KR101630740B1 - Mutant 3-Hydroxybutyrate Dehydrogenase - Google Patents

Abstract

The present invention relates to a mutant 3-hydroxybutyrate dehydrogenase and a gene thereof. More specifically, the present invention relates to an enzyme and a gene thereof mutated in two of 3-hydroxybutyrate dehydrogenase genes isolated from Alcaligenes faecalis , and converting levulinic acid into 4-hydroxyvaleric acid.

Description

The mutant 3-hydroxybutyrate dehydrogenase {Mutant 3-Hydroxybutyrate Dehydrogenase}

The present invention relates to a mutant 3-hydroxybutyrate dehydrogenase (3HBDH) and its gene. More specifically, the present invention relates to the use of an Alcaligenes < RTI ID = 0.0 > relates to a 3-hydroxybutyrate dehydrogenase enzyme gene and the mutant gene were separated from the two positions of faecalis), unlike the wild-type aminolevulinic acid at a high yield (levulinic acid) 4-hydroxy-valeric acid (4- hydroxyvaleric acid).

In recent industrial biotechnology field, it is attracting attention to produce various useful chemicals and biofuels by using renewable biomass. Of the renewable biomass, cellulose-based biomass is easy to obtain in a wide range of locations, is inexpensive compared to grain biomass, and is also free from food problems because it is not used as food. Which is particularly suitable for use. A common method of using cellulosic-based biomass is by using a cellulose enzyme. Cellulase hydrolyzes cellulose into sugar, and the resulting sugar can be fed into the feed of industrial microorganisms to produce industrially useful products. However, due to the structural characteristics of cellulose, which is difficult to decompose, the efficiency of cellulase is low, and the high price of cellulase is also a hindrance to practical application.

On the other hand, recently, a method using levulinic acid or 4-oxopentanoic acid as an alternative route using cellulosic-based biomass has received attention. Levulinic acid is a versatile synthetic intermediate that is relatively easy to obtain by treating cellulose as an acid, and can be used to make other useful chemicals such as nylon, synthetic rubber, plastics, and pharmaceuticals. Levulinic acid has also been recognized for its application value as one of the 12 building block chemicals designated by the Department of Energy (DOE) in the United States.

The levulinic acid obtained from cellulose can be converted into a variety of useful chemicals through subsequent conversion reactions. From a chemical structural point of view, levulinic acid is a five-carbon gamma-ketoacid, which has a ketone group and a carboxyl group. 4-hydroxyvaleric acid or 4-hydroxypentanoic acid is produced when the ketone group of levulinic acid is reduced to a hydroxyl group (Martin et al., 2009) 4-Hydroxyvaleric acid can be usefully used as a unit of bio-polyester, a precursor of bio-fuel, and the like (Corma et al., 2007).

Since most levulinic acid conversion reactions that produce 4-hydroxyvaleric acid by reducing levulinic acid are performed through the use of a chemical catalyst under high-temperature and high-pressure conditions, there is a high energy consumption and a high risk of operation, And low environmental friendliness. Enzymes have the advantage of being environmentally friendly in low-temperature, low-pressure and low-energy conditions, but enzymes that make levulinic acid a native substrate have never been known.

 Martin, C. H., Prather, K.L.J., 2009. High-titer production of monomeric hydroxyvalerates from levulinic acid in Pseudomonas putida. J. Biotechnol. 139 (1), 61.67.  Corma Canos, A., Iborra, S., Velty, A., 2007. Chemical routes for transformation of biomass into chemicals. Chem. Rev. 107 (6), 2411.2502.

The present invention been made in view of the above problems, an alkaline paper Ness par faecalis (Alcaligenes The present invention provides an enzyme that is biosynthesized by mutating two of 3-hydroxybutyrate dehydrogenase genes isolated from E. faecalis .

It is another object of the present invention to provide the mutated 3-hydroxybutyrate dehydrogenase gene.

It is still another object of the present invention to provide a plasmid comprising the mutated 3-hydroxybutyrate dehydrogenase gene.

The mutant 3-hydroxybutyrate dehydrogenase of the present invention is characterized by comprising the amino acid sequence of SEQ ID NO: 1 in order to achieve the above-mentioned object.

In addition, the 3-hydroxybutyrate dehydrogenase can convert levulinic acid to 4-hydroxyvaleric acid.

