CN118185886A - For activating CO2Formate dehydrogenase mutants of (C) - Google Patents
For activating CO2Formate dehydrogenase mutants of (C) Download PDFInfo
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Abstract
The invention relates to a formate dehydrogenase mutant for activating CO 2, the amino acid sequence of which is mutated from the sequence shown in SEQ ID NO.1, and one or more groups of amino acid residues are mutated as follows: 158 bits, 235 bits, 270 bits, 274 bits. The invention provides a gene and a protein sequence of the formate dehydrogenase mutant, a constructed expression vector, a genetic engineering strain and application of the formate dehydrogenase mutant in CO 2 activation. The expression vector and the genetically engineered bacterium are constructed to induce and express the formate dehydrogenase mutant, and the pure enzyme in vitro catalyzes CO 2 to produce the formate. The formate dehydrogenase mutant developed by the invention has excellent oxidation resistance and catalytic activity, and the relative enzyme activity is 743% of that of the original enzyme, thereby being beneficial to efficient activation of CO 2.
Description
Technical Field
The invention belongs to the technical field of biology, and in particular relates to a formate dehydrogenase mutant for activating CO 2, which comprises an amino acid sequence, a nucleotide sequence, an expression vector and a recombinant engineering strain constructed by the formate dehydrogenase mutant, and application of the formate dehydrogenase mutant, the expression vector and the recombinant engineering strain in activating CO 2.
Background
Biocatalysis to convert CO 2 to methanol has the potential to achieve CO 2 activation and economical conversion, during which the formate dehydrogenase FDH plays a central role, a bottleneck of which is the catalytic activity and stability of the bio-enzyme preparation. In the multienzyme cascade of conversion of CO 2 to methanol, the rate of catalysis of formate- > CO 2 (K m＝3.3mM,Vmax =0.02 mM/min) was much higher than that of CO 2 - > formic acid (K m＝30-50mM,Vmax =0.002 mM/min) using commercial formate dehydrogenase CbFDH derived from candida boidinii. Thus, the targeted efficient conversion of CO 2 to formic acid and other products presents challenges.
Currently, to improve the activity and stability of formate dehydrogenase FDH, the development of efficient formate dehydrogenase by protein engineering has become an important way to increase the activation efficiency of CO 2. Kim et al obtained a 5.8-fold higher formate yield than commercial formate dehydrogenase of Candida boidinii by expressing the formate dehydrogenase gene in Thiobacillus sp.KNK65MA (Plos One,2014,9, e 103111). Tishkov et al obtained mutants with 1.5-fold improved thermostability over wild-type formate dehydrogenase by comparing the amino acid sequences of 9 different sources of formate dehydrogenase in combination with the three-level structural analysis of the Pseudomonas sp.101 biological enzyme preparation (FEBS Letters,1999,445,183-188) by mutagenesis of the 5 serine residue positions 131, 160, 168, 184 and 228 of Pseudomonas sp.101 formate dehydrogenase. Patent CN202011179775.1 utilizes a short peptide recognition model PPR bioinformatics platform to firstly establish a complete formate dehydrogenase database and a short peptide sequence library (6987 protein sequences, 53 PPR groups) to successfully predict the functions of different types of formate dehydrogenases, and autonomously create a novel efficient formate dehydrogenase FDHPa derived from Paracoccus sp.mku1, the affinity and catalytic activity of which to CO 2 are 1.6 and 22.8 times that of commercial formate dehydrogenase CbFDH respectively.
By independently developing or modifying the formate dehydrogenase, the catalytic activity and stability of the enzyme are improved, and the CO 2 activation efficiency is improved, however, due to the complexity of a CO 2 activation reaction system, the structural stability and catalytic activity of the existing formate dehydrogenase and/or mutants thereof in a buffer solution are still limited, and the yield of the formate is far from the requirement of large-scale biological manufacturing, so that the further improvement of the performance of the formate dehydrogenase for activating CO 2 is important. Based on the background and the advantages, the invention carries out rational and irrational design on the high-efficiency formate dehydrogenase independently created in the earlier stage, obtains the formate dehydrogenase mutant with further improved CO 2 activation and reduction efficiency, and improves the technical support for economically converting CO 2 into methanol.
