CN116376855A - Rare earth-dependent thermostable alcohol dehydrogenase mutant and application thereof - Google Patents
Rare earth-dependent thermostable alcohol dehydrogenase mutant and application thereof Download PDFInfo
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Abstract
The invention discloses a rare earth-dependent thermostable alcohol dehydrogenase mutant and application thereof, and relates to the technical field of enzyme engineering. Compared with a wild type (62.51 ℃), the rare earth element-dependent alcohol dehydrogenase mutant has improved stability, wherein the stability of the mutant S181T/S242T/A401G/G445P (72.07 ℃) is improved by 9.5 ℃, and the enzyme activity (0.53U/mg) of the mutant is slightly higher than that of the wild type (0.5U/mg). The invention constructs a rare earth element-dependent alcohol dehydrogenase mutant, and the rare earth enzyme mutant catalyst with HMF activity and high stability is used for catalyzing FDCA through HMF conversion. The invention is hopeful to change the technical monopoly situation of single enzyme source in the process of catalyzing HMF to FDCA by single enzyme at present, and has pushing effect on realizing green manufacturing of FDCA.
Description
Technical Field
The invention relates to the technical field of enzyme engineering, in particular to a rare earth-dependent thermostable alcohol dehydrogenase mutant and application thereof.
Background
2,5-furandicarboxylic acid (2, 5-furandicarboxylic acid, FDCA) has the most potential use as an important bio-based platform compound for the synthesis of renewable polyethylene 2,5-furandicarboxylic acid (PEF) as a substitute for terephthalic acid (PTA), and is widely used in the production of a variety of bio-based high molecular polymers (e.g., polyamides, polyesters, polyurethanes, etc.). At present, FDCA is mainly synthesized by adopting a chemical method, and O is utilized at high temperature and high pressure by using a noble metal catalyst 2 Oxidized 5-Hydroxymethylfurfural (HMF) preparation. Mainly comprises the following two steps: firstly, oxidizing an aldehyde group of an HMF side chain to generate carboxyl to obtain 5-hydroxymethyl-2-furancarboxylic acid (HFCA), and then oxidizing the hydroxymethyl of the HFCA side chain to generate aldehyde groups (FFCA) and FDCA. However, the chemical process for producing FDCA requires the participation of organic solvents and high concentrations of alkali, is costly and is severely contaminated.
The method for converting HMF to FDCA by using biological enzyme has the characteristics of high selectivity and mild condition, and is widely focused. Enzymes currently catalyzing oxidation reactions of HMF mainly comprise xanthine oxidase, laccase, lipase and the like, but due to the different selectivities of enzymes to HMF intermediates, direct catalysis of HMF to FDCA is generally difficult to achieve by single enzymes.
Rare earth element-dependent alcohol dehydrogenases (Lanthanide-dependent alcohol dehydrogenases, ln-ADH) are alcohol dehydrogenases which catalyze the conversion of alcohols or aldehydes into aldehydes or acids in the absence of oxygen, and are mainly distributed in methyl metabolizing bacteria, but are also found in non-methyl metabolizing bacteria such as Pseudomonas putida KT2440 and the like. Ln-ADH is selective for oxidized substrates, and is primarily directed to primary alcohols or aldehydes for dehydrogenation reactions. The molecular mass of Ln-ADH is typically around 130 KD. Ln-ADH has a binding site for pyrroloquinoline quinone (PQQ) and a rare earth ion (La-Lu, etc.), wherein the rare earth ion functions as a Lewis acid and PQQ participates in the oxidation reaction therein as a cofactor. During the dehydrogenation reaction, the cofactor PQQ in Ln-ADH is reduced to PQQH2, and then hydrogen protons are transferred to the hydrogen acceptor, thus completing the hydrogen transfer, i.e., the dehydrogenation process. Oxygen is not needed to participate in the dehydrogenation reaction, hydrogen peroxide is not generated, and alcohol can be directly converted into acid under certain conditions. Ln-ADH has thus potential for application in the single-enzyme conversion of HMF to FDCA.