In addition, the 3-hydroxybutyrate dehydrogenase can be biosynthesized by mutating the one isolated from Alcaligenes faecalis .

Meanwhile, the 3-hydroxybutyrate dehydrogenase gene of the present invention is characterized in that the 3-hydroxybutyrate dehydrogenase is encoded.

In addition, the gene may include the gene sequence of Sequence Listing 2.

On the other hand, the recombinant plasmid of the present invention is characterized by containing the gene.

The present invention has the effect of purifying the 3-hydroxybutyrate dehydrogenase under the conditions of low temperature and low pressure in the conventional reaction conditions of high temperature and high pressure by imparting the ability to convert levulinic acid, which did not show activity in the wild type. As a result, the process is safer, the environment friendliness is enhanced, and facility investment and energy consumption are reduced.

1 is a structural reaction scheme of a reaction catalyzed by a wild-type 3-hydroxybutyrate dehydrogenase and a mutant 3-hydroxybutyrate dehydrogenase of the present invention, respectively.
FIG. 2 is a graph showing the conversion of wild-type 3-hydroxybutyrate dehydrogenase and the mutant 3-hydroxybutyrate dehydrogenase of the present invention, respectively, over time in the catalysed reaction. FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, numerous specific details, such as specific elements, are set forth in order to provide a thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without these specific details, It will be obvious to those who have knowledge of. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

The present invention aims at producing 4-hydroxyvaleric acid by reducing levulinic acid. Most of the levulinic acid conversion reactions are carried out through the use of chemical catalysts under high temperature and high pressure conditions. Therefore, there are drawbacks such as high energy consumption, high risk of operation, high facility investment cost, and low environmental friendliness. In the case of using the enzyme, since the reaction can be carried out environmentally friendly under a low-temperature and low-pressure safe and low energy consumption condition, the present invention aims to develop a reduction reaction of levulinic acid using an enzymatic biological reaction.

However, since enzymes that make levulinic acid a native substrate are not known, it is necessary to improve existing enzymes using protein engineering. 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis (3-hydroxybutyrate dehydrogenase from Alcaligenes Faecalis , EC 1.1.1.30) uses reduction reaction with levulic acid (C5) and acetoacetate (C4) with one structural difference of carbon as the main substrate. However, this enzyme does not reduce levulinic acid. Therefore, in the present invention, the substrate specificity of 3-hydroxybutyrate dehydrogenase is improved to reduce levulinic acid to produce 4-hydroxyvaleric acid. Finally, a rational design method based on the analysis of the protein structure of the enzyme was applied to detect the presence of double mutant enzymes (eg, 144-histidine in leucine and 187-tryptophan in phenylalanine) (H144L / W187F double mutant). The enzyme reduced levulinic acid to yield 4-hydroxyvaleric acid at a conversion of 58%.

Specifically, the mutant 3-hydroxybutyrate dehydrogenase of the present invention is characterized in that it comprises the amino acid sequence of Sequence Listing 1.

[Sequence Listing 1]

MLKGKKAVVT GSTSGIGLAM ATELAKAGAD VVINGFGQPE DIERERSTLE SKFGVKAYYL 60

NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAALPIMQ KQGWGRIINI ASALGLVASV NKSAYVAAKH GVVGLTKVTA LENAGKGITC 180

NAICPGFVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGGAAVFLSS 240

AAADQMTGTT LSLDGGWTAR

The present invention also provides a gene coding for the 3-hydroxybutyrate dehydrogenase, which comprises the gene sequence of SEQ ID NO: 2.

[Sequence Listing 2]

