WO2015130119A1 - Methanol dehydrogenase variant exhibiting increased reduction activity - Google Patents

Abstract

The present invention relates to: a methanol dehydrogenase (MDH) variant which is derived from Bacillus methanolicus and has increased reduction activity; a polynucleotide encoding the enzyme; an expression vector comprising the polynucleotide; a transformant into which the expression vector is introduced; a method for preparing the MDH variant by culturing the transformant; a method for producing methanol from formaldehyde by using the MDH variant; and a use of the MDH variant for producing methanol from formaldehyde. The production yield of methanol can be increased by using the MDH variant of the present invention since the reduction activity is improved to be more than that of a conventional MDH, and thus the MDH variant could be widely applied to a more effective preparation of methanol.

Description

Mutant Methanol Dehydrogenase Showing Increased Reducing Activity

The present invention relates to a mutant methanol dehydrogenase which exhibits increased reducing activity, and more particularly, to a mutant methanol dehydrogenase (MDH) derived from Bacillus metanoliqus and having an increased reducing activity. A polynucleotide encoding an enzyme, an expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, a method of culturing the transformant to produce the mutated methanol dehydrogenase, the mutant methanol dehydrogenase It relates to a method for producing methanol from formaldehyde and the use of the mutated methanol dehydrogenase for producing methanol from the formaldehyde.

Formaldehyde is an organic compound that is used in a variety of industrial and biomedical applications. 50% of the total production is used to make condensation product resin, and thermosetting resin and thermoplastic resin, polyacetal, etc., and 40% is used as fiber, leather, paint solvent, etc. as a synthetic intermediate. About 1.5% of the remaining production is made of 35% aqueous solution (formalin) and used as a disinfectant, sample preservative and preservative. It has a large market, accounting for 1.2% of the United States and Canada's GDP, and the annual total production of formaldehyde in 2010 amounts to 29 million tons. However, despite these widely used substances, there are many restrictions on their use due to the toxicity of formaldehyde to humans. As a colorless gas at room temperature, it is mainly absorbed through inhalation and skin contact. Since it is metabolized in a very short time, it is difficult to detect it in the mucous membrane or blood immediately after exposure. As a stimulant to the eyes and respiratory tract, it causes primary irritant and allergic dermatitis, and breathing becomes difficult at concentrations of 10 to 20 ppm, and can lead to airway damage, pulmonary edema, pneumonia, and death. In 2005, the International Agency for Research on Cancer (IARC) designated formaldehyde as a group 1 carcinogen for humans.

Methanol, on the other hand, is becoming increasingly valuable as it is recognized as the next-generation alternative fuel for petroleum resources. Although it is superior to existing gasoline or diesel oil in terms of performance and environmental pollution, it is not widely distributed due to economic feasibility. However, the situation is changing with rising oil prices and tightening greenhouse gas emission regulations in accordance with the Kyoto Protocol. China selected methanol as an alternative fuel for automobiles in 2006, and commercialization of MTO (Methanol to Olefin) and MTP (Methanol to Propylene) facilities is underway. In addition, the United States has been actively researching alternative energy using methanol by promising to reduce gasoline consumption by 20% and increase alternative fuel supply over the next 10 years. In order to use hydrogen energy, which is attracting attention as an energy source with less environmental pollution, a medium for supplying hydrogen to a fuel cell is required.Methanol has a high hydrogen conversion efficiency, safety, and low temperature operation characteristics. The use is also expanding.

On the other hand, methanol dehydrogenase (MDH) is an enzyme that reversibly catalyzes the reaction of oxidizing methanol to convert formaldehyde or again to reduce formaldehyde to methanol. Among them, Bacillus methanolicus, which belongs to the Gram-positive bacteria, belongs to the NAD (P) + dependent group, and is known to exist mainly in the cytoplasm rather than the periplasm. It is also known to exhibit activity in a single subunit. The MDH is used for the industrial production of methanol, and studies are being actively conducted to further increase the production yield of methanol.