The rare earth enzyme from the methyl metabolizing bacteria is difficult to realize the correct expression in the escherichia coli host, and the in-vitro catalysis application of the rare earth enzyme is limited. Pseudomonas putida KT2440 has similar characteristics to the Escherichia coli, so that the rare earth enzyme derived from KT2440 can be expressed and purified normally in the Escherichia coli, and has higher activity. However, rare earth enzyme derived from pseudomonas putida KT2440 has the problems of low activity, low stability and the like, and the industrial application of the rare earth enzyme is limited. There is no case of using rare earth element-dependent alcohol dehydrogenase to realize HMF single enzyme catalysis to FDCA. Therefore, the stability of the rare earth element-dependent alcohol dehydrogenase is improved, and the application of the rare earth element-dependent alcohol dehydrogenase in industry is facilitated.
Disclosure of Invention
The invention aims to provide a rare earth-dependent thermostable alcohol dehydrogenase mutant with high activity and stability and application of the mutant in catalyzing 5-hydroxymethylfurfural to generate 2,5-furandicarboxylic acid. The site to be mutated of the rare earth element-dependent alcohol dehydrogenase is determined by a computer protein design method, and mutants with improved stability and/or improved HMF activity are obtained by screening.
The specific technical scheme is as follows:
the wild rare earth dependent alcohol dehydrogenase is derived from Pseudomonas putida (Pseudomonas putida) KT2440, the amino acid sequence of which is shown as SEQ ID NO.1, and the encoding gene of which is shown as SEQ ID NO. 2. The mutant is modified by site-directed mutagenesis, so that the thermal stability of the protein is improved. Wherein the site of the mutation is one or more of the following: specifically, the mutation at (1) position 181, (2) position 242, (3) position 401 and (4) position 445 is any one of the following:
(1) Mutation of amino acid 181 from valine to threonine;
(2) Mutation of amino acid 242 from serine to threonine;
(3) Glycine at amino acid 401 is mutated from alanine;
(4) Mutation of glycine to proline at amino acid 445;
(5) Mutation of amino acid 445 from glycine to methionine;
(6) Mutation of valine to threonine at amino acid 181 and mutation of glycine to proline at amino acid 445;
(7) Mutation of valine to threonine at amino acid 181 and mutation of serine to threonine at amino acid 242;
(8) Mutation of valine to threonine at amino acid 181 and glycine at amino acid 401;
(9) Mutation of serine to threonine at amino acid 242 and glycine at amino acid 401;
(10) Mutation of serine to threonine at amino acid 242 and mutation of glycine to proline at amino acid 445;
(11) The 401 st amino acid is mutated from alanine to glycine and the 445 th amino acid is mutated from glycine to proline;
(12) Mutation of amino acid 181 from valine to threonine, mutation of amino acid 242 from serine to threonine, mutation of amino acid 401 from alanine;
(13) Mutation of valine to threonine at amino acid 181, serine to threonine at amino acid 242, and glycine to proline at amino acid 445;
(14) Mutation of serine to threonine at amino acid 242, glycine to alanine at amino acid 401, and glycine to proline at amino acid 445;
(15) Mutation of valine to threonine at amino acid 181, mutation of serine to threonine at amino acid 242, mutation of alanine to glycine at amino acid 401, mutation of glycine to proline at amino acid 445.
The stability of the mutants S181T, S242T, A401G, G445P, G445M, S181T/G445P, S181T/S242T, S181T/A401G, S242T/G445P, A401G/G445P, S181T/S242T/A401G, S181T/S242T/G445P, S181T/A401G/G445P, S242T/A401G/G445P and S181T/S242T/A401G/G445P is higher than that of the wild type WT. Wherein the enzyme activity and stability of the mutants S181T/G445P, S181T/S242T/A401G, S242T, S181T/S242T/A401G/G445P, S242T/G445P and S181T/S242T are higher than those of wild type WT.