atgttaaaag gaaaaaaagc agttgttacc ggtagcacct caggtatcgg actggcaatg 60

gcaactgagc tggcaaaagc tggcgctgat gttgtcatta atgggtttgg ccagccagaa 120

gacattgaac gtgaaagaag caccctggag tctaaattcg gtgtaaaagc gtattatctc 180

aatgcggatc tttccgacgc ccaagctacg cgtgatttta tagcaaaagc agcagaagct 240

ctgggtggtc ttgatattct ggttaataat gcaggtatcc aacacacagc accgatcgaa 300

gagtttcctg ttgataaatg gaatgcaata attgcactga acttaagcgc agtatttcat 360

ggaactgcag cagcactgcc aataatgcaa aaacagggtt gggggcgcat cattaatatt 420

gcttcggcac atggtctagt tgctagtgta aacaaatcag catacgttgc tgcaaagcat 480

ggtgttgttg gccttaccaa ggttacagca ttggaaaacg ctggcaaagg tattacatgt 540

aacgcaattt gcccgggatg ggttcggact ccgctggtgg aaaagcagat tgaagcaata 600

tcacagcaga aagggatcga tatcgaggca gcagctcgtg agctgctcgc agaaaaacag 660

ccttctttgc aatttgttac ccccgaacag ttagggggtg cggcagtctt cttatcttcc 720

gcagctgcag accagatgac gggaacgaca cttagtttgg atggtggctg gacggcacgc

Furthermore, the present invention also covers recombinant plasmids containing the gene.

Hereinafter, embodiments of the present invention will be described.

Example

reagent

The 3HBDH gene was commercially synthesized from Bioneer (Daejeon, Korea). Restriction enzymes (NdeI, XhoI) and DNA ligase were purchased from Enzynomics (Daejeon, Korea). Acceptance Escherichia coli Top 10 and E. coli BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA) and Novagen (Madison, WI, USA), respectively. Levulinic acid, lithium acetoacetate, NADH and ammonium formate were purchased from Sigma (St. Louis, Mo., USA). (Vasavada, M., Carpenter, CE, Cornforth, DP, Ghorpade, V., 2003. Sodium levulinate and sodium lactate effects as a substrate for enzyme analysis, on microbial growth and stability of fresh pork and turkey sausages. J. Muscle Foods 14 (2), 119.129).

Positioning mutations, expression and purification of enzymes

The 3HBDH gene was cloned into the pET-22b (+) vector (Novagen, USA) for the production of recombinant plasmids for expression of proteins with C-terminal 6-histidine tags. Mutations were made with the modified QuickChange (TM) site-directed mutagenesis protocol (Zheng et al., 2004). For protein expression, wild-type and mutant plasmids were transformed into recipient E. coli BL21 (DE3). Cells were induced by the addition of 1 mM IPTG (isopropyl bD-1-thiogalactopyranoside) and cultured at 20 ° C for 3 hours for 3HBDH expression. The wild-type and mutant enzymes were purified using a Ni-NTA agarose column and the purity was determined by 12% SDS-PAGE. Enzyme concentrations were determined by the Bradford method (Bradford, 1976).

Enzyme analysis and kinetics  analysis

For enzyme activity assay, the reduction of acetoacetate and levulinic acid was monitored by measuring the rate of oxidation of NADH using a UV spectrophotometer (Varian, USA) at 340 nm. Activity was determined in 50 mM sodium phosphate (pH 6.0) supplemented with 2.7 μM 3HBDH, 0.20 mM NADH and 0.60 mM levulinic acid at 30 ° C. for the selection of mutants with catalytic activity on levulinic acid. For kinetic analysis of 3HBDH, K _m and k _cat values for each substrate were measured at an optimal pH of 50 mM sodium phosphate buffer (pH 6.0) containing 0.25 mM NADH at 30 ° C.

Enzyme reactions and HPLC  analysis

The enzyme reaction was initiated by the addition of 12 μM 3HBDH (final concentration) to 50 mM sodium phosphate buffer (pH 6.0) containing 30 mM NADH and 12 mM levulinic acid at 30 ° C. Reaction samples were obtained at defined time intervals and were obtained from Martin and Prather (Martin, CH, Prather, KLJ, 2009. High-titer production of monomeric hydroxyvalerate from levulinic acid in Pseudomonas putida, J. Biotechnol. (Valentin, HE, Schonebaum, A., Steinbuchel, A., 1992. Identification of 4-hydroxyvaleric acid as a constituent of biosynthetic polyhydroxyalkanoic acids from bacteria. Appl. Microbiol. Biotechnol. 36 (4), 507.514) And analyzed by HPLC as a modification method. HPLC samples were analyzed on a Finnigan Surveyor Plus HPLC system (Thermo Scientific) equipped with an Agilent ZORBAX SB-Aq reversed phase column (4.6 x 250 mm, 5 μm).