For example, WO 85/01063 discloses NDA dependent MDH which can form a conjugate with NADH dependent MDH to increase methanol reducing activity, and US Pat. No. 6280972 discloses an activation which can increase the reducing activity of MDH. I am disclosed. In addition, the research on the mutant MDH to increase the reducing activity of the MDH itself is actively progressed, but there was a disadvantage that the level of increase of the reducing activity is weak.

The present inventors have diligently researched to develop a mutant MDH capable of increasing methanol productivity by increasing the reducing activity exhibited by the conventional MDH. As a result, the mutant MDH obtained by causing a random mutation in MDH derived from Bacillus metanolicus was studied. It was confirmed that exhibits up to twice the reducing activity of the conventional MDH, to complete the present invention.

One object of the present invention is to provide mutated methanol dehydrogenase (MDH) to have enhanced reducing activity.

Another object of the present invention is to provide a polynucleotide encoding the variant MDH.

Still another object of the present invention is to provide an expression vector comprising the polynucleotide.

Still another object of the present invention is to provide a transformant into which the expression vector is introduced.

Still another object of the present invention is to provide a method of preparing a variant MDH comprising culturing the transformant and recovering the variant MDH therefrom.

It is another object of the present invention to provide a method for producing methanol from formaldehyde using the above-mentioned MDH.

Another object of the present invention is to provide the use of the mutated methanol dehydrogenase for producing methanol from the formaldehyde.

When the mutated MDH of the present invention is used, since the reducing activity is improved compared to the conventional MDH, the production yield of methanol can be increased, and thus it can be widely used for the production of more effective methanol.

1 is a graph showing a result of comparing the growth rate of the E. coli BL21 (DE3) strain cultured in a medium containing formaldehyde of various concentrations (0, 1, 2, 3, 4 or 5mM) over time .

Figure 2 is a graph showing the change in proliferation level with the cultivation time of the strain expressing wild-type or mutant MDH cultured in a medium containing formaldehyde, vector represents a positive control group, WT represents a negative control group, mt1 represents variant 1, mt2 represents variant 2, and mt3 represents variant 3.

Figure 3 is an electrophoresis picture showing the change in the expression of mutant MDH expressed from each variant prepared in Example 5-1, U represents a culture without the addition of IPTG, I is the solid content of the culture to which the IPTG is added S represents the water-soluble content of the culture to which IPTG was added.

Figure 4 is a graph showing the change in reducing activity with the passage of the reaction time of each variant purified in Example 5-3.

The present inventors conducted various studies to develop a mutant MDH that increased the reducing activity of MDH itself, the mutant MDH obtained by causing a random mutation in MDH derived from Bacillus methanolicus shows an increased reducing activity It was confirmed. Specifically, three variants of MDH derived from MDH of Bacillus methanolicus were obtained, one with three amino acids substituted, the other with six amino acids substituted, and the last one with 18 amino acids substituted. It is. As a result of culturing the mutant strains expressing the three mutant MDHs in a medium containing formaldehyde, it was confirmed that the less the number of amino acid substitutions, the improved resistance to toxicity caused by formaldehyde. The analysis was attributed to the reducing activity. Thus, six amino acid substitutions were prepared by combining three amino acid substitutions of the mutant MDHs, which were determined to have the best reducing activity, and comparing the reducing activities thereof, the two amino acids were substituted (F213V, F289L). It was confirmed that MDH showed the best reducing activity.

Therefore, the variant MDH provided by the present invention may be utilized to increase the productivity of methanol.

As an embodiment for achieving the above object, the present invention is a variant introduced into the wild-type methanol dehydrogenase (MDH) consisting of the amino acid sequence of SEQ ID NO: 1, mutated methanol dehydrogenation, the reduction activity is increased than the MDH Provide enzymes.

The term "methanol dehydrogenase (MDH)" of the present invention is also referred to as methanol dehydrogenase, and the oxidation activity and formaldehyde to form formaldehyde by oxidizing methanol to release two electrons to form two electrons By binding to means an enzyme capable of reversibly showing the reducing activity to produce methanol. In this case, NADH may be used as the electron donor or acceptor. The specific amino acid sequence of the methanol dehydrogenase or the nucleotide sequence information of the gene encoding the same can be obtained from a known database such as GenBank of NCBI (GenBank Accession No. AAA25380.1, AAA88366.1, WP_003599114.1, etc.).