Wherein S181T represents: mutation of amino acid 181 from valine to threonine; S181T/G445P: mutation of valine to threonine at amino acid 181 and mutation of glycine to proline at amino acid 445; and the other is the same.
The invention also provides application of the rare earth-dependent heat-stable alcohol dehydrogenase mutant in catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
The invention also provides a gene for encoding the rare earth dependent thermostable alcohol dehydrogenase mutant.
The invention also provides application of the gene in catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid. The invention also provides an expression vector containing the gene.
The invention also provides a genetic engineering bacterium for expressing the rare earth dependent thermostable alcohol dehydrogenase mutant.
The invention also provides application of the genetically engineered bacterium in catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
The invention also provides a preparation method of the 5-hydroxymethyl-2-furancarboxylic acid, which uses the rare earth dependent thermostable alcohol dehydrogenase mutant to catalyze the oxidation reaction of the 5-hydroxymethyl furfural to prepare the 5-hydroxymethyl-2-furancarboxylic acid.
Specifically, wet thalli or supernatant obtained by ultrasonic crushing of wet thalli obtained by fermenting recombinant genetic engineering bacteria containing the wild-type rare earth element-dependent alcohol dehydrogenase or mutant encoding genes are used as catalysts, enzymes purified by nickel affinity chromatography are used as substrates, PQQ is used as a coenzyme, rare earth ions are used as active centers, phenazine Ethyl Sulfate (PES) or Phenazine Methyl Sulfate (PMS) are used as electron transfer bodies, dichlorophenol indophenol (DCPIP) or N, N, N ', N' -tetramethyl p-phenylenediamine (WB) is used as a hydrogen acceptor, 25mM imidazole is used as an activator, and the reaction is carried out in Tris-HCl buffer solution with pH of 8.0 at 30 ℃.
The invention has the beneficial effects that:
compared with a wild type (62.51 ℃), the rare earth element-dependent alcohol dehydrogenase mutant has improved stability, wherein the stability of the mutant S181T/S242T/A401G/G445P (72.07 ℃) is improved by 9.6 ℃ compared with that of the wild type, and the enzyme activity (0.53U/mg) of the mutant is slightly higher than that of the wild type WT (0.5U/mg).
The invention constructs a rare earth element-dependent alcohol dehydrogenase mutant, and the rare earth enzyme mutant catalyst with HMF activity and high stability is used for catalyzing FDCA through HMF conversion. The invention is hopeful to change the technical monopoly situation of single enzyme source in the process of catalyzing HMF to FDCA by single enzyme at present, and has pushing effect on realizing green manufacturing of FDCA.
Drawings
FIG. 1 is a three-dimensional structural model of rare earth element-dependent alcohol dehydrogenase PedH (6 ZCW. Pdb).
FIG. 2 is a schematic representation of the production of FDCA by PedH catalysis of HMF.
FIG. 3 shows point mutations and ΔT thereof m And (5) a value graph.
Detailed Description
Reagents for upstream genetic engineering: e.coli BL21 (DE 3), plasmid pET28a, etc. used in the examples of the present invention were purchased from Novagen; the gene synthesis, primer synthesis and sequence sequencing work of PedH were completed by the company of Optimuno engineering, inc. of the family of Prinsepia.
Reagents for catalytic reactions: tris and hydrochloric acid were purchased from national pharmaceutical groups chemical reagent limited; PQQ (pyrroloquinoline quinone) and PMS (phenazine methosulfate) are purchased from taixi (Shanghai) chemical industry development limited; neodymium chloride and HMF (5-hydroxymethylfurfural) were purchased from Aba Ding Shiji (Shanghai) Inc.; DCPIP (2, 6-dichlorophenol indophenol) was purchased from Shanghai Michelin Biochemical technologies Co.