Simulation of molecular docking

Molecular docking simulations were performed by partial protein modeling using the SYBYL-X package (Tripos, ver. 1.2). The crystal structure of 3HBDH (pdb code: 3VDR) was used to determine the optimum docking position of levulinic acid in the binding pocket. Surflex docking of the substrate was performed using Run-Multiple ligand option of Surflex-Dock and ΔG _b (free bond energy) value was calculated.

3 HBDH Rational Design and Mutation Research

Since the wild-type enzyme does not exhibit catalytic activity on levulinic acid (Table 2), an engineering approach to the substrate specificity of 3HBDH was made for enzymatic reduction of levulinic acid. The binding properties of the wild type 3HBDH to native substrate (acetoacetate) were analyzed and a strategy for mutagenesis was proposed as shown in Table 1. According to this strategy, six single mutations (Gln94Asn, His144Leu, Lys152Ala, Gln196Asn, Trp187Phe and Trp257Phe) were selected for substrate specificity manipulation of 3HBDH (Table 1).

Binding characterization and mutation strategies Substrate binding residue Analysis of binding characteristics Mutation strategy Mutation Gln94, His144,
Lys152,
Gln196 Hydrogen bond formation with carboxyl group and / or ketone group of acetoacetate Selective mutagenesis with homologous and / or homologous hydrophobic residues to reconstruct the hydrogen bonding network between residues and levulinic acid Gln94Asn, Gln196Asn
(Single-ended)
His144Leu, Lys152Ile
(With a homogeneous hydrophobicity) Trp187, Trp257 Formation of hydrophobic interaction with carbon atoms of acetoacetate Mutagenized to small hydrophobic residues, to further secure binding pocket space, to maintain desirable hydrophobic interactions Trp187Phe,
Trp257Phe
(With small hydrophobicity) Ser142, Tyr155 Direct association with catalysis Excluded from mutant candidates

In vitro activity assays for the six selected single mutants selected above for the selection of mutants with catalytic activity for levulinic acid were performed with levulinic acid, with the result that the His144Leu mutation was the only catalytic activity (Table 2 ).

The results of molecular docking and the activity of the 3HBDH mutation on levulinic acid 3HBDH activation
(μmol / min / mg) ΔG _b ^a
(kcal / mol) d ₁
(A) d ₂
(A)   Wild type ND ^b     -8.3 4.9 5.9   Gln94Asn    N.D. NA ^c N.A. N.A.   His144Leu    0.035     -9.1 3.4 3.0   Lys152Ile    N.D.     -6.7 4.3 5.3   Trp187Phe    N.D.     -8.5 4.1 4.7   Gln196Asn    N.D.     -8.0 4.9 6.0   Trp257Phe    N.D.     -7.2 4.2 5.5   His144Leu / Trp187Phe    0.390    -10.1 3.4 3.0

^a bond free energy

^{b Not} measurable due to very low activity

^c No docking (no ligand binding to enzyme)

According to the reaction mechanism of 3HBDH obtained from alkaline jinestae paeallis, the distance (d _{1 , std} = 3.8 Å) between the C4 atom of NADH and the C3 atom of acetoacetate and the oxygen atom of Tyr155 and the ketone group oxygen of acetoacetate The distance between atoms (d _{2 , std} = 3.1 Å) was found to be important for catalysis. At this point, for the reduction of levulinic acid by the engineered 3HBDH, the distance (d ₁ ) between the C4 atom of NADH and the C4 atom of levulinic acid and the distance between the oxygen atom of Tyr155 and the ketone group oxygen atom of levulinic acid (D ₂ ) of the _stiffness should be d _{1 , std} and d _{2 , std} value _, respectively. This prediction was applied to the evaluation of the 3HBDH mutation along with the molecular docking simulation.

Molecular docking simulations for six 'single' mutations were performed, from which d ₁ , d ₂ and ΔG _b values were obtained. Negative ΔG _b values with large absolute values indicate strong binding affinities between the enzyme and the substrate (Table 2). Of the docking results, only His144Leu showed suitable d ₁ (3.4 Å) and d ₂ (3.0 Å) values and a desirable ΔG _b value (-9.1 kcal / mol). Based on these results, the His144Leu mutation was found to have activity against levulinic acid, and as a result, was selected as the initial mutation for further activity improvement.