Disclosure of Invention
The invention aims to provide a formate dehydrogenase mutant for activating CO 2, which comprises an amino acid sequence of formate dehydrogenase, a nucleotide sequence for encoding the same, a gene expression vector and a recombinant engineering strain.
To achieve the purpose, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a formate dehydrogenase mutant for activating CO 2, the formate dehydrogenase mutant having any one of the amino acid sequences shown in (I) or (II):
(I) The amino acid sequence is mutated from the sequence shown in SEQ ID NO.1, and one or more amino acid residues selected from the following amino acid residues are mutated: 159, 235, 270, 274, and the amino acid residue is Ile, gly, leu, met after mutation;
(II) the formate dehydrogenase mutant has an amino acid sequence with the sequence SEQ ID NO.1 of more than or equal to 99 percent;
Preferred formate dehydrogenase mutants in the present invention are preferably characterized in that the amino acid mutations of the formate dehydrogenase are amino acid residues shown below: lie at 159, and/or Gly at 235, and/or Leu at 270, and/or Met at 274; and/or mutations of two or more amino acid residues at the above positions.
Preferably, the formate dehydrogenase mutants are one set of positions 159Ile or 274Met, two sets of positions 159Ile and 274Met, and three sets of positions 159Ile and 235Gly and 274Met.
Preferably, the amino acid sequence of the formate dehydrogenase mutant is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.
In a second aspect, a nucleotide encoding the formate dehydrogenase mutant of claim 1 or 2, preferably a nucleotide sequence encoding the formate dehydrogenase mutant shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5; more preferably, the nucleotide sequences as shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No. 9.
In a third aspect, the invention also provides a gene expression vector. The gene expression vector includes: a nucleotide sequence encoding an amino acid sequence according to the first aspect or a nucleotide sequence according to the second aspect.
The expression vector may be various expression vectors commonly used in the art for expressing a gene of interest in E.coli. Preferably, the gene expression vector is a pET plasmid, preferably a petdet plasmid.
In a fourth aspect, the present invention also provides a method for constructing a gene expression vector according to the third aspect, comprising the steps of:
The sequence of SEQ ID NO.1 was constructed between BamHI/SacI cleavage sites of pETDuet plasmid by gene synthesis to construct plasmid pETDuet-FDH. Designing a primer based on the single-site of the first aspect, and obtaining a single-site expression vector by PCR (polymerase chain reaction) amplification of a sequence by taking pETDuet-FDH as a template; the multi-site expression vector construction method is the same, and single-site expression vectors are used as templates for construction and are constructed in sequence.
The construction method specifically comprises the following steps of:
Designing a primer based on the single-site design of the first aspect, namely designing a primer with the length of 17-25bp by taking the mutation site shown as SEQ ID NO.6 or SEQ ID NO.7 as the center, and amplifying the whole plasmid sequence by PCR by taking pETDuet-FDH as a template to obtain the single-site expression vector. Two site expression vector construction: the expression vector containing SEQ ID NO.6 sequence is used as a template, the mutation site of SEQ ID NO.7 is used as the center, and a primer with the length of 17-25bp is designed and constructed through PCR amplification. Three-site expression vector: the expression vector containing SEQ ID NO.8 sequence is used as a template, the mutation site of SEQ ID NO.9 is used as the center, and a primer with the length of 17-25bp is designed and constructed through PCR amplification.
In a fifth aspect, a recombinant engineering bacterium for activating CO 2, comprising a gene expression vector according to the third aspect and/or a nucleotide encoding a formate dehydrogenase mutant according to the first aspect and/or a nucleotide according to the second aspect. The engineering bacteria can be E.coli BL21 (DE 3).
