JP7294672B2 - Method for producing unsaturated uronic acid reductase and alginic acid derivative - Google Patents

Description

The present invention relates to the production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) reductase, which is an unsaturated uronic acid, and alginic acid derivative from alginic acid. 2-keto-3-deoxy-D-gluconic acid (2-keto-3-deoxy-D-gluconate: KDG), etc.

Looking at the percentage of world production by type of aquatic plants, brown algae account for 68.6% of the 7 million tons, and alginic acid is the largest constituent polysaccharide, which accounts for about 30%. Some brown algae, such as kelp and wakame seaweed, are used as food, but most of them are unused resources. Brown algae do not require arable land, fresh water, or fertilizer, and exist naturally in a gel-like state. Compared to terrestrial biomass, brown algae are attracting attention for their advantages such as easier enzymatic reactions. In particular, Japan is surrounded by the sea, boasting one of the largest ocean areas in the world. Moreover, since alginic acid is the most abundant polysaccharide constituting brown algae, the use of alginic acid is important.

Alginic acid is a linear polymer in which two types of uronic acids, β-D-mannuronic acid and its C5 epimer α-L-guluronic acid, are linked by glycosidic bonds. It is composed of a (M) block, a poly (G) block bound with α-L-guluronic acid, and a poly (M G) block bound alternately with β-D-mannuronic acid and α-L-guluronic acid. (Fig. 1), its molecular weight is about 200,000 to 2,000,000. Alginic acid is degraded by endo-type and exo-type alginate lyases to unsaturated uronic acid, which is an alginate monosaccharide, and 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) is generated by cleaving the pyranose ring. do. After that, DEH is reduced by DEH reductase (DehR; DEH reductase) and its coenzyme NADH or NADPH to produce 2-keto-3-deoxy-D-gluconate (KDG) (Fig. 2). . KDG is metabolized to pyruvate through the Entner-Doudoroff pathway, which is very similar to glycolysis. On the other hand, KDG is also produced by microorganisms that metabolize pectin contained in the cell walls and middle leaves of land plants. However, production from alginic acid contained in brown algae in the sea using metabolism-related enzymes of alginate-utilizing microorganisms requires fewer types of enzymes than production from pectin, making it easier to produce KDG. can. However, in terms of the number of research reports, the route to produce KDG from pectin was overwhelmingly numerous, and the route to produce KDG from alginic acid was very few.

The present inventor's research has made it possible to produce DEH from alginic acid (Patent Document 1). Therefore, it is considered that mass production of KDG will become possible if DEH reductase can be mass produced. Both DEH and KDG are intermediate metabolites of alginate-utilizing microorganisms, and it is difficult to extract them from natural products. DEH and KDG are monosaccharides or derivatives from monosaccharides and are rare sugars. DEH is not on the market at the moment, and KDG is very expensive although it is on the market. Alginic acid oligosaccharides before reaching DEH from alginic acid have various functionalities such as human epidermal keratinocyte activating action, ET-1 and MMP-9 production inhibitory action, and fibroblast proliferation promoting agent. It is used as an anti-aging effect in cosmetics raw materials and functional foods. Since DEH and KDG are also expected to have similar effects, a method for mass-producing these substances at low cost has been desired.

If it becomes possible to mass-produce KDG from brown algae, it can be expected to be applied to biorefineries. KDG becomes pyruvate through the Entner-Doudoroff pathway, and from pyruvate, various organic acids, alcohols, and fatty acids can be produced. Farming brown algae does not require large areas of land, water, or fertilizer, so the use of brown algae as biomass does not pose a significant food or fuel conflict. If the KDG production route from brown algae is established, it can be expected to be used as biomass that can be procured domestically. Alginic acid and its metabolites are also used as a carbon source for ethanol fermentation by genetic recombination of E. coli and yeast. DEH reductase is a key enzyme for alginate metabolism in bacteria because, unlike DEH, many bacteria can assimilate KDG.

There are only a few known DEH reductases, and papers identifying KDG by LC / MS are A1-R (Non-Patent Documents 1 and 2), FlRed (Non-Patent Document 3), HdRed (Non-Patent Document There are only 3 examples of 4). Furthermore, among the above three enzymes, only A1-R has been subjected to kinetic analysis. Thus, DEH reductase is essential for mass production of KDG, but information on DEH reductase has been limited.

