CN118726326A

CN118726326A - A phenolic acid decarboxylase mutant and its preparation and application

Info

Publication number: CN118726326A
Application number: CN202410754413.2A
Authority: CN
Inventors: 任鑫坤; 甘忠文; 曹家南; 董佳敏; 王子妍
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2024-06-12
Filing date: 2024-06-12
Publication date: 2024-10-01
Anticipated expiration: 2044-06-12
Also published as: CN118726326B

Abstract

The invention discloses a phenolic acid decarboxylase mutant and preparation and application thereof, wherein the mutant is obtained by mutating at least one amino acid in the 40 th site, 92 th site and 131 th site of an amino acid sequence shown in SEQ ID No. 1; isoleucine Ile at position 40 to valine Val; isoleucine Ile at position 92 to threonine Thr, valine Val, or any one of them; valine Val at position 131 is mutated to leucine Leu. The phenolic acid decarboxylase mutant is modified by a site-directed mutagenesis method, so that the amino acid sequence is changed, the change of the structure and the function of the protein is realized, the enzyme activity is improved, and the catalytic activity of ferulic acid is greatly improved relative to that of the original enzyme.

Description

Phenolic acid decarboxylase mutant and preparation and application thereof

Technical Field

The invention relates to the technical fields of enzyme engineering and genetic engineering, in particular to a phenolic acid decarboxylase mutant and preparation and application thereof.

Background

The chemical name of the 4-vinyl guaiacol (4-vinylguaiacol, 4 VG) is 2-methoxy-4-vinyl phenol, has volatility, unique fermentation aroma and higher olfactory recognition degree, and is an important flavor substance in soy sauce, white spirit, wine and beer. GB 2760-1996 specifies that 4VG is a food flavor that allows use, while natural 4VG is also used as an intermediate in the production of natural vanillin.

The existing production methods of 4-vinyl guaiacol include chemical methods and biological transformation methods. For example, chinese patent publication No. CN101885669a discloses a "process for preparing 4-vinylguaiacol", which comprises reacting quinoline with ferulic acid at high temperature in the presence of a blocking agent p-diphenol, then repeatedly washing with hydrochloric acid and benzene, and finally refining to volatilize benzene to obtain 4-vinylguaiacol. The chemical process is relatively complex in steps, the process involves a number of organic solvents, and the chemically produced 4-vinylguaiacol cannot be used to produce high value-added natural fragrances downstream. For example, chinese patent publication No. CN110184315B discloses a "method for preparing high concentration 2-methoxy-4 vinylphenol", which uses recombinant escherichia coli containing phenolic acid decarboxylase BaPAD derived from bacillus atrophaeus as a catalyst, converts ferulic acid into 4-vinylphenol by non-oxidative decarboxylation in the presence of water/n-octanol two phases, solving the problem that phenolic acid decarboxylase has low activity in the presence of higher concentration 4-vinylguaiacol. The bioconversion process for producing 4-vinylguaiacol requires a large amount of organic solvent for extraction, which increases the cost of production and generates a large amount of waste liquid during production and product purification.

Natural 4-vinylguaiacol is a precursor substance for producing high-value-added natural compound vanillin, and thus it is required to enhance the yield of natural 4-vinylguaiacol produced by a biological method in a conventional aqueous phase to satisfy industrial production. Under the conventional reaction conditions, the enzyme activity is low, and the conversion rate is low, which is an important factor for limiting the production of natural 4-vinyl guaiacol.

Disclosure of Invention

The invention aims to: the first object of the invention is to provide a phenolic acid decarboxylase mutant, which improves the catalytic activity of the phenolic acid decarboxylase through site-directed mutagenesis, and meets the industrial production requirement of 4-vinyl guaiacol under the conventional reaction conditions; a second object of the present invention is to provide a method for producing the phenolic acid decarboxylase mutant; the third object of the invention is to provide the application of the phenolic acid decarboxylase mutant in the catalysis preparation of 4-vinyl guaiacol.

The technical scheme is as follows: the phenolic acid decarboxylase mutant disclosed by the invention is characterized in that at least one amino acid in the 40 th site, the 92 th site and the 131 th site of an amino acid sequence shown in SEQ ID No.1 is replaced; isoleucine Ile at position 40 to valine Val; isoleucine Ile at position 92 to threonine Thr, valine Val, or any one of them; valine Val at position 131 is mutated to leucine Leu.

The invention constructs the phenolic acid decarboxylase mutant with high activity by site-directed mutagenesis of the amino acid of the phenolic acid decarboxylase active center from Lactiplantibacillus plantarum (STRAIN ATCC BAA-793/NCIMB 8826/WCFS 1).