In a sixth aspect, the present invention also provides a method for preparing formic acid by using the recombinant engineering bacteria according to the fifth aspect, the method comprising the following steps:
And (3) culturing the recombinant engineering bacteria, centrifuging and re-suspending the induced bacterial liquid to obtain bacterial suspension, performing ultrasonic crushing, and then performing protein purification to obtain a free formate dehydrogenase mutant, mixing the free enzyme with a buffer solution containing coenzyme NADH, introducing CO 2 for reaction, and detecting NADH consumption and formic acid concentration after stopping the reaction by 8M urea.
In a preferred embodiment of the present invention, the reaction temperature is 25 to 40 ℃, for example, 25 ℃, 30 ℃, 35 ℃, 40 ℃ or the like, and the reaction time is 0.5 to 3 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours or the like, preferably 0.5 to 1 hour.
The concentration of NADH in the mixture is preferably 0.001 to 5mM, and may be, for example, 0.001mM, 0.002mM, 0.005mM, 0.01mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.5mM, 0.6mM, 0.8mM, 1mM, 1.5mM, 2mM, 3mM, 4mM, 5mM, etc., preferably 0.5 to 1mM.
Preferably, the concentration of formate dehydrogenase in the mixed solution is 0.20mg/mL; ;
Preferably, the buffer is 100mM phosphate buffer, pH 7.0;
preferably, the temperature of the catalytic reaction is 25 ℃;
preferably, the catalytic reaction is carried out under stationary conditions;
preferably, after the induced bacterial liquid is centrifuged, the method further comprises a freezing operation, wherein the freezing operation is as follows: freezing at-80deg.C for more than 1 hr.
In the invention, after CO 2 is activated, the method for detecting NADH consumption comprises the following steps:
NADH standard solutions with concentrations of 0.01, 0.05, 0.10, 0.25, 0.50, 0.75 and 1.00mM are prepared in 100mM buffer solution with pH of 7.0, OD values at 340nm are detected by a multifunctional microplate detector to prepare an NADH standard curve, and the consumption of NADH in the reaction solution is determined according to the standard curve.
In a seventh aspect, the invention also provides the use of a formate dehydrogenase mutant according to the first aspect, a nucleotide according to the second aspect, a gene expression vector according to the third aspect or a recombinant engineering strain according to the fifth aspect for the activation of CO 2.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The invention provides a formate dehydrogenase mutant capable of efficiently activating CO 2, which can be obtained by constructing an expression vector and carrying out induced expression after genetic engineering bacteria, and has higher catalytic efficiency of 743% of original enzyme when free enzyme of the formate dehydrogenase mutant catalyzes and activates CO 2;
(2) The residual enzyme activity of the formate dehydrogenase mutant provided by the invention is more than 50% after the formate dehydrogenase mutant is placed in 150mM H 2O2 for 1H, and the formate dehydrogenase mutant has excellent oxidation resistance; the catalytic activity of the formate dehydrogenase mutant in the oxidation direction is lower than that of the original enzyme, especially mutant SEQ ID NO.9, and the initial reaction rate and activity of the formate dehydrogenase mutant in the oxidation direction are the lowest, so that the formate dehydrogenase mutant is favorable for efficiently activating CO 2 and has potential application prospect.
Drawings
FIG. 1 is a comparison of formate dehydrogenase mutant enzyme activities.
FIG. 2 shows the oxidation resistance of formate dehydrogenase mutants.
FIG. 3 shows the catalytic activity of formate dehydrogenase mutant in the oxidation direction.
FIG. 4 shows formate yield of formate dehydrogenase mutants.
Detailed Description
The technical solution of the present invention will be further described with reference to the following specific embodiments, and it should be apparent to those skilled in the art that the following examples are only simple examples of the present invention and do not represent or limit the scope of the claims of the present invention.
Example 1
The embodiment provides a construction method of a genetic engineering strain.
