccac ggagttctgg cagcgcctcg ccgacctgtg gaacgccggc 360
ttcaccccgg tcatcccgcg cgacggcacg gtcagcgcga gcggcgacct ccagccgctg 420
gcgcatgtcg cgctggcctg cgccgggcac ggcgaggcgt gggtccgcga cgagcaggac 480
cgctggaccc gccgcccggc cgccgaggcc ctcgccgggc tcggcgccga accgctcgtc 540
tggccggtgc gcgaggcgct cgccttcgtc aacggcaccg gcgtcggcct ggccgtcgcc 600
atcctcaacc agcgctccgc ggtccgcctg gtccgcgccg tcgccaccct caccgcccgc 660
ctgaccgacc tgctcggcgg caacgcggag cactacgacg agggcgtcgg ccaggcccgc 720
aaccagctcg gccagctgga ggtggcccgc tggatccgcg ccgaaatccc cgccggccac 780
cgccgcgacg agcgccgccc gctccaggag ccgtacagcc tgcgctgcgc cccgcaggtg 840
ctcggcgccg tcctggacca gctcaccacc gccggcgaga tcctgctccg cgaggccaac 900
ggctgcaccg acaaccccct cacctacgag gaccgggtgc tgcacgccgg caacttccac 960
gcgatgccgg tcggtttcgc ctccgagcag accggtctgg ccatgcacat ggccgcgtat 1020
ctggcggagc gtcagctcgg gctggtggtc aaccccacca ccaacggcga cctgccgatc 1080
atgctcaccc cgcgggccgg ccgcggctgc ggtctggccg gtgtgcagat cagcgcgacc 1140
tcgttcatct cccggatccg ccagctggtc acccccgcct cgctgaccac cctgccgacc 1200
aacggctgga atcaggacca cgtcccgatg gccctcaacg gcgccaacgg cgtcggcgag 1260
gccctggagc tgggctggct cgccgtcggc tcgctcgcgc tggccgccgc ccagctggcc 1320
gtgatgaccg gcaaggcgga gtccgccacc ggcgtctggg ccgaactcgc ccggatctcc 1380
cccgcgttgg acgccgaccg cccgatggcc ggcgaggtgc gcgccgccgc cgagctgttc 1440
cgcgaccacg cggagcgcca gctgaccggc gccgcctga 1479
Claims (10)
1. The tyrosine ammonia lyase recombinant strain is characterized in that the tyrosine ammonia lyase recombinant strain is a recombinant plasmid with a nucleotide sequence shown as SEQ ID NO.1 and/or SEQ ID NO. 2.
2. The method for constructing the recombinant tyrosine ammonia lyase as claimed in claim 1, which comprises the following steps:
extracting a target gene with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2;
connecting a target gene to a plasmid to obtain a recombinant plasmid;
and introducing the recombinant plasmid into the bacteria to obtain the tyrosine ammonia lyase recombinant bacteria.
3. The method for constructing the recombinant tyrosine ammonia lyase as claimed in claim 2, wherein the step of connecting the target gene to a plasmid to obtain a recombinant plasmid comprises the following steps:
amplifying a target gene and connecting the amplified target gene to a first plasmid to obtain a recombinant vector;
carrying out enzyme digestion treatment on the recombinant vector to obtain a target gene fragment;
and connecting the target gene fragment to a second plasmid to obtain a recombinant plasmid.
4. A method for producing tyrosine ammonia lyase, characterized by culturing the recombinant tyrosine ammonia lyase of claim 1 to obtain a cell; crushing the thalli, performing centrifugal separation, and collecting supernatant; and separating and purifying the supernatant to obtain the tyrosine ammonia lyase.
5. The method for producing tyrosine ammonia lyase according to claim 4, wherein the culturing of the recombinant tyrosine ammonia lyase to obtain a cell comprises the steps of: in LB medium containing 5-70. mu.g/mL kanamycin, the culture is expanded to OD at 100-500 rpm and 25-45 ℃600When the concentration reaches 0.4-0.6, adding 80-320 mu M isopropyl- β -D-1-thiogalactopyranoside into the culture medium, and continuously culturing for 8-16 hours to obtain the thalli.
6. A tyrosine ammonia lyase prepared by the method for preparing the tyrosine ammonia lyase of claim 4 or 5.
7. Use of the tyrosine ammonia lyase of claim 6 for the conversion of a precursor of a phenylpropanoid compound.
8. The application of the tyrosine ammonia lyase in the conversion of the phenylpropanoid compound precursor as claimed in claim 7, wherein the tyrosine ammonia lyase is added into a phosphate buffer solution with the pH value of 7.0-12.0 in an amount of 0.01-0.05 mol, a glycine alkaline buffer solution in an amount of 40-60 mol and tyrosine or phenylalanine in an amount of 10-20 mol are added, the mixture is uniformly mixed and cultured at the temperature of 30-70 ℃ for not less than 1 hour, then the mixture is treated in a boiling water bath for 8-15 min and then centrifuged to obtain a supernatant, and 3- (4-hydroxyphenyl) -2-acrylic acid or 3-phenyl-2-acrylic acid is obtained.
9. The use of the recombinant tyrosine ammonia lyase strain of claim 1 in the transformation of phenylpropanoid compound precursors.
10. The recombinant tyrosine ammonia lyase strain as claimed in claim 9, which is used for conversion of phenylpropanoid compound precursorThe application is characterized in that the first tyrosine ammonia lyase recombinant bacteria and/or the second tyrosine ammonia lyase recombinant bacteria are cultured in an LB culture medium containing 5-70 mu g/mL kanamycin at the rpm of 100-500 and the temperature of 25-45 ℃ until OD is reached600Adding 80-320 mu M isopropyl- β -D-1-thiogalactopyranoside into a culture medium when the concentration reaches 0.4-0.6, continuously culturing for 8-16 hours, separating and cleaning, collecting recombinant bacteria, transferring the recombinant bacteria into a phosphate buffer solution with the pH value of 7-12, adding 0.01-0.05 molar volume of tyrosine or phenylalanine, uniformly mixing, reacting for 4-8 hours at the temperature of 28-37 ℃ at 100-500 rpm, adding 100-300 molar volume of hydrochloric acid into a reaction solution, centrifuging, and taking a supernatant to obtain 3- (4-hydroxyphenyl) -2-acrylic acid or 3-phenyl-2-acrylic acid.
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| CN116731945A (en) * | 2023-07-10 | 2023-09-12 | 苏州优信合生技术有限公司 | A recombinant Escherichia coli expressing tyrosine ammonia lyase SeSam8 and its application |
| CN116855434A (en) * | 2023-07-10 | 2023-10-10 | 苏州优信合生技术有限公司 | A recombinant Escherichia coli and its application in preparing tyrosinemia drugs |
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