WO2017/175694

Takase, R., Ochiai, A., Mikami, B., Hashimoto, W., and Murata, K. (2010) Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1. Biochim. Biophys. Acta, 9, 27 Takase, R., Mikami, B., Kawai, S., Murata, K., and Hashimoto, W. (2014) Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism. J. Biol. Chem., 289, 33198-33214 Inoue, A., Nishiyama, R., Mochizuki, S., and Ojima, T. (2015) Identification of a 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase, FlRed, in an alginolytic bacterium Flavobacterium sp. strain UMI-01. Mar. Drugs, 13, 493-508. Mochizuki, S., Nishiyama, R., Inoue, A., and Ojima, T. (2015) A novel aldo-keto reductase, HdRed, from the pacific abalone Haliotis discus hannai, which reduces alginate-derived 4-Deoxy-L -erythro-5-hexoseulose uronic acid to 2-keto-3-deoxy-D-gluconate. J. Biol. Chem., 290, 30962-30974 Tanaka, R., Cleenwerck, I., Mizutani, Y., Iehata, S., Shibata, T., Miyake, H., Mori, T., Tamaru, Y., Ueda, M., Bossier, P., and Vandamme, P. (2015) Formosa haliotis sp. nov., a brown-alga-degrading bacterium isolated from the gut of the abalone Haliotis gigantea. Int. J Syst. Tanaka, R., Mizutani, Y., Shibata, T., Miyake, H., Iehata, S., Mori, T., Kuroda, K., and Ueda, M. (2016) Genome Sequence of Formosa haliotis Strain MA1 , a Brown Alga-Degrading Bacterium Isolated from the Gut of Abalone Haliotis gigantea. Genome Announc., 4, 01312-01316 Takase, R., Mikami, B., Kawai, S., Murata, K., and Hashimoto, W. (2014) Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism. J. Biol. Chem., 289, 33198-33214

Reiji Tanaka, one of the inventors of the present invention, isolated a new species of seaweed body-disintegrating bacterium Formosa haliotis MA1 strain from the digestive tract of abalone, and demonstrated that it has a high ability to assimilate alginic acid (non Patent document 5). As a result of whole genome analysis of this strain, two genes sdr1 and sdr2, which are presumed to be short-chain dehydrogenases/reductases (SDR), were discovered in the alginate degrading enzyme gene cluster (Fig. 3). However, the functions of these genes when expressed as proteins remained unclear. Therefore, in order to investigate whether sdr1 acts as a DEH reductase, we constructed a recombinant SDR1, identified the reaction products, and investigated their enzymatic properties.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for efficiently producing KDG using an enzyme that reduces DEH to produce KDG.

In the method for producing KDG according to the invention for solving the above problems, the amino acid sequence shown in SEQ ID NO: 2 is added to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH), which is an unsaturated uronic acid. 2-keto-3-deoxy-D-gluconate, which is an alginic acid derivative, by contacting a polypeptide having the amino acid sequence or a polypeptide having an amino acid sequence having at least 85% homology with the amino acid sequence and having DEH reducing activity (KDG) is manufactured.
According to this invention, KDG can be produced from DEH with a high efficiency of 90% or more.
In addition, a plasmid for expressing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) reductase according to another invention comprises a DNA having the nucleotide sequence set forth in SEQ ID NO: 1, and the amino acid sequence set forth in SEQ ID NO: 2. or a DNA encoding a polypeptide having DEH-reducing activity and having an amino acid sequence having at least 85% homology with the amino acid sequence set forth in SEQ ID NO: 2 is incorporated in an expressible form. It is characterized by
According to the present invention, DEH reductase can be produced using appropriate hosts (microorganisms (Escherichia coli, yeast, etc.), cultured cells (animal cells, plant cells, insect cells, etc.), individual organisms, etc.).