In the present invention, the MDH may be interpreted as an MDH derived from Bacillus methanolicus, the MDH is not particularly limited to this, preferably a wild type MDH having a amino acid sequence of SEQ ID NO: 1 or a poly of SEQ ID NO: 2 Wild type MDH with an amino acid sequence expressed from nucleotides.

As used herein, the term "methanol dehydrogenase (MDH)" means an MDH that is mutated from MDH derived from Bacillus methanolicus and exhibits enhanced reducing activity.

In the present invention, the variant MDH is not particularly limited, but may be MDH consisting of an amino acid sequence substituted with one or a plurality of amino acids in the wild type MDH having the amino acid sequence of SEQ ID NO: 1, preferably SEQ ID NO: L40P, S42G, I53T, D72E, D80G, V81A, F82L, K83E, K83I, L107S, V132A, K165E, I198T, T207K, T210A, F213V, F213S, E233G, L259S, H261R, V281A, F289L at the amino acid sequence of 1 , K344R, A352T, F356S, Q371R and the like can be MDH consisting of an amino acid sequence comprising a single or a combination of substituted amino acids, more preferably amino acids that can be expressed from the polynucleotide of SEQ ID NO: 12 to 19 MDH consisting of a sequence or amino acid sequence of SEQ ID NO: 2 to 9, most preferably from the amino acid sequence of SEQ ID NO: 5 or polynucleotide of SEQ ID NO: 15 It can be an MDH consisting of an amino acid sequence that can be represented.

In addition, the variant MDH may include a polypeptide having a sequence in which at least one amino acid residue is different from the aforementioned amino acid sequence. Amino acid exchanges in proteins and polypeptides that do not alter the activity of the enzyme as a whole are known in the art. The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity against heat, pH, etc. of the protein by variation or modification on the amino acid sequence.

Finally, the mutant MDH may be prepared by a chemical peptide synthesis method known in the art, or the gene encoding the domain may be amplified by PCR (polymerase chain reaction) or synthesized by a known method and cloned into an expression vector for expression. Can be prepared.

According to one embodiment of the present invention, a random mutation is induced in a polynucleotide (SEQ ID NO: 2) encoding a wild type MDH derived from Bacillus methanolicus (Bacillus methanolicus), to obtain each of the mutated polynucleotides, This was introduced into E. coli to obtain each transformant, and then the transformant obtained was inoculated in a medium containing formaldehyde and cultured to obtain a transformant resistant to formaldehyde (Example 2). As a result of analyzing the amino acid sequence of each MDH contained in the obtained transformant, three amino acids were substituted in the wild type MDH (F213V, F289L, F356S) of the variant MDH (SEQ ID NO: 2), wild type MDH 6 Variant MDH (SEQ ID NO: 3) in the amino acid substituted form (S42G, K83I, L107S, L259S, S313G, A352T) and 18 amino acid substituted in the wild type MDH (L40P, I53T, D72E, D80G, V81A, F82L, Variant MDH (SEQ ID NO: 4) of K83E, V132A, K165E, I198T, T207K, T210A, F213S, E233G, H261R, V281A, K344R, Q371R was excavated (Example 3). After inoculating and culturing the strain strain containing each variant MDH in a medium containing 2% formaldehyde, it was confirmed that the variant MDH of SEQ ID NO: 8 shows the best resistance to formaldehyde. It was analyzed that it was due to the activity of reducing the formaldehyde represented by MDH to methanol, and it was determined that the mutated MDH of SEQ ID NO: 2 showed the relatively best reducing activity (FIG. 2). Thus, by combining three substituted amino acids (F213V, F289L, F356S) contained in the variant MDH of SEQ ID NO: 2, SEQ ID NO: 5 (F213V + F289L), 6 (F289L + F356S), which is six variants MDH, A variant MDH consisting of the amino acid sequences of 7 (F213V + F356S), 8 (F213V), 9 (F289L) and 10 (F356S) was prepared and their reducing activity was compared with that of wild type MDH and variant MDH of SEQ ID NO: 2. As a result, it was confirmed that the remaining five variant MDH except for the variant MDH of SEQ ID NO: 10 (F356S) showed better reducing activity than that of the wild type MDH and the variant MDH of SEQ ID NO: 2, among which two amino acids of SEQ ID NO: 5 It was confirmed that this substituted form of MDH (F213V + F289L) showed better reducing activity than wild type MDH as well as all other variants MDH (FIG. 4).