Based on formate dehydrogenase mutant gene delta FDH (SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO. 9), designing a primer by taking the mutation sites of SEQ ID NO.6 and SEQ ID NO.7 as the center, amplifying the whole plasmid by PCR by taking pETDuet-FDH as a template to obtain point mutants 1 and 2, and constructing two-point and multi-point mutants 3 and 4 by taking the point mutant plasmid as the template in sequence. And respectively transferring into E.coli BL21 (DE 3) chassis cells to construct genetic engineering strains for formic acid synthesis, which are still named as delta FDH1, delta FDH2, delta FDH3 and delta FDH4.
Example 2
This example provides a method for preparing a free formate dehydrogenase mutant.
The engineering strains Δfdh1, Δfdh2, Δfdh3, Δfdh4 obtained in example 1 were cultured overnight at 37 ℃ in 5mL of LB medium to which 100mg/L of ampicillin was added to obtain a seed solution.
The seed solution is transferred into 50mL of LB culture medium added with 100mg/L of ampicillin antibiotics according to the transfer amount of 1% by volume, cultured at 37 ℃, induced by adding IPTG with the final concentration of 0.1mM when OD 600 is 0.6, continuously cultured at 20 ℃ for 20 hours, centrifuged at 4000rpm, and the thalli are collected and frozen at-80 ℃.
Buffer A (20 mM potassium phosphate buffer, 500mM NaCl,20mM imidazole, pH 7.4 adjusted), buffer B (20 mM potassium phosphate buffer, 500mM NaCl,500mM imidazole, pH 7.4 adjusted) and stock solution (50 mM potassium phosphate buffer, 10% glycerol, pH 7.4 adjusted) were pre-chilled at 4 ℃. Re-suspending frozen thallus at-80deg.C with pre-cooled Binding buffer, crushing with ultrasonic wave at 55%, ultrasonic processing for 2s, suspending for 3s, centrifuging at 12000rpm at 4deg.C for 20min to obtain supernatant, filtering the supernatant with 0.22 μm filter membrane, purifying with 5mL Histrap purification column AKTA protein purifier, replacing the preservation solution with 5mL HiTrap Desalting desalting column, obtaining pure enzyme dissolved in the preservation solution, measuring the concentration of purified formate dehydrogenase mutant with BCA protein quantification method, and preserving at-80deg.C.
Example 3
The mutant catalytic activity obtained by the purification of example 2 was compared in this example.
In 1000. Mu.L of the catalytic system, the substrate was CO 2, the concentration of coenzyme NADH was 1mM, the addition amount of the formate dehydrogenase mutant was 0.2mg/mL, and the buffer used in the system was 100mM phosphate buffer (pH 7.0). The reaction was stopped by adding 10%8M urea at 25℃for 1 hour, and the supernatant was centrifuged at 12000rpm for 2 minutes to measure the NADH consumption. The results of the catalysis of the mutants are shown in FIG. 1, wherein the enzyme activities of the mutants ΔFDH1, ΔFDH2, ΔFDH3 and ΔFDH4 are 121.63%, 143.85%, 743.04% and 358.88% of the initial enzyme activities, respectively, and the enzyme activities of the mutants ΔFDH3 are the highest.
Example 4
The formate dehydrogenase mutants Δfdh3 and Δfdh4 were compared for their oxidation resistance in this example.