Further, in a method for producing DEH reductase according to another invention, the DEH reductase expression plasmid is for E. coli, and after incorporating the plasmid into E. coli, an enzyme induction step of expressing DEH reductase, induction and an enzyme purification step of purifying the DEH reductase that has been purified.
According to the present invention, using E. coli, DEH reductase can be rapidly produced in large amounts at a low cost.
In addition, a method for producing KDG according to another invention provides (a) to alginic acid, (1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 5 or having at least 90% homology with the amino acid sequence a polypeptide having an amino acid sequence and exo-alginate lyase activity; and (2) a polypeptide having the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 90% homology with the amino acid sequence. and (3) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 85% homology with said amino acid sequence, 4-deoxy-L- an enzyme addition step of contacting a polypeptide having erythro-5-hexoseulose uronic acid (DEH) reducing activity; and an enzymatic action step in which the activity is maintained at a temperature at which the DEH reduction activity works.

Amino acid sequence shown in SEQ ID NO: 5 (MSTENKSRSNLFPLDEPKAGRLTIQYGPLETTTLIENPPRFSWLPVIEDGATYALRISTDPEYSAANTLLFSGIQLNFFTPDAPLAAGTWYWSYAQCDASGKPVTEWSTSRRITLDEGLPQTPLAPRKTRFDAATRAHPRLWMDGGRLEQFRKDVAADPTHCTWSTFFEGSVLPWMDRDIIEEPVGYPDHKRVAKIWRKVYIECQEL MYAIRHLAVGGQVTQDAAMLARAKEWLLSAARWNPAGTTSRAYTDEWAFRVNLALAWGYDWLYDQLDEDERTLVRTALLERTRQTADHLMRHASIHLFPFDSHAVRAVSAVLIPACIALLDDEPEAEDWLNYAVEFLFTVYSPWGDHDGGWAEGPHYWMTGMAYLIDAANLLRGWSGIDLYQRPFFQKTGDFPLYTKAPDTRRATFGDDSTMGDLPAIKVGYNLRQYAG VTGNGAYQWYYDEILRTNPGTEMAFYNWGWWDFRFDEMLYRTDFPIVEAVPPADEDALRWFKGIGWVAIQHRMQAPDEHVQFVFKSSPYGSISHSHGDQNAFCLSAFGEDLAIQSGHYVAFNSTMHQNWRRQTLSKNAILIDGKGQYAGKDKAIAMQSTGKVNIAEDRGDHIFLQGDATEAYRTLSPEVRSVVRDVYFVNREYFVIVDAID ADTPVSIDWRLHANAPFNLGDSSFRYTGEKAGFYGQILWSEAGPAELTQETGFPDVDPSEIEGLPVSTCLTARFPKSTRHRIATLIVPYALDAPRRIFSFLDQGYDCDLYFTDANDNSFRVIVPKTFDVGTPGIKNN) is disclosed in Patent Document 1 as a polypeptide having exo-alginate lyase activity.

In addition, the amino acid sequence shown in SEQ ID NO: 6 (MSLKLRTFCLAGTATIFVALPSTYALAAGTGACTGVSQLAIVSASDDGTFDDIYAPEFAIDGEFGPSSRWSSLGEGKQLVLDLGEPQTVSEVGLAWYKGNERTSSFTLEASNDGENWMPLMDRTESAGKSEAVEKYSFDATEARYVRVTGMGNSASGWNSLYEAQVFGCGSGEIAATGDGSGEVKEADVSAYGLRTDVPPSENFDLTHWKLTLPADRDNDG KVDEIEEEELQGWSDPRFFYTDPATGGMVFRTAPDGKTTSGSHYTRSELREMIRGGDKSIATRVDDGTPNKNNWVFSTAPEEAQALAGGVDGTMTATLAVNHVTRTGESGKIGRVIIGQIHAMDDEPIRLYYRKLPTNKYGSIYFAHEPVGGDDDLVNVIGDRGSDIDNPADGIALDEVFSYEIKVTSEEKDGELHPILNVSITRDDGTVVKAEPYDMFESGYSTDKDF MYFKAGAYSQNNSITWPDFDQVTFYALDVTHGE) is disclosed in Patent Document 1 as a polypeptide having endo-alginate lyase activity.
According to this invention, KDG can be produced from alginic acid simply by adding three kinds of enzymes.