Academic of the use of enzyme Activity units (U)World general definition: the amount of enzyme required for 1. Mu. MDCPI was consumed per minute in the reaction system. And quantitatively analyzing the consumption of DCPIP in the reaction system by using an enzyme-labeled instrument, and calculating to obtain the specific enzyme activities of the original rare earth dependent alcohol dehydrogenase and the rare earth dependent alcohol dehydrogenase mutant to the substrate 5-hydroxymethylfurfural. The reaction system comprises an appropriate amount of enzyme solution, 15. Mu.M PQQ and 15. Mu.MNDCl 3 10mM 5-hydroxymethylfurfural, 200. Mu.L of the total system. The reaction medium was Tris HCl buffer (100 mM pH 8.0) containing 1mM PMS and 200. Mu.M DCPIP.
FIG. 2 is a schematic representation of the production of FDCA by the catalysis of HMF by PedH, which may be used in each link.
Example 1
Construction of wild-type rare earth element-dependent alcohol dehydrogenase recombinant bacteria.
The rare earth element-dependent alcohol dehydrogenase PedH (the amino acid sequence is shown as SEQ ID NO.1, the three-dimensional structure model is shown as figure 1) from pseudomonas putida KT2440 is taken as a template, the codon optimization is carried out according to the codon preference of E.coli, and the nucleotide sequence of the enzyme is synthesized by the method of total gene synthesis by the Suzhou gold intelligent company, which is shown as SEQ ID NO. 2. The coding sequence of a 6X his-tag is added at the end of the PedH gene sequence and cloned between the digestion sites BamHI and HindIII of the vector pET28a (+) to obtain a recombinant expression plasmid pET28b-PedH. And (3) transforming the recombinant plasmid into E.coli BL21 (DE 3) competent cells by adopting a heat shock method, and obtaining the wild rare earth element dependent alcohol dehydrogenase recombinant strain after sequencing and verification are correct.
Example 2
Design of rare earth element-dependent alcohol dehydrogenase to be mutant.
High performance mutants of PedH were predicted by applying Fireprot (https:// loschmidt. Chemi. Mux. Cz/Fireproteweb /) and PROSS (https:// PROSS. Weizmann. Ac. Il/step/PROSS-ters /) online tools. The results given by the two online tools are combined, and 4 potential mutation sites are obtained by centralized screening according to the composed single-point mutation.
Example 3
Construction of rare earth element-dependent alcohol dehydrogenase mutants.
Primers were designed according to the mutants obtained in example 2, and site-directed mutagenesis was performed on PedH.
The specific method comprises the following steps:
1. full plasmid PCR
The pET28a-PedH plasmid is used as a template, an upstream primer and a downstream primer (table 1) covering mutation sites are designed to carry out full plasmid PCR, and recombinant genes are obtained, wherein the recombinant genes are obtained by mutation of AGC (S181T), mutation of AGC (S242T), mutation of GCA (A401G), mutation of GGT (G445P) and mutation of GGT (ATG 445M) in a nucleotide sequence shown by SEQ ID NO. 2.
TABLE 1 primers for site-directed mutagenesis construction
PCR amplification system:
PCR amplification conditions:
1) Pre-denaturation: 98 ℃ for 3min;
2) Denaturation: 98 ℃ for 10s; annealing: 15s at 60 ℃; extension: 72 ℃ for 1min; cycling for 33 times;
3) Rear extension: 72 ℃ for 5min;
4) Preserving at 4 ℃.
2. Transformation and validation
And directly converting the PCR product into E.coli BL21 (DE 3) competent cells by adopting a heat shock method, and obtaining rare earth-dependent alcohol dehydrogenase mutant strains named mutant strain S181T, mutant strain S242T, mutant strain A401G, mutant strain G445P and mutant strain G445M after sequencing verification is correct.
Example 4
Strain culture and protein expression and purification.
Single colonies of the recombinant bacteria (original rare earth-dependent alcohol dehydrogenase recombinant bacteria and rare earth-dependent alcohol dehydrogenase mutant strain) sequenced correctly in example 3 were picked up and cultured in 5mL LB liquid medium (containing 50. Mu.g/mL kanamycin) at 37℃for 12 hours with shaking. The medium was transferred to 50mL of LB liquid medium containing 50. Mu.g/mL kanamycin and 10g/L alpha-lactose at an inoculum size of 2%, and cultured with shaking at 30℃for 12 hours.