Preferably, the amino acid sequence of the mutant is any one of sequences shown in SEQ ID No. 2-5.

The invention provides a gene for encoding the phenolic acid decarboxylase mutant protein.

Preferably, the nucleotide sequence of the gene for encoding the phenolic acid decarboxylase mutant protein is shown as SEQ ID No. 7-10.

The cloning vector or the expression vector containing the genes disclosed by the invention.

The invention also relates to a recombinant vector of the phenolic acid decarboxylase mutant encoding gene and recombinant genetic engineering bacteria prepared by the recombinant vector transformation. The recombinant vector of the present invention is not limited as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and/or eukaryotic cells, and the vector may be various vectors conventional in the art, such as various plasmids, phage or viral vectors, etc., preferably a pET22b (+) plasmid is used as an expression vector, and escherichia coli is used as an expression host (escherichia coli C43 cells or escherichia coli BL 21).

The preparation method of the phenolic acid decarboxylase mutant comprises the following steps:

(1) Designing point mutation primers, taking plasmids with phenolic acid decarboxylase wild genes as templates, respectively carrying out PCR (polymerase chain reaction) by adopting the point mutation primers, and purifying to obtain mutant gene fragments and linearization plasmids;

(2) Connecting the mutant gene fragment with a linearization plasmid to construct an expression vector, and transferring the expression vector into host bacteria for induced expression;

(3) Collecting host bacteria expressing the phenolic acid decarboxylase mutant, re-suspending the bacterial cells, crushing the cells, centrifuging and taking the supernatant to obtain crude enzyme liquid containing the phenolic acid decarboxylase mutant.

Preferably, the point mutation primer is:

preferably, the PCR reaction system is:

The phenolic acid decarboxylase mutant disclosed by the invention is applied to the catalytic preparation of 4-vinyl guaiacol.

The invention also provides an application method of the phenolic acid decarboxylase mutant in catalyzing ferulic acid to synthesize 4-vinyl guaiacol, and the application method is preferable: the method comprises the steps of taking wet thalli of recombinant engineering bacteria containing phenolic acid decarboxylase mutant encoding genes after induced expression as a catalyst, taking ferulic acid as a substrate, reacting in a reaction system consisting of potassium phosphate buffer solution with pH value of 5.8-6.8 (preferably 6.75) at 300-500 rmp (preferably 400 rpm) and 15-20 ℃ for at least 8 hours to obtain reaction liquid containing 4-vinyl guaiacol after the reaction is finished, and separating and purifying the reaction liquid to obtain the 4-vinyl guaiacol.

The potassium phosphate buffer solution system is an aqueous solution with a pH of 7.5, which is composed of 1M of dimethyl hydrogen phosphate and potassium dihydrogen phosphate, and the pH is 6.75 after the substrate is added according to the substrate concentration/buffer solution concentration=3/5. The pH of the reaction system can be controlled to be 6.75 by a buffer solution, and the pH of the reaction system can be stabilized to be 6.75 by continuously adjusting the pH.

Preferably, the catalyst is used in an amount such that the OD600 = 20 in the reaction solution of the wet cells, and the substrate is initially added at a concentration of 400-800 (preferably 600 mM).

Preferably, the wet cell is prepared as follows: inoculating recombinant engineering bacteria containing the gene encoding the phenolic acid decarboxylase mutant into a liquid LB culture medium containing ampicillin sodium with the final concentration of 100ug/mL, and culturing for 8 hours at 37 ℃ to obtain seed liquid; inoculating the seed solution into a sterile TB medium containing 100ug/L ampicillin at an inoculum size of 2% by volume, culturing at 37 ℃ for about 8-12h, adding isopropyl thio-beta-D-galactoside (IPTG) at a final concentration of 0.1-1.0 mM (preferably 0.5 mM) to the medium when the cell OD600 is 0.6, performing induced expression at 18 ℃ for 16-24 h, centrifuging at 4 ℃ at 4000rpm for 10-20 min, and suspending the wet bacterial body by using a reaction buffer;