Experiments were performed using the mutants obtained by purification in example 2, by placing ΔFDH3 and ΔFDH4 in 150mM H 2O2 solution, placing at 25℃for 0, 1 and 3 hours, adding 0.2mg of mutant enzyme to 1000. Mu.L of a reaction system further comprising 5mM coenzyme NADH and 100mM phosphate buffer (pH 7.0), reacting 50mM NaHCO 3 as a substrate at 25℃for 1 hour, stopping the reaction by adding 10%8M urea, centrifuging at 12000rpm for 2 minutes, and collecting the supernatant to detect the remaining amount of NADH. Since H 2O2 has a certain oxidation effect on the coenzyme NADH, the 1000. Mu.L reaction system of the control group comprises the same amount of H 2O2, 0.2mg mutant enzyme, 5mM coenzyme NADH and 100mM phosphate buffer (pH 7.0) as that of the experimental group, and no substrate is added in the control group. The oxidation resistance of mutant enzyme is represented by calculating the difference between the residual amounts of NADH in the control group and the experimental group to obtain the NADH consumption, and comparing the NADH consumption with the NADH consumption for 0H to calculate the residual relative enzyme activity, and the result is shown in FIG. 2, the residual enzyme activity of mutant DeltaFDH 4 after being placed in 150mM H 2O2 solution for 1H is more than 50%, 54.2%, and the original enzyme FDH is completely inactivated; after 3h of standing, residual enzyme activities of ΔFDH3 and ΔFDH4 still remain 30% and 44%.
Example 6
In this example, the catalytic activities of formate dehydrogenase mutants Δfdh3 and Δfdh4 in the oxidation direction were compared.
The mutant obtained by the purification of example 2 was used for the experiment, and 5mM HCOOH was added as a substrate, the concentration of coenzyme NAD + was 1mM, the addition amount of formate dehydrogenase was 0.2mg/mL, and the buffer used for the system was 100mM phosphate buffer (pH 7.0) in 500. Mu.L of the catalytic system. After mixing the system, the mixture was rapidly dispensed into 200. Mu.L/well 96-well plates, and the NADH yield was measured by kinetic method, once every minute at 340nm, and continuously for 10min. In addition, the same reaction system was used to detect NADH production using kinetic methods, scanning every 2min at 340nm, again for 1h continuous scanning until the reaction equilibrated. As a result, as shown in FIG. 3, the formate dehydrogenase mutant had HCOOH oxidative activity ordered as FDH > ΔFDH3> ΔFDH4, and the ΔFDH3 oxidative direction was lower at an initial reaction rate of 51.2% of FDH, but the oxidative activity was gradually increased during reaction 1h, and the final oxidative activity was still lower than FDH, but relatively higher. The initial reaction rate of the delta FDH4 in the oxidation direction is only 20.6% of that of the FDH, the catalytic activity in the oxidation direction is rapidly increased after the reaction is carried out for 10min, the catalytic activity in the oxidation direction is obviously lower than that of the FDH after the reaction is carried out for 1h while the catalytic activity in the oxidation direction is basically stable and unchanged although the catalytic activity is greatly fluctuated after the reaction reaches the highest level at 20 min.
Example 7
This example is based on the comparison of formate dehydrogenase mutant enzyme activities of example 3 to determine the formate yield of the optimal mutant.
The mutants obtained by purification in example 2 were used for experiments, in 1000. Mu.L of the catalytic system, the substrate was CO 2, the concentration of coenzyme NADH was 1mM, the addition amount of formate dehydrogenase was 0.2mg/mL, and the buffer used in the system was 100mM phosphate buffer (pH 7.0). The reaction was stopped by adding 10%8M urea at 25℃for 1 hour, and centrifuged at 12000rpm for 2 minutes, and the supernatant was collected and examined for formic acid yield by a chromogenic reaction. The results are shown in FIG. 4, and the optimal formate yield of the mutant is 0.23mg/mL.
Claims (9)
1. A formate dehydrogenase mutant for activating CO 2, characterized in that the formate dehydrogenase mutant has any one of the amino acid sequences shown in (I) or (II):
(I) The amino acid sequence is mutated from the sequence shown in SEQ ID NO.1, and one or more amino acid residues selected from the following amino acid residues are mutated: 159, 235, 270, 274, and the amino acid residue is Ile, gly, leu, met after mutation;
(II) the formate dehydrogenase mutant has an amino acid sequence with more than or equal to 99% of the amino acid sequence of (I).