Therefore, it was confirmed that the mutant MDH provided by the present invention is a novel protein showing better reducing activity than the wild type MDH, and it can be seen that the use of the mutant MDH can increase the productivity of methanol.

As another embodiment for achieving the above object, the present invention provides a polynucleotide encoding the variant MDH, a variant MDH expression vector comprising the polynucleotide, a transformant having the expression vector introduced and the transformant It provides a method for producing the variant MDH using.

The polynucleotide provided in the present invention includes a nucleotide sequence encoding the mutated MDH provided in the present invention, preferably may be a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 5, 6, 7 or 18.

The polynucleotide may be mutated by one or more bases by substitution, deletion, insertion, or a combination thereof so long as the mutant MDH expressed therefrom can exhibit better reducing activity than the wild type MDH. When chemically synthesizing a nucleotide sequence, synthesis methods well known in the art may be used, for example, those described in Engels and Uhlmann, Angew Chem Int Ed Eng., 37: 73-127, 1988. , Triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on a solid support, and the like.

As used herein, the term "expression vector" refers to a gene construct which is a recombinant vector capable of expressing a peptide of interest in a host cell of interest, and which contains essential regulatory elements operably linked to express the gene insert. The expression vector includes expression control elements such as initiation codon, termination codon, promoter, operator, etc. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide and when the gene construct is administered, Must be functional and must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

As used herein, the term "operably linked" refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. . For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. Operative linkage with expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can employ enzymes commonly known in the art.

In addition, the expression vector may include a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture. Specific initiation signals may also be required for efficient translation of inserted nucleic acid sequences. These signals include ATG start codons and contiguous sequences. In some cases, an exogenous translational control signal must be provided that can include an ATG start codon. These exogenous translational control signals and initiation codons can be various natural and synthetic sources. Expression efficiency can be increased by the introduction of appropriate transcriptional or translation enhancing factors.

In addition, the expression vector may further include a protein tag that can be removed using an endopeptidase, in order to facilitate the detection of the fusion protein.

As used herein, the term "tag" refers to a molecule that exhibits quantifiable activity or properties, and refers to a polypeptide fluorescent substance such as a chemical fluorescent substance such as fluorescein, fluorescent protein (GFP) or related proteins. It may be a fluorescent molecule including; It may be an epitope tag such as a Myc tag, a flag tag, a histidine tag, a leucine tag, an IgG tag, a straptavidin tag, or the like. In particular, when an epitope tag is used, a peptide tag preferably consisting of 6 or more amino acid residues, more preferably 8 to 50 amino acid residues, may be used.

In the present invention, the expression vector may include a nucleotide sequence encoding a variant MDH provided in the present invention described above, wherein the vector used is not particularly limited as long as it can produce a variant MDH of the present invention. , Preferably plasmid DNA, phage DNA and the like, more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus Subtilis derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal viral vectors (retrovirus) (retrovirus), adenovirus (adenovirus), vaccinia virus (vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the expression vector is expressed differently depending on the host cell expression amount and formula, it is preferable to select the host cell most suitable for the purpose.

The transformant provided by the present invention may be prepared by introducing the expression vector provided by the present invention into a host and transforming the same, and expressing a polynucleotide included in the expression vector to produce a mutant MDH of the present invention. have. The transformation can be carried out by a variety of methods, as long as it can produce a mutant MDH of the present invention exhibiting the effect of improving a variety of cellular activities to a high level, CaCl ₂ precipitation method, CaCl ₂ Hanahan method, the electroporation method, calcium phosphate precipitation method, plasma fusion method, stirring method using silicon carbide fiber, agrobacterial mediated trait which improved efficiency by using reducing material called DMSO (dimethyl sulfoxide) in precipitation method Conversion methods, transformation methods with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation methods and the like can be used. In addition, as long as the host used in the preparation of the transformant can also produce the fusion protein of the present invention, there are no particular limitations, such as bacterial cells such as E. coli , Streptomyces, Salmonella typhimurium; Yeast cells, such as Saccharomyces cerevisiae and ski-irradiated caromyces pombe; Fungal cells such as Pchia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, bow melanoma cells; Or plant cells. For example, in the present invention, E. coli BL21 (DE3) strain was used as a host.