The phenolic acid decarboxylase mutant can be catalyzed in a whole cell form, and can also be catalyzed by crude enzyme liquid of cell disruption or pure enzyme of complete disruption. In addition, the phenolic acid decarboxylase may be prepared as an immobilized enzyme or as an immobilized cell form enzyme using specific immobilization techniques.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) The phenolic acid decarboxylase mutant is modified by a site-directed mutagenesis method based on the phenolic acid decarboxylase amino acid shown in SEQ ID NO.1, so that the amino acid sequence is changed, the change of the protein structure and function is realized, the phenolic acid decarboxylase mutant with the mutation is obtained, the enzyme activity is improved, and the catalytic activity on ferulic acid is greatly improved relative to the original enzyme; (2) The mutant I40V/I92T/V131L can catalyze substrate ferulic acid under the condition of water phase normal temperature, the yield of the obtained phenolic acid decarboxylase mutant is 69.4 percent after 10 hours of reaction under the condition of OD < 600 > = 20 and the substrate concentration is 600mM, and the yield is 5.61 times of that of the parent enzyme; (3) The phenolic acid decarboxylase mutant has high enzyme activity, is used for catalyzing ferulic acid to synthesize 4-vinyl guaiacol, has simple production process and mild reaction conditions, and is environment-friendly in production process, thereby being more beneficial to realizing industrial production.

Drawings

FIG. 1 shows the conversion profile of the mutant I40V/I92T/V131L whole-cell catalyst of example 3 with OD ₆₀₀ = 20 at different temperatures versus 600mM ferulic acid;

FIG. 2 shows the conversion profile of the mutant I40V/I92T/V131L whole-cell catalyst of example 3 with OD ₆₀₀ = 20 at different pH values versus 600mM ferulic acid;

FIG. 3 is a graph of conversion of 600mM ferulic acid by the original 2GC9 strain with OD ₆₀₀ =20, the example 1 mutant I40V, the example 2 mutant I40V/I92T, the example 3 mutant I40V/I92V, the example 4 mutant I40V/I92T/V131L whole cell catalyst.

Detailed Description

The technical scheme of the invention is further described below by referring to examples.

Example 1

The phenolic acid decarboxylase mutant disclosed by the invention is a phenolic acid decarboxylase mutant I40V with a sequence shown as SEQ ID No.2, wherein the mutant is characterized in that the 40 th isoleucine of a phenolic acid decarboxylase starting enzyme sequence shown as SEQ ID No.1 is mutated into valine (I40V);

the preparation method of the mutant I40V is as follows:

1. Recombinant plasmid construction

The plasmid pET-22b (+), E.coli BL21 (DE 3), both of which were deposited by the applicant, were commercially available. The phenolic acid decarboxylase wild-type gene from Lactiplantibacillus plantarum (STRAIN ATCC BAA-793/NCIMB 8826/WCFS 1) was synthesized by the company Jin Weizhi (Suzhou). The site-directed mutant sequence of the enzyme was constructed by the applicant by a PCR method. The primers used to introduce the mutation sites are shown in Table 1. The template for the point mutation PCR is pET-22b (+) plasmid with phenolic acid decarboxylase wild type gene sequence.

TABLE 1 mutant Gene fragment primers and linearization plasmid primers

Note that: the primer is underlined and marked with a mutation site, "F" represents the upstream primer and "R" represents the downstream primer.

TABLE 2PCR reaction System

TABLE 3PCR reaction conditions

After the PCR product is verified by nucleic acid electrophoresis, the PCR product is purified by using a DNA gel recovery kit. A reaction system as shown in Table 4 was prepared in which the mutated gene fragment was a gene fragment in which isoleucine at position 40 was mutated to valine and the linearized plasmid was a linearized plasmid comprising the gene fragment in which isoleucine at position 40 was mutated to valine. And the reaction is carried out for 1h at 37 ℃ for one-step cloning, so as to construct the expression plasmid of the mutant enzyme. The gene sequence of the mutant enzyme is shown as SEQ ID No. 2.

TABLE 4 one-step cloning System

2. Recombinant strain construction and crude enzyme solution preparation

The recombinant plasmid was transformed into E.coli DH 5. Alpha. Competent cells, which were plated on LB solid medium containing 100. Mu.g/mL ampicillin, and cultured at 37℃for 12 hours. All single colonies grown on the surface of the plate medium were scraped into a test tube containing 5mL of LB liquid medium containing 100. Mu.g/mL ampicillin, and shake-cultured at 37℃for 12 hours at 200 rmp. Plasmid was extracted using a plasmid extraction kit and the recombinant plasmid was stored in a-20℃refrigerator. The gene sequence of the mutant is shown as SEQ ID No. 7.