Amino acid sequence homology of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, more preferably 95%, 96%, 97% , 98% or 99%, or 100% identical.
A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or a polypeptide comprising an amino acid sequence having at least 85% or more homology with the amino acid sequence and having DEH-reducing activity naturally has DEH-reducing activity.
A polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is known as SDR1, but its activity remained unknown. This time, the present inventor's research revealed for the first time that DEH has the activity of reducing to KDG.
A polypeptide having an amino acid sequence having at least 85% homology with the amino acid sequence shown in SEQ ID NO: 2 has a DEH reduction activity of 30% or more, preferably Has a DEH reduction activity of 50% or more or 70% or more.

The DNA encoding a polypeptide having the above amino acid sequence and having DEH-reducing activity includes the nucleotide sequence of SEQ ID NO: 1 itself, a polypeptide having the amino acid sequence of SEQ ID NO: 2, or at least 85% of the amino acid sequence. It means a DNA that encodes a polypeptide having an amino acid sequence with 10% or more homology and having DEH-reducing activity. In DNA, three consecutive bases code for one amino acid, and depending on the type of amino acid, multiple codes may correspond. As for the DNA sequence, an appropriate sequence can be designated by referring to the genetic code, as long as it encodes the above-mentioned polypeptide. However, it is preferable to designate a vector with good protein expression according to the stability and expression system of the expression vector used for expression. When using a vector that expresses in bacteria (including E. coli), the DNA shown in SEQ ID NO: 1 can be used as the DNA encoding the amino acid sequence of SEQ ID NO: 2.

A polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is a novel enzyme having DEH reduction activity. This amino acid sequence showed only about 20-30% homology with the known amino acid sequences of various enzymes belonging to the SDR superfamily. The enzymatic activity was sufficiently strong compared with the conventional ones.
A polypeptide having an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO: 2 has a DEH reduction activity of 30% or more, preferably Has a DEH reduction activity of 50% or more or 70% or more.
SEQ ID NO: 7 shows the DNA sequence of sdr2, and SEQ ID NO: 8 shows the amino acid sequence of SDR2. A polypeptide having the amino acid sequence shown in SEQ ID NO: 8 is known as SDR2, but its activity remained unknown. It is highly likely that this polypeptide also has the activity of reducing DEH to KDG. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or a polypeptide comprising an amino acid sequence having at least 85% homology with the amino acid sequence and having DEH reduction activity has SDR1 or an activity similar to that of SDR1. It can be used similarly to polypeptides. Alternatively, a DNA having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8 or a polypeptide having an amino acid sequence having at least 85% homology with the amino acid sequence set forth in SEQ ID NO: 8 and having DEH reduction activity. As with sdr1, the encoding DNA can be used as a DEH reductase expression plasmid by incorporating it into a microorganism (such as E. coli) in an expressible form. In that case, the DNA shown in SEQ ID NO: 7 can be used as the DNA encoding the amino acid sequence of SEQ ID NO: 8.

In the present invention, "alginic acid" means a material used as a starting material for producing KDG. ) is included.
In the present invention, "contacting" the polypeptide means that the substrate (DEH or alginic acid) and the polypeptide are mixed in the form of a solid, an aqueous solution, or a suspension in a state in which the polypeptide exhibits an enzymatic activity. means to The substrate and the three polypeptides may be in different forms prior to mixing.

In the present invention, the "temperature at which alginate lyase activity and DEH reduction activity work" refers to a predetermined time (e.g., 72 hours or less, preferably 24 hours) without losing the alginate lyase activity and DEH reduction activity of the polypeptide. hereinafter, more preferably 3 hours or less) means a temperature at which it can act. Temperature conditions include other reaction conditions (for example, conditions such as alginic acid concentration, polypeptide concentration, type of aqueous solution (water containing no components other than alginic acid and polypeptide, aqueous solution containing appropriate salt, etc.)). can be determined as appropriate.
When three types of polypeptides (endo-type alginate lyase, exo-type alginate lyase, and DEH reductase) are brought into contact with alginic acid, the three types of polypeptides can be brought into contact simultaneously or in an appropriate order. can also When they are brought into contact in an appropriate order, (1) first, alginic acid, endo-alginate lyase, and exo-alginate lyase are mixed and treated under appropriate reaction conditions (temperature, pH, etc.) to obtain a large number of ( After (most) alginic acid is converted to DEH, (2) DEH reductase is added and treated under other appropriate reaction conditions (temperature, pH, etc.) to efficiently reduce DEH to KDG.