The transformant may be used in the method for producing the mutant MDH provided by the present invention. Specifically, the method of producing a mutant MDH provided by the present invention comprises the steps of (a) culturing the transformant to obtain a culture; And, (b) recovering the variant MDH of the present invention from the culture.

As used herein, the term "culture" means a method of growing microorganisms under appropriately artificially controlled environmental conditions.

In the present invention, the method of culturing the transformant may be performed using a method well known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the mutated MDH of the present invention, but may be continuously cultured in a batch process or an injection batch or repeated fed batch process (fed batch or repeated fed batch process). .

The medium used for culturing should meet the requirements of the particular strain in an appropriate manner while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used include mixed sugars of glucose and xylose as the main carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, coconut Oils such as oils and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, suitable precursors to the culture medium may be used. The raw materials described above may be added batchwise, fed-batch or continuous in a suitable manner to the culture in the culture process, but is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

In addition, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Inject oxygen or an oxygen-containing gas (eg air) into the culture to maintain aerobic conditions. The temperature of the culture is usually 27 ° C to 37 ° C, preferably 30 ° C to 35 ° C. Incubation is continued until the maximum amount of D-type lactic acid is obtained. For this purpose it is usually achieved in 10 to 100 hours. In general, type D lactic acid is excreted in the culture medium, but may be contained in the cells in some cases.

In addition, the step of recovering the mutant MDH from the culture can be carried out by methods known in the art. Specifically, the recovery method is not particularly limited as long as it can recover the produced MDH of the present invention, preferably centrifugation, filtration, extraction, spraying, drying, augmentation, precipitation, crystallization, electrophoresis, Fractional solubilization (eg ammonium sulfate precipitation), chromatography (eg ion exchange, affinity, hydrophobicity and size exclusion) can be used.

As another embodiment for achieving the above object, the present invention provides a method for producing methanol by reducing formaldehyde using the above-mentioned MDH.

Specifically, the methanol production method of the present invention comprises the steps of: (a) adding the mutated methanol dehydrogenase to a mixture containing formaldehyde and an electron donor to obtain a reaction product; And (b) recovering methanol from the reaction product. At this time, the electron donor is not particularly limited to this, but preferably NADH may be used, and the reaction buffer may be further included in the mixture. The reaction buffer is not particularly limited thereto, but is preferably a neutral phosphate buffer. Can be used.

As another embodiment for achieving the above object, the present invention provides the use of the mutated methanol dehydrogenase for producing methanol from the formaldehyde.

As mentioned above, MDH is an enzyme that reversibly catalyzes the reaction of oxidizing methanol to convert formaldehyde or again to reduce formaldehyde to methanol. Since the mutant MDH provided by the present invention is superior in reducing activity to reduce formaldehyde to methanol than wild type MDH, it can be utilized as a use for producing methanol from formaldehyde more effectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Mutation Library Construction Using Wild-type Methanol Dehydrogenase Expression Vector Cloning and Direct Evolution

Polynucleotide encoding the wild type methanol dehydrogenase (SEQ ID NO: 11) to express in wild Escherichia coli Methanol Dehydrogenase (MDH) (SEQ ID NO: 1) derived from Bacillus methanolicus (SEQ ID NO: 11) Was synthesized (Bionia, Daejeon). The synthesized polynucleotide was cloned into the pET21b vector to obtain an expression vector, and the expression vector was used as a template for direct evolution to construct a mutation library. By using induced mutation within the gene to MDH, primers for ^{Diversity ® PCR Random Mutagenesis Kit (Clontech} , USA) by performing polymerase chain reaction (PCR), a mutation library was constructed. At this time, PCR conditions are once for 30 seconds at 94 ℃; 25 times (94 ° C 30 seconds, 68 ° C 1 minute); It was performed under one reaction condition at 68 ° C. for 1 minute.