Additional mutation studies for selection of improved mutants

In addition to the mutations His144Leu, 15 are of the additional production of the double mutations for the 6 combinations of single mutations (C _{2 6),} was tested in vitro activity assays (on the results shown). Only one double mutant (His144Leu / Trp187Phe) exhibited enzyme activity in levulinic acid (Table 2), showing about a 11-fold increase in activity compared to a single mutant (His144Leu) (Table 2). From these results, His144Leu / Trp187Phe was selected as the best double mutation for enzymatic reduction of levulinic acid.

Structure review and dynamical analysis

In three mutants (His144Leu, Trp187Phe and His144Leu / Trp187Phe), kinetic parameters for acetoacetate and levulinic acid were determined (Table 3). Kinetic studies have supported that the His144Leu mutation has an initial activity on levulinic acid. Structural review of the docking results also showed that the presence of a group between His144 and levulinic acid in the wild-type enzyme, in which the substrate is inadequate to convert to a product, is destroyed by mutation, To a desirable combination.

enzyme
Acetoacetate Levulinic acid K _m
(mM) k _cat
(Min ^-1 ) k _cat / K _m
(Min ^-1 M ^-1 ) K _m
(mM) k _cat
(Min ^-1 ) k _cat / K _m
(Min ^-1 M ^-1 ) Wild type 10.47 ± 1.17 293.87 ± 19.46 28.172 ± 1305 ND ^a  N.D.  N.D. His144Leu 2.42 ± 0.13 47.24 + 1.89 19.527 ± 879 16.98 ± 0.59 1.65 + 0.08 97.15 + - 5.34 Trp187Phe 48.70 + - 5.00 492.02 + - 33.60 9534 ± 590  N.D.  N.D.  N.D. His144Leu
/ Trp187Phe 2.53 + - 0.21 50.79 ± 2.26 20.111 ± 833 8.25 ± 0.15 4.77 ± 0.19 578.03 + - 11.94

^{a Not} measurable due to very low activity

The Trp187Phe mutation alone did not show catalytic activity on levulinic acid (Table 3). However, the double mutant (His144Leu / Trp187Phe) showed an approximately 6-fold increase in catalytic efficiency (k _cat / K _m) than a single His144Leu mutation (Table 3). The increased catalytic efficiency (k _cat / K _m ) of double mutants to levulinic acid was due to an increase in both binding affinity (decrease in K _m ) and number of catalytic reactions (increase in k _cat ) (Table 3). The increased binding affinity can also be explained by the absolute value increase of? G _b (-9.1 to -10.1) obtained in the docking simulation (Table 2). Structure review shows that the binding pocket for the preferred combination of additional mutations in Trp187Phe initially single mutation (His144Leu) of the levulinic acid at a distance of d ₁ and d _2, and have more space, which is the catalyst efficiency .

In the mountains of Lesbouli  4- Hydroxy valeric acid Enzymatic  transform

HPLC analysis showed peaks for 4-hydroxyvaleric acid and levulic acid at retention times of 11.0 and 11.5 minutes, respectively, and enzymatic conversion of levulinic acid by wild-type and His144Leu / Trp187Phe mutations was performed. After 24 hours, about 57% of levulinic acid was converted to 4-hydroxyvaleric acid in the double mutation, but no conversion was observed in the wild type. As a result, it was confirmed that the modified 3HBDH had a novel catalytic activity for levulinic acid.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course it is possible. Accordingly, the scope of the present invention should not be construed as being limited to the above-described embodiments, but should be determined by equivalents to the appended claims, as well as the following claims.