According to the present invention, it is possible to provide a method for efficiently producing KDG from DEH. KDG can also be produced by contacting alginic acid with three types of enzymes. Therefore, a biorefinery (energy production and production of useful substances) can be easily constructed from brown algae.

1 is a diagram showing the chemical structure of alginic acid; FIG. FIG. 2 shows the chemical structures of DEH and KDG; FIG. 2 shows the structure of the alginate-degrading enzyme gene cluster possessed by the Formosa haliotis MA1 strain. FIG. 2 shows the base sequence of sdr1 (top) and the amino acid sequence of SDR1 (bottom). FIG. 3 is a plasmid map of pET-22b(+)-SDR1. FIG. 2 is a photograph of agarose gel electrophoresis after the sdr1 gene was amplified from the genomic DNA of the F. haliotis MA1 strain by PCR. Lane M indicates a 1 kb DNA Ladder (NEW ENGLAND Biolabs), Lane 1 indicates the amplified sdr1 gene. Fig. 2 is an agarose gel electrophoresis photograph obtained by examining the presence or absence of the sdr1 gene by colony direct PCR. Lane M indicates 1 kb DNA Ladder (NEW ENGLAND Biolabs), and Lane 1 and 2 indicate pET-22b(+)-SDR1. Fig. 10 is an SDS-PAGE photograph of SDR1 transformants cultured and SDR1 induced with IPTG. Lane M is a protein marker (Nacalai Tesque), Lane 1 is pET-22b (+) transformant +0 mM IPTG (no IPTG), Lane 2 is pET-22b (+) transformant +0.1 mM IPTG, Lane 3 is pET-22b(+) transformant +1mM IPTG, Lane 4 is SDR1 transformant +0mM IPTG (no IPTG), Lane 5 is SDR1 transformant +0.1mM IPTG, Lane 6 is SDR1 transformant +1mM IPTG indicate. FIG. 3 is a photographic diagram showing the results of SDS-PAGE of the bacterial cell fraction, and the insoluble and soluble fractions after sonication. Lane M is a protein marker, Lane 1 is a bacterial cell fraction, Lane 2 is an insoluble fraction, and Lane 3 is a soluble fraction. FIG. 3 is an SDS-PAGE photograph after SDR1 was purified by nickel affinity chromatography. Lane M is a protein marker, Lane 1 is a flow-through fraction, Lane 2 is a wash fraction, and Lane 3 to Lane 22 are elution fractions. FIG. 2 is a photographic diagram showing the results of subjecting an eluted fraction of SDR1 to SDS-PAGE after dialysis and 10-fold dilution. Lane M is a protein marker and Lane 1 is purified SDR1. FIG. 4 is a graph showing the results of reacting SDR1 with DEH in the presence of NADH or NADPH and monitoring the absorption wavelength of 340 nm possessed by NADH or NADPH. FIG. 4 is a photograph showing the results of subjecting the SDR1 reaction solution to TLC. Lane M is DEH, Lane 1 is NADH, and Lane 2 is the reaction solution. It is a total ion chromatogram when the reaction product was analyzed by LC/MS. It is a SIM chromatogram (m/z 177) when the reaction product was analyzed by LC/MS. In FIG. 15, it is the mass spectrum of the peak with a retention time of 3.166 minutes. 1 is a graph showing the results of examining the relationship between DehR activity and pH. 1 is a graph showing the results of examining the relationship between DehR activity and temperature.