2. The formate dehydrogenase mutant according to claim 1, characterized in that the amino acid sequence of the formate dehydrogenase mutant is selected from one or more of the following groups of sites, mutated to the amino acid residues indicated below: lie at 159, and/or Gly at 235, and/or Leu at 270, and/or Met at 274.
Preferably, the formate dehydrogenase mutants are one set of positions 159Ile or 274Met, two sets of positions 159Ile and 274Met, and three sets of positions 159Ile and 235Gly and 274Met.
Preferably, the amino acid sequence of the formate dehydrogenase mutant is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.
3. A nucleotide sequence encoding the formate dehydrogenase mutant according to claim 1 or 2, preferably as shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5; more preferably, the nucleotide sequences as shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No. 9.
4. A gene expression vector for the heterologous expression of a formate dehydrogenase mutant, the gene expression vector comprising: an amino acid sequence encoding the formate dehydrogenase mutant according to claim 1 or 2 or a nucleotide sequence according to claim 3;
Preferably, the gene expression vector is a pET plasmid, preferably a petdet plasmid.
5. The method for constructing a gene expression vector according to claim 4, comprising the steps of:
The sequence of SEQ ID NO.1 was constructed between BamHI/SacI cleavage sites of pETDuet plasmid by gene synthesis to construct plasmid pETDuet-FDH. Designing a primer based on the single-site of the claim 1 or 2, and obtaining a single-site expression vector by PCR (polymerase chain reaction) amplification sequence by taking pETDuet-FDH as a template; the construction method of the multi-site expression vector is the same, and the single-site expression vector is used as a template for construction.
6. Recombinant engineering bacterium for synthesizing a formate dehydrogenase mutant, comprising the gene expression vector of claim 4 and/or the amino acid sequence encoding the formate dehydrogenase mutant of claim 1 or 2 or the nucleotide sequence of claim 3. The engineering bacteria can be E.coli BL21 (DE 3).
7. A method for activating CO 2 using the formate dehydrogenase mutant expressed according to claim 6, said method comprising the steps of:
And (3) culturing the recombinant engineering bacteria, centrifuging and re-suspending the induced bacterial liquid to obtain bacterial suspension, performing ultrasonic crushing, and then performing protein purification to obtain a free formate dehydrogenase mutant, mixing the free enzyme with a buffer solution containing coenzyme NADH, introducing CO 2 for reaction, and detecting NADH consumption and formic acid concentration after stopping the reaction by 8M urea.
8. The method according to claim 7, wherein the concentration of NADH in the reaction system is 0-10 mM, the addition amount of formate dehydrogenase is 0-2.0 mg/mL, the temperature of the system is 25-40 ℃ and the time is 0.5-3 h;
Preferably, the time of the mixing reaction is 0.5-1 h;
Preferably, the concentration of NADH in the mixture is 1-5 mM;
preferably, the concentration of formate dehydrogenase in the mixed solution is 0.20mg/mL; ;
Preferably, the buffer is phosphate buffer with a pH of 7.0;
preferably, the temperature of the catalytic reaction is 25 ℃;
preferably, the catalytic reaction is carried out under stationary conditions;
preferably, the method comprises the steps of:
(1) Centrifuging the bacterial liquid obtained after the recombinant engineering bacteria are cultured and induced, re-suspending the bacterial liquid after freezing to obtain bacterial suspension, and centrifuging the bacterial suspension at 8000-12000 rpm for 20min after the bacterial suspension is cracked by an ultrasonic cell disruption instrument to obtain crude enzyme liquid;
(2) And (3) separating and purifying the formate dehydrogenase mutant in the crude enzyme solution by using an AKTA protein purifier, and quantifying the free enzyme by using a BCA protein quantification method.
9. Use of a formate dehydrogenase mutant amino acid sequence according to claim 1 or 2, a nucleotide sequence according to claim 3, a gene expression vector according to claim 5 or a recombinant engineering strain according to claim 6 for CO 2 activation.
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