forward primer: 5'-AAGAAGGAGATATACATATGACA-3 '(SEQ ID NO: 21)

reverse primer: 5'-ATACTCGAGCAGAGCGTTTTTG-3 '(SEQ ID NO: 22)

Example 2: Selection of MDH Mutants with Higher Formaldehyde Resistance than Wild-type

First, the E. coli BL21 (DE3) strain having the wild type MDH gene was inoculated into a medium containing formaldehyde at various concentrations (0, 1, 2, 3, 4 or 5 mM), and then cultured to grow the growth rate according to the concentration of formaldehyde. Was compared (FIG. 1). 1 is a graph showing a result of comparing the growth rate of the E. coli BL21 (DE3) strain cultured in a medium containing formaldehyde of various concentrations (0, 1, 2, 3, 4 or 5mM) over time . As shown in Figure 1, it was confirmed that the growth rate of E. coli significantly reduced when formaldehyde or more than 2mM in the medium.

Next, the mutant library constructed in Example 1 was introduced into the E. coli BL21 (DE3) strain to obtain respective transformants, and the transformants were 3 mM of formaldehyde and empicillin (50 µg / ml). Inoculated in a 2YT liquid medium (tryptone 16g, yeast extract 10g, NaCl 5g / 1L) and then shaken at 37 ℃. When the 600 nm absorbance of the culture medium reached 0.4, 0.1 mM IPTG was added to express MDH, and then cultured at 37 ° C. for 24 hours to obtain a culture solution, and the obtained culture solution was smeared onto solid medium containing empicillin. And cultured to select single colonies resistant to formaldehyde.

Example 3: Sequencing of Mutant MDHs

Single colonies obtained in Example 2 were each inoculated in 2YT liquid medium containing empicillin, shaken at 37 ° C. for 12 hours, and plasmid DNA was recovered from the culture, thereby encoding the polyimide MDH contained therein. The nucleotide sequence and the amino acid sequence of the protein expressed therefrom were analyzed. As a result, variant 1, variant 2, and variant 3 were identified which contain polynucleotides encoding variant MDH, each of which comprises a variant polynucleotide of SEQ ID NOs: 12-14 and is expressed from SEQ ID NO: 2 It was confirmed that a variant having an amino acid sequence of 4 to 4 can express MDH. Among the variant MDHs, the variant MDH having the amino acid sequence of SEQ ID NO: 2 is a form in which 3 amino acids are substituted in the wild type MDH (F213V, F289L, F356S), and the variant MDH having the amino acid sequence of SEQ ID NO: 3 is 6 in the wild type MDH Variants of amino acids (S42G, K83I, L107S, L259S, S313G, A352T), and the variant MDH having the amino acid sequence of SEQ ID NO: 4 has 18 amino acid substitutions in the wild type MDH (L40P, I53T, D72E, D80G). , V81A, F82L, K83E, V132A, K165E, I198T, T207K, T210A, F213S, E233G, H261R, V281A, K344R, Q371R).

Example 4: Analysis of Reducing Activity of Mutant MDH

Expression vectors comprising the mutated polynucleotides of SEQ ID NOs: 12 to 14 were introduced into E. coli BL21 (DE3) to obtain respective transformants, which were inoculated in 2YT medium to which empicillin was added, respectively, at 37 ° C. Shake culture. When the absorbance (O.D. 600) of the culture reached 0.4, 0.1mM IPTG was added to the culture and further incubated for 1 hour to express MDH. Then, 3 mM of formaldehyde was added to each of the cultures, and the absorbance (O.D. 600) of the cultures was measured at intervals of 2-4 hours (FIG. 2). In this case, E. coli BL21 (DE3), which was not transformed, was used as a positive control group, and E. coli BL21 (DE3), into which an expression vector capable of expressing wild-type MDH, was used as a negative control group.