Next, embodiments of the present invention will be described with reference to the drawings. can be implemented in
<Construction of Formosa haliotis-derived SDR1 expression system and purification system>
Short-chain dehydrogenases/reductases (SDRs) are one of the largest protein superfamilies known today. The SDR superfamily has approximately 250 amino acid residues and includes diverse enzymes that utilize NAD(P)H. However, the identity of amino acid residues between superfamilies is low at 20-30%. All structurally known SDRs to date have a central β-sheet of 6-7 strands sandwiched between three α-helices and a single domain consisting of a Rossman fold. Currently, more than 180,000 SDR members are registered in the Universal Protein Resource (Uniprot) database. Many of these proteins have amino acid sequences deduced from genes, and most of them remain unknown because their substrate properties and enzymatic activities have not been evaluated.

As shown in FIG. 3, genes sdr1 and sdr2 presumed to be SDR were found in the alginase gene cluster possessed by the Formosa haliotis MA1 strain (Non-Patent Documents 5 and 6). As a result of subjecting the amino acid sequences of sdr1 and sdr2 to multiple alignment with the amino acid sequences of other SDRs, catalytic four residues (Asn-Lys-Tyr-Ser (SEQ ID NO: 9)), which are specific sequence motifs of SDR A cofactor-binding sequence motif (Thr-Gly-XXX-Gly-X-Gly) in the Rossman fold was conserved. Thus, the sdr1 gene belongs to the 'classical' C-type SDR, an oxidoreductase with conserved cofactor-binding sequence motif Thr-Gly-XXX-Gly-X-Gly. presumed to be NADPH-dependent DEH reductase has been reported to have a basic pocket that stabilizes the 2' phosphate group of the adenosine nucleotide of NADPH at the active site. NADPH-dependent DEH reductase has basic amino acids in the pocket. This portion of the sdr1 gene contains acidic amino acids, while the portion of the sdr2 gene contains neutral amino acids. Based on these considerations, it was speculated that sdr1 and sdr2 act as NADH-dependent SDRs that use NADH, which has a hydroxyl group at the 2'-position of adenosine nucleotide, as a coenzyme.
Therefore, in order to investigate whether the sdr1 and sdr2 genes act as DEH reductase, we constructed an expression system and a purification system for SDR1 and SDR2, and prepared recombinants of SDR1 and SDR2. In addition, activities were measured using recombinant SDR1, SDR2, NADH and NADPH to evaluate which coenzyme SDR1 and SDR2 use. Data for SDR1 are described below as a representative example.

1. experimental method
(1) Subcloning of sdr1
FIG. 4 shows the base sequence of the coding sequence region of SDR1 and the primary structure of the protein obtained based on the genome analysis derived from the F. haliotis MA1 strain. The base sequence of sdr1 is shown in SEQ ID NO: 1, and the amino acid sequence of protein SDR1 is shown in SEQ ID NO: 2, respectively. A PCR method was performed to construct a large-scale expression system of SDR1 in E. coli. Formosa_SDR1_F (SEQ ID NO: 3: gggCATATGagtaaagtagcagtaataaccggtgc) and Formosa_SDR1_R (SEQ ID NO: 4: ggggCTCGAGgatacctaaagcttgaccaccaccaa) were used as primers. Capital letters in SEQ ID NOS: 3 and 4 indicate the recognition sequences of restriction enzymes NdeI and XhoI, respectively.
As a PCR reaction solution, 32 μL of purified water, 5 μL of 10×PCR buffer for KOD-Plus-Neo, 5 μL of 2 mM dNTPs, 3 μL of 25 mM MgSO ₄ , 1.5 μL of 10 μM Formosa_SDR1_F, 1.5 μL of 10 μM Formosa_SDR1_R, 1 μL of template DNA. and 1 μL of KOD-Plus-Neo were added to make 50 μL. The PCR reaction conditions were heat denaturation at 94°C for 2 minutes, followed by "heat denaturation at 98°C for 10 seconds, annealing at 50°C for 30 seconds, and extension reaction at 68°C for 23 seconds". The target gene was amplified by performing 30 cycles of amplification reaction, which is regarded as one cycle. KOD-Plus-Neo (TOYOBO) was used as the DNA polymerase. Genomic DNA of the F. haliotis MA1 strain (provided by Dr. Reiji Tanaka) was used as the template DNA. To purify SDR1 using His-tag as a primer, the His-tag sequence in the expression vector pET-22b(+) plasmid vector (Merck) was used. The stop codon of SDR1 was deleted and His-tag was added. A NdeI restriction enzyme site was added to the sense strand of the template DNA, and an XhoI restriction enzyme site was added to the antisense strand. As a result of analyzing the presence of a signal peptide using Signal P 4.1 Server, no signal peptide was present in SDR1.
After subjecting the PCR product to agarose gel electrophoresis and confirming that the PCR product corresponds to the theoretical base number of the target gene, the band was excised from the gel and purified using Wizard SV Gel and PCR Clean-up System (Promega). to purify the PCR product.