<110> SNU R & DB FOUNDATION <120> Mutant 3-Hydroxybutyrate Dehydrogenase <130> M12-9241-FD <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Mutant of 3-Hydroxybutyrate Dehydrogenase from Alcaligenes          Faecalis <400> 1 Met Leu Lys Gly Lys Lys Ala Val Val Thr Gly Ser Thr Ser Gly Ile   1 5 10 15 Gly Leu Ala Met Ala Thr Glu Leu Ala Lys Ala Gly Ala Asp Val Val              20 25 30 Ile Asn Gly Phe Gly Gln Pro Glu Asp Ile Glu Arg Glu Arg Ser Thr          35 40 45 Leu Glu Ser Lys Phe Gly Val Lys Ala Tyr Tyr Leu Asn Ala Asp Leu      50 55 60 Ser Asp Ala Gln Ala Thr Arg Asp Phe Ile Ala Lys Ala Ala Glu Ala  65 70 75 80 Leu Gly Gly Leu Asp Ile Leu Val Asn Asn Ala Gly Ile Gln His Thr                  85 90 95 Ala Pro Ile Glu Glu Phe Pro Val Asp Lys Trp Asn Ala Ile Ile Ala             100 105 110 Leu Asn Leu Ser Ala Val Phe His Gly Thr Ala Ala Ala Leu Pro Ile         115 120 125 Met Gln Lys Gln Gly Trp Gly Arg Ile Ile Asn Ile Ala Ser Ala Leu     130 135 140 Gly Leu Val Ala Ser Val Asn Lys Ser Ala Tyr Val Ala Ala Lys His 145 150 155 160 Gly Val Val Gly Leu Thr Lys Val Thr Ala Leu Glu Asn Ala Gly Lys                 165 170 175 Gly Ile Thr Cys Asn Ale Ile Cys Pro Gly Phe Val Arg Thr Pro Leu             180 185 190 Val Glu Lys Gln Ile Glu Ala Ile Ser Gln Gln Lys Gly Ile Asp Ile         195 200 205 Glu Ala Ala Ala Arg Glu Leu Leu Ala Glu Lys Gln Pro Ser Leu Gln     210 215 220 Phe Val Thr Pro Glu Gln Leu Gly Gly Ala Ala Val Phe Leu Ser Ser 225 230 235 240 Ala Ala Ala Asp Gln Met Thr Gly Thr Thr Leu Ser Leu Asp Gly Gly                 245 250 255 Trp Thr Ala Arg             260 <210> 2 <211> 780 <212> DNA <213> Artificial Sequence <220> <223> Mutant of 3-Hydroxybutyrate Dehydrogenase from Alcaligenes          Faecalis <400> 2 atgttaaaag gaaaaaaagc agttgttacc ggtagcacct caggtatcgg actggcaatg 60 gcaactgagc tggcaaaagc tggcgctgat gttgtcatta atgggtttgg ccagccagaa 120 gacattgaac gtgaaagaag caccctggag tctaaattcg gtgtaaaagc gtattatctc 180 aatgcggatc tttccgacgc ccaagctacg cgtgatttta tagcaaaagc agcagaagct 240 ctgggtggtc ttgatattct ggttaataat gcaggtatcc aacacacagc accgatcgaa 300 gagtttcctg ttgataaatg gaatgcaata attgcactga acttaagcgc agtatttcat 360 ggaactgcag cagcactgcc aataatgcaa aaacagggtt gggggcgcat cattaatatt 420 gcttcggcac atggtctagt tgctagtgta aacaaatcag catacgttgc tgcaaagcat 480 ggtgttgttg gccttaccaa ggttacagca ttggaaaacg ctggcaaagg tattacatgt 540 aacgcaattt gcccgggatg ggttcggact ccgctggtgg aaaagcagat tgaagcaata 600 tcacagcaga aagggatcga tatcgaggca gcagctcgtg agctgctcgc agaaaaacag 660 ccttctttgc aatttgttac ccccgaacag ttagggggtg cggcagtctt cttatcttcc 720 gcagctgcag accagatgac gggaacgaca cttagtttgg atggtggctg gacggcacgc 780                                                                          780

Claims (6)

3-hydroxybutyrate dehydrogenase consisting of the amino acid sequence of Sequence Listing 1. The method according to claim 1,
Wherein the 3-hydroxybutyrate dehydrogenase converts levulinic acid to 4-hydroxyvaleric acid. 3. The 3-hydroxybutyrate dehydrogenase according to claim 1, wherein the 3-hydroxybutyrate dehydrogenase converts levulinic acid to 4-hydroxyvaleric acid. The method according to claim 1,
Wherein the 3-hydroxybutyrate dehydrogenase is a mutant in which the 3-hydroxybutyrate dehydrogenase is mutated from Alcaligenes faecalis . A gene of 3-hydroxybutyrate dehydrogenase encoding the enzyme of claim 1. The method of claim 4,
The gene of 3-hydroxybutyrate dehydrogenase gene is characterized in that the gene consists of the gene sequence of Sequence Listing 2. A recombinant plasmid comprising the gene of claim 4.

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