After the completion of the culture, the bacterial liquid was centrifuged at 4000 Xg at 4℃for 15 minutes, and the supernatant was discarded to collect the bacterial cells. After washing the cells with 100mM Tris-HCl buffer pH 8.0, they were resuspended in Tris-HCl buffer and sonicated in an ice-water bath until clear. The cell disruption solution is centrifuged for 20min at 8000 Xg and 4 ℃, the supernatant is taken and subjected to affinity chromatography by a nickel column, impurities are washed by a washing buffer (100 mM Tris-HCl buffer, 150mM NaCl,50mM imidazole, pH 8.0), target proteins are eluted by an eluting buffer (100 mM Tris-HCl buffer, 150mM NaCl,250mM imidazole, pH 8.0), the separated target proteins are placed in an ultrafiltration centrifuge tube for full desalting and concentration, and pure target proteins are obtained, and the SDS-PAGE detects the theoretical protein molecular weight of PedH (63.0 kDa).
Example 5
T of rare earth element-dependent alcohol dehydrogenase m And (5) measuring a value.
The enzyme solution concentrations of the rare earth element-dependent alcohol dehydrogenase and the mutant were uniformly adjusted to 0.5mg/ml, and added to a high-precision quartz glass capillary group, and T was measured by using a protein stability analyzer (Prometaus NT. Plex) m The values, results of the assay are shown in FIG. 3, where T m The single point mutation with the increased value was S181T (3.03 ℃ C., increased), S242T (0.66 ℃ C., increased), A401G (4.71 ℃ C., increased), G445P (1.77 ℃ C., increased), G445M (0.16 ℃ C.).
Example 6
Construction of rare earth element-dependent alcohol dehydrogenase combination mutants.
In order to further improve the transformation effect of the thermal stability, the invention further combines on the basis of the single-point mutant so as to obtain a combined mutant with more obvious effect. Of the single point mutants, the mutants obtained in example 5 were used: the S181T mutant, the S242T mutant, the A401G mutant and the G445P mutant have obviously improved stability, so that the 4 single-point mutants are further combined to obtain the mutants with better stability.
By using the same method as in example 1, the combination mutation was performed to obtain 11 combination mutants having stability further improved than that of the single-point mutant, the genes encoding the 11 combination mutants were respectively ACC-mutated and GGT-mutated to CCT (S181T/G445P), ACC-mutated to ACC (S181T/S242T), AGC-mutated to ACC-mutated to GCA-mutated to GGT (S181T/A401G), AGC-mutated to ACC-mutated to GGT (S242T/A401G), AGC-mutated to ACC-mutated to CCT (S242T/G445P), GCA-mutated to GGT-mutated to CCT (A401G/G445P), ACC-mutated to GGT (S181T/S242T/A401G), AGC-mutated to ACC-mutated to GCT (S181T/S242T/G445P), AGC-mutated to ACC-mutated to GCT, AGC-mutated to GCT (S181T/S242T/G445P), GCA-mutated to ACC, AGC-mutated to GCT and GCT (ACC 181T/G445P), and GCA-mutated to GCT (ACC) and GCT-mutated to GCT).
And (3) carrying out combined mutant protein expression and purification on the escherichia coli vector of the combined mutant after construction according to the same method as that of the example 4, and obtaining the pure enzyme solution of the combined mutant.
The enzyme solution concentrations of the rare earth element-dependent alcohol dehydrogenase, the single-point mutant and the combined mutant were uniformly adjusted to 0.5mg/ml, and the mixture was added to a high-precision quartz glass capillary tube group, and T was measured by using a protein stability analyzer (Prometheus NT.plex) m Values, results are shown in Table 2, T for all mutants m The values were all higher than for wild-type WT.
TABLE 2
Example 7
Enzyme activity determination of rare earth element-dependent alcohol dehydrogenase.