Figure 2 is a graph showing the change in proliferation level with the cultivation time of the strain expressing wild-type or mutant MDH cultured in a medium containing formaldehyde, vector represents a positive control group, WT represents a negative control group, mt1 represents variant 1, mt2 represents variant 2, and mt3 represents variant 3. As shown in Figure 2, all strains show a pattern of stationary phage due to toxicity of formaldehyde and then regrowth, wherein variant 1 is the fastest regrowth in 17 hours, variant 2 is Regrowth occurred in 21 hours, and variant 3 performed regrowth in 23 hours. However, no positive control and no negative control were performed.

From the above results, it was analyzed that the mutant MDH has better activity of reducing formaldehyde to methanol than the wild type MDH, and among these, the mutant MDH having the amino acid sequence of SEQ ID NO: 2 expressed in Variant 1 shows the best reducing activity.

Example 5 Construction and Selection of Variant MDH Using Variant MDH in Variant 1

Variant MDH having the amino acid sequence of SEQ ID NO: 2, which was analyzed as having the best reducing activity in Example 4, was substituted with three phenylalanines with valine, leucine, and serine (F213V, F289L, and F356S). Since the phenylalanine is known to weaken the fluidity in the protein including a ring substituent, the mutant MDH was expected to show a higher level of fluidity than the wild type MDH. Thus, by using the recombinant PCR method, by combining the three substitution sites with a polynucleotide encoding the wild type MDH (SEQ ID NO: 11), to obtain a polynucleotide encoding each variant MDH, the variant MDH expressed therefrom To compare the activity of, to select the best variant mutant MDH.

Example 5-1 Construction of Mutant Strains That Express Mutant MDH

An expression vector comprising the polynucleotide of SEQ ID NO: 11 obtained in Example 1 was used as a template, and a pair of primers for introducing F213V (SEQ ID NOs: 23 and 24), a pair of primers for introducing F289L (SEQ ID NOs: 25 and 26) or F356S Recombinant PCR was performed using introduction primer pairs (SEQ ID NOs: 27 and 28) to prepare respective polynucleotides, to obtain expression vectors into which the prepared polynucleotides were introduced. It was introduced into BL21 (DE3) to prepare each strain strain (variants 4 to 9) (Table 1).

F213V forward primer: 5'-TACTGATGCAGTTGCAATTCAAGCA-3 '(SEQ ID NO: 23)

F213V reverse primer: 5'-GAATTGCAACTGCATCAGTAACTGG-3 '(SEQ ID NO: 24)

F289L forward primer: 5'-CGTTTGCGCACTCAACCTAATTGCT-3 '(SEQ ID NO: 25)

F289L reverse primer: 5'-TTAGGTTGAGTGCGCAAACGTGTGG-3 '(SEQ ID NO: 26)

F356S forward primer: 5'-AAAAACGCATCCGAAGACGTATGTA-3 '(SEQ ID NO: 27)

F356S reverse primer: 5'-ACGTCTTCGGATGCGTTTTTCGCTA-3 '(SEQ ID NO: 28)

Table 1 Variant MDH Created Using Variant MDH in Variant 1

Variant	F213V	F289L	F356S	SEQ ID NO:	Remarks
Negative Control	×	×	×	One	Wild type MDH
Positive control group	○	○	○	2	MDH of variant 1
4	○	○	×	5
5	×	○	○	6
6	○	×	○	7
7	○	×	×	8
8	×	○	×	9
9	×	×	○	10

Example 5-2: Production of Mutant MDH

The control prepared in Example 5-1 and each of the variants (variants 4 to 9) were inoculated in 5 ml of minimal liquid medium (2YT, 50 µg / ml empicillin), incubated at 37 ° C, and grown. Inoculated in 100 ml of minimal liquid medium and incubated at 37 ° C. until absorbance (OD 600) was 0.4. Subsequently, 0.1mM IPTG was added to the cultures, and shake cultured at 18 ° C. for 48 hours, thereby expressing each of the mutant MDHs and confirming the expression through SDS-PAGE (FIG. 3). Figure 3 is an electrophoresis picture showing the change in the expression of mutant MDH expressed from each variant prepared in Example 5-1, U represents a culture without the addition of IPTG, I is the solid content of the culture to which the IPTG is added S represents the water-soluble content of the culture to which IPTG was added. As shown in FIG. 3, it was confirmed that the mutant MDH was overexpressed in each variant by IPTG addition.