The recombinant plasmid was transformed into E.coli BL21 (DE 3) host bacteria and spread on the surface of LB solid medium containing 100. Mu.g/mL ampicillin, and cultured at 37℃for 8 hours. Individual E.coli colonies were picked and inoculated into 3mL of LB liquid medium containing 100. Mu.g/mL of ampicillin, and cultured overnight at 37℃as seed solutions. The seed solution was inoculated at 5% into a 250mL Erlenmeyer flask containing 50mL of TB medium, and cultured at 37℃and 200 rpm. The TB liquid culture medium comprises 12g/L of yeast powder, 12g/L of tryptone, 4ml/L of glycerol, 12.5g/L of dipotassium hydrogen phosphate and 2.3g/L of potassium dihydrogen phosphate. When the culture was completed for 8 hours, IPTG was added to a final concentration of 0.5mM, the culture temperature was set at 18℃and the culture was continued for 16 hours. The fermentation broth was centrifuged and the cells were collected and resuspended in 1M phosphate buffer pH 7.5 as whole cell catalyst for mutant I40V.

Example 2

The phenolic acid decarboxylase mutant disclosed by the invention is a phenolic acid decarboxylase mutant I40V/I92T with a sequence shown as SEQ ID No.3, wherein the mutant is formed by mutating isoleucine at position 40 of a phenolic acid decarboxylase starting enzyme sequence shown as SEQ ID No.1 into valine (I40V) and mutating isoleucine at position 92 into threonine (I92T).

The preparation method of the mutant I40V/I92T is as follows:

The rest is the same as in example 1, except that the PCR template is the I40V mutant of example 1, the mutant gene primers are as follows:

TABLE 5 mutant Gene fragment primers and linearization plasmid primers

Example 3

The phenolic acid decarboxylase mutant disclosed by the invention is a phenolic acid decarboxylase mutant I40V/I92V with a sequence shown as SEQ ID No.4, wherein the mutant is formed by mutating the 40 th isoleucine of a phenolic acid decarboxylase zymogen starting enzyme sequence shown as SEQ ID No.1 into valine (I40V) and mutating the 92 th isoleucine into valine (I92V).

The preparation method of the mutant I40V/I92V is as follows:

TABLE 6 mutant Gene fragment primers and linearization plasmid primers

Example 4

The phenolic acid decarboxylase mutant is shown as a phenolic acid decarboxylase mutant I40V/I92T/V131L with a sequence shown in SEQ ID No.5, wherein the mutant is formed by mutating the 40 th isoleucine of an amino acid sequence shown in SEQ ID No. 1 into valine (I40V), mutating the 92 th isoleucine into threonine (I92T) and mutating the 131 th valine into leucine (V131L).

The preparation method of the mutant I40V/I92T/V131L is as follows:

The rest is the same as in example 1, except that the PCR template is the I40V/I92T mutant of example 2, the mutant gene primers are as follows:

TABLE 7 mutant Gene fragment primers and linearization plasmid primers

Inoculating recombinant engineering bacteria containing the gene encoding the phenolic acid decarboxylase I40V/I92T/V131L mutant into a liquid LB culture medium containing ampicillin sodium with a final concentration of 100ug/mL, and culturing for 8 hours at 37 ℃ to obtain seed liquid; inoculating the seed solution into a sterilized TB medium containing 100ug/L ampicillin at an inoculum concentration of 2% by volume, culturing at 37 ℃ for 12 hours, adding isopropyl thio-beta-D-galactoside (IPTG) at a final concentration of 0.5mM to the medium when the cell OD600 is 0.6, performing induced expression at 18 ℃ for 16 hours, centrifuging at 4 ℃ at 4000rpm for 10 minutes, and using the reacted buffer to suspend the wet cell body weight as a whole cell catalyst.

Example 5 catalytic performance test

1. Catalytic Performance test of the phenolic acid decarboxylase mutant enzyme I40V/I92T/V131L prepared in example 4

Determination of the catalytic yields of the phenolic acid decarboxylase mutant enzyme I40V/I92T/V131L whole-cell catalyst prepared in example 4 at different temperatures

Recombinant engineering wet thalli containing the phenolic acid decarboxylase mutant I40V/I92T/V131L coding gene obtained in the example 4 is used as a catalyst, the reaction system is formed by a potassium phosphate buffer solution with the catalyst OD ₆₀₀ =20 and the substrate ferulic acid concentration of 600mM and the pH value of 6.75, the reaction is carried out at 400rpm at 10 ℃,15 ℃,20 ℃,25 ℃,30 ℃ and 35 ℃ overnight (> 8 h) respectively, a reaction solution containing 4-vinylguaiacol is obtained after the reaction is finished, 500ul of the reaction solution is extracted by 500ul of ethyl acetate after the reaction is finished, and then the reaction solution is centrifuged at 1,2000rpm for 1min, and 300 ul of supernatant is detected by gas chromatography.

Gas phase analysis conditions: agilent HP-5 column, gas phase procedure was 60℃2min,55℃/min to 250℃and 45℃/min to 300℃for 2min. 4-vinylguaiacol retention time = 3.40min.