Quantitative analysis by enzyme-labeled instrumentThe specific enzyme activities of the original rare earth element-dependent alcohol dehydrogenase and the rare earth element-dependent alcohol dehydrogenase mutant to the substrate 5-hydroxymethylfurfural are obtained through calculation of the amount of the consumed DCPIP in the reaction system. The reaction system contains proper amount of enzyme solution, 1.5 mu M PQQ and 1.5 mu MNDCl 3 10mM substrate, 25mM imidazole, total system 200. Mu.L. The reaction medium was Tris-HCl buffer (100 mM, pH 8.0) containing 1mM PMS and 200. Mu.M DCPIP. The results of the specific enzyme activity measurements are shown in Table 2, wherein the activities of the mutants, S181T/G445P, S181T/S242T/A401G, S242T, S181T, S181T/S242T/A401G/G445P, S242T/G445P, and S181T/S242T are higher than those of the wild-type WT.
Claims (8)
1. A rare earth dependent thermostable alcohol dehydrogenase mutant, characterized in that it is obtained by mutation of a wild type rare earth dependent alcohol dehydrogenase from pseudomonas putida (Pseudomonas putida), the amino acid sequence of which is shown in SEQ ID No.1, and the specific mutation is any one of the following:
(1) Mutation of amino acid 181 from valine to threonine;
(2) Mutation of amino acid 242 from serine to threonine;
(3) Glycine at amino acid 401 is mutated from alanine;
(4) Mutation of glycine to proline at amino acid 445;
(5) Mutation of amino acid 445 from glycine to methionine;
(6) Mutation of valine to threonine at amino acid 181 and mutation of glycine to proline at amino acid 445;
(7) Mutation of valine to threonine at amino acid 181 and mutation of serine to threonine at amino acid 242;
(8) Mutation of valine to threonine at amino acid 181 and glycine at amino acid 401;
(9) Mutation of serine to threonine at amino acid 242 and glycine at amino acid 401;
(10) Mutation of serine to threonine at amino acid 242 and mutation of glycine to proline at amino acid 445;
(11) The 401 st amino acid is mutated from alanine to glycine and the 445 th amino acid is mutated from glycine to proline;
(12) Mutation of amino acid 181 from valine to threonine, mutation of amino acid 242 from serine to threonine, mutation of amino acid 401 from alanine;
(13) Mutation of valine to threonine at amino acid 181, serine to threonine at amino acid 242, and glycine to proline at amino acid 445;
(14) Mutation of serine to threonine at amino acid 242, glycine to alanine at amino acid 401, and glycine to proline at amino acid 445;
(15) Mutation of valine to threonine at amino acid 181, mutation of serine to threonine at amino acid 242, mutation of alanine to glycine at amino acid 401, mutation of glycine to proline at amino acid 445.
2. Use of a rare earth-dependent thermostable alcohol dehydrogenase mutant according to claim 1 for catalyzing the production of 5-hydroxymethyl-2-furancarboxylic acid from 5-hydroxymethylfurfural.
3. A gene encoding the rare earth-dependent thermostable alcohol dehydrogenase mutant of claim 1.
4. Use of the gene according to claim 3 for catalyzing the production of 5-hydroxymethyl-2-furancarboxylic acid from 5-hydroxymethylfurfural.
5. An expression vector comprising the gene of claim 3.
6. A genetically engineered bacterium expressing the rare earth-dependent thermostable alcohol dehydrogenase mutant of claim 1.
7. The use of the genetically engineered bacterium of claim 6 in catalyzing the production of 5-hydroxymethyl-2-furancarboxylic acid from 5-hydroxymethylfurfural.
8. A preparation method of 5-hydroxymethyl-2-furancarboxylic acid is characterized in that the rare earth-dependent thermostable alcohol dehydrogenase mutant of claim 1 is used for catalyzing 5-hydroxymethyl furfural to perform oxidation reaction, and 5-hydroxymethyl-2-furancarboxylic acid is prepared.
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