Example 5-3 Purification of Mutant MDH

Centrifugation (5000rpm, 15 minutes, 4 ℃) of the culture of each variant cultured in Example 5-2 to recover each cell, and the recovered cells were buffered (20mM Tris, 0.5M NaCl, pH 7.5 Suspension to give a suspension, to which was added a proteinase inhibitor (complete mini, Roche, USA). The suspension was sonicated (10 mm diameter 750 W microtip, 20% amplitude, 4 ° C., 6 minutes) to break up each cell to obtain a lysate, and the lysate was centrifuged (10000 rpm, 20 minutes, 4 ° C.), respectively. Supernatant of was obtained. Each obtained supernatant was subjected to HisTrap FF column chromatography to adsorb variant MDH, eluting variant MDH by adding 250 mM imidazole (pH 7.5) buffer solution to purify variant MDH.

Example 5-4 Analysis of Reducing Activity of Mutant MDH

MDH consumes NADH as a cofactor when reducing formaldehyde to methanol. Thus, the amount of NADH consumed during the reduction reaction was measured, and the reduction activity of each variant MDH purified in Example 5-3 was compared.

Specifically, 0.6mM NADH and 50mM formaldehyde were added to 500µl of 100mM KH ₂ PO ₄ (pH 7.5) buffer, followed by adding 10 µg of MDH purified in Example 5-3 for 2 hours. While reacting, the absorbance was measured at 340 nm to calculate the change in content of NADH contained in the reactants (FIG. 4). Figure 4 is a graph showing the change with the reaction time of the reduction activity of each variant MDH purified in Example 5-3. As shown in Figure 4, among the variant MDH (SEQ ID NOs: 5 to 10) expressed in variants 4 to 9 except for the variant MDH (SEQ ID NO: 10) expressed in variant 9 (SEQ ID NOs: 5 to 9) Were found to show better reducing activity than the wild type MDH (SEQ ID NO: 1) and variant MDH (SEQ ID NO: 2) expressed in Variant 1. In particular, among the five mutant MDHs (SEQ ID NOs: 5 to 9), the mutated MDH of SEQ ID NO: 5 showed the best reducing activity, whereas the mutated MDH of SEQ ID NO: 10 showed a lower level of reducing activity than the wild type MDH. It was.

In addition, based on the time taken for the cofactor (NADH) contained in the reaction solution to be reduced by half, it was confirmed that the mutated MDH of SEQ ID NO: 5 exhibits a reducing activity approximately twice that of the wild type MDH. .

Claims (12)

1. The mutant methanol dehydrogenase, wherein the mutation is introduced into the wild type methanol dehydrogenase (MDH) consisting of the amino acid sequence of SEQ ID NO: 1, the reducing activity is increased than the MDH.
2. The method of claim 1,

   A mutated methanol dehydrogenase, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-9.
3. A polynucleotide encoding the variant methanol dehydrogenase of claim 1.
4. The method of claim 3,

   A polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12-19.
5. An expression vector comprising the polynucleotide of claim 3.
6. A transformant transformed by introducing the expression vector of claim 5.
7. (a) culturing the transformant of claim 6 to obtain a culture; And,

   (b) recovering the mutated methanol dehydrogenase of claim 1 from the culture, the method of producing the mutated methanol dehydrogenase of claim 1.
8. (a) reacting the mutated methanol dehydrogenase of claim 1 or 2 with a mixture comprising formaldehyde and an electron donor to obtain a reaction product; And

   (b) recovering methanol from the reaction product.
9. The method of claim 8,

   Wherein said electron donor is NADH.
10. The method of claim 8,

    Wherein said mixture further comprises a reaction buffer.
11. The method of claim 10,

    The reaction buffer is a neutral phosphate buffer.
12. Use of the mutated methanol dehydrogenase of claim 1 for producing methanol from formaldehyde.

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