The test results are shown in FIG. 1, and the enzyme has better catalytic efficiency in the range of 15-20 ℃.

2. Enzyme Activity assay of the mutant phenolic acid decarboxylase prepared in example 4 under different pH conditions

Recombinant engineering wet thalli containing the phenolic acid decarboxylase mutant I40V/I92T/V131L coding gene obtained in example 4 is used as a catalyst, in a reaction system consisting of a catalyst OD ₆₀₀ =20, substrate ferulic acid concentration is 600mM, the reaction is carried out at 400rpm at 15 ℃ overnight (> 8 h) respectively in a potassium phosphate buffer solution with pH value equal to 5.25,5.75,6.25,6.75 and 7.75, a reaction solution containing 4-vinylguaiacol is obtained after the reaction is finished, 500ul of the reaction solution is extracted with 500ul of ethyl acetate after the reaction is finished, and then the reaction solution is centrifuged at 1,2000rpm for 1min, 300 ul of supernatant is detected by gas chromatography, and the gas chromatography detection conditions are the same as the above.

The test results are shown in fig. 2. The enzyme has better catalytic efficiency in the range of pH=5.75-6.75.

3. Performance test of phenolic acid decarboxylase mutant enzyme

The phenolic acid decarboxylase enzyme starting enzyme 2GC9 strain and the example 1 mutant I40V, the example 2 mutant I40V/I92T, the example 3 mutant I40V/I92V, the example 4 mutant I40V/I92T/V131L were tested and compared for catalytic efficiency on ferulic acid under whole cell catalysis conditions, OD ₆₀₀ = 20, substrate concentration 600mM, ph 6.75, temperature 15 ℃.

The result shows that compared with the original strain, the catalytic efficiency of the mutant I40V/I92T/V131L is improved by 4-5 times, and the mutant I40V/I92T/V131L can catalyze and generate 425mM vanillin under the substrate condition of 600Mm, thereby having extremely high industrial application prospect.

Claims

1. A phenolic acid decarboxylase mutant, characterized in that the mutant is a mutant in which at least one of the three positions 40, 92 and 131 of the amino acid sequence shown in SEQ ID No.1 is substituted; the isoleucine Ile at position 40 is mutated to valine Val; the isoleucine Ile at position 92 is mutated to any one of threonine Thr and valine Val; the valine Val at position 131 is mutated to leucine Leu.

2. The phenolic acid decarboxylase mutant according to claim 1, characterized in that the amino acid sequence of the mutant is any one of the sequences shown in SEQ ID No. 2 to 5.

3. A gene encoding the phenolic acid decarboxylase mutant protein according to any one of claims 1 to 2.

4. The gene encoding the phenolic acid decarboxylase mutant protein according to claim 3, characterized in that the nucleotide sequence of the gene is shown as SEQ ID No. 7 to 10.

5. A cloning vector or expression vector containing the gene according to claim 3.

6. The method for preparing the phenolic acid decarboxylase mutant according to claim 1, characterized in that it comprises the following steps:

(1) Designing point mutation primers, using a plasmid carrying a wild-type gene of phenolic acid decarboxylase as a template, and using the point mutation primers to perform PCR reactions respectively, and obtaining mutant gene fragments and linearized plasmids after purification;

(2) connecting the mutant gene fragment to the linearized plasmid to construct an expression vector, and then transferring the expression vector into the host bacteria for inducing expression;

(3) Collecting host bacteria expressing the phenolic acid decarboxylase mutant, resuspending the bacteria and breaking the cells, and centrifuging and collecting the supernatant to obtain a crude enzyme solution containing the phenolic acid decarboxylase mutant.

7. The method for preparing a phenolic acid decarboxylase mutant according to claim 6, characterized in that the point mutation primers are: I40V_F, I40V_R, I92T_F, I92T_R, I92V_F, I92V_R, V131L_F, V131L_R.

8. The method for preparing a phenolic acid decarboxylase mutant according to claim 6, wherein the PCR reaction system is:

9. Use of the phenolic acid decarboxylase mutant according to claim 1 or 2 in catalyzing the preparation of 4-vinylguaiacol.

10. The use according to claim 9, characterized in that the use method comprises: using wet bacteria of the recombinant engineering bacteria containing the gene encoding the phenolic acid decarboxylase mutant after induction of expression as a catalyst, reacting at 15 to 20°C for at least 8 hours in a reaction system consisting of a potassium phosphate buffer solution with a pH of 5.8 to 6.8 and taking ferulic acid as a substrate, obtaining a reaction solution containing 4-vinylguaiacol after the reaction is completed, separating and purifying the reaction solution to obtain 4-vinylguaiacol.