(2) Construction of expression plasmid The purified PCR product and pET-22b(+) plasmid vector (Merck) were treated with NdeI (New England Biolabs) and XhoI (New England Biolabs) at 37°C for 3 hours with restriction enzymes. rice field. After the reaction solution was subjected to agarose gel electrophoresis to confirm the size of each DNA after cleavage, the band was excised from the gel and purified using Wizard SV Gel and PCR Clean-up System (Promega).
A ligation reaction solution was adjusted so that the molar ratio of the vector and the insert, which is a PCR product, was 1:5, and the mixture was reacted at 16°C for 3 hours using Ligation high Ver.2 (TOYOBO). Transform 1 μL of the ligation product into E. coli DH5α and add 5 mL of LB agar medium containing 100 μg/mL ampicillin (1.0% (w/v) tryptone, 0.5% (w/v) yeast extract, 1.0% (w/v) NaCl) and statically cultured overnight at 37°C.
In order to confirm whether the ligation was successful, colony direct PCR was performed using the colonies on the plate as templates. As a PCR reaction solution, add 79.4 μL of purified water, 2.8 μL of T7 promoter primer, 2.8 μL of T7 terminator primer and 85 μL of 2×Quick taq HS DyeMix to make 170 μL, and distribute it to 17 PCR tubes. bottom. A colony was picked with a sterilized toothpick, and each colony was placed in each PCR tube and used for colony direct PCR. As the PCR reaction conditions, after heat denaturation at 94°C for 2 minutes, "heat denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension reaction at 68°C for 45 seconds" were performed. Twenty-five cycles of the amplification reaction were performed as one cycle to amplify the target gene. As a primer, a T7 universal primer was used. Quick taq™ HS DyeMix (TOYOBO) was used as the polymerase. A master plate of each colony was prepared at the same time as the colony direct PCR.
The PCR product was subjected to agarose gel electrophoresis, and the target gene-inserted colony master plate was inoculated into an LB liquid medium containing 100 µg/mL ampicillin and cultured overnight at 37°C with shaking. Plasmids were extracted from the cultures using the Wizard Plus SV Minipreps DNA Purification System (Promega). This plasmid was named pET-22b(+)-SDR1 (Fig. 5).

(3) SDR1 expression
Escherichia coli BL21 (DE3) was transformed with pET-22b(+)-SDR1, inoculated onto LB agar medium containing 100 μg/mL ampicillin, and statically cultured overnight at 37°C. The formed single colony was inoculated into each of three 5 mL LB liquid media containing 100 μg/mL ampicillin and cultured with shaking at 37° C. and 160 rpm. When the turbidity (OD600) of the culture medium reached 0.4-0.6, isopropyl-β-thiogalactopyranoside (IPTG) was added to the medium to 0, 0.1 and 1 mM, and cultured overnight with shaking. .
After culturing, 200 μL of each culture solution was collected in an Eppendorf tube and centrifuged at 15,000×g for 3 minutes. The supernatant was removed, 200 μL of 50 mM Tris-HCl buffer was added to each suspension, and the cells were centrifuged again under the same conditions. The supernatant was removed, and 20 μL of 50 mM Tris-HCl buffer was added and suspended. 20 μL of 2×SDS sample buffer was added to suspend and boiled for 3 minutes. Each sample was subjected to Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) prepared on a 15% polyacrylamide gel to examine the expression of the target protein SDR1.

(4) Mass culture of SDR1 and confirmation of its solubilization The colonies on the LB agar medium prepared for examining the expression of SDR1 in (3) above were inoculated into 5 mL of LB liquid medium containing 100 µg/mL ampicillin, Shaking culture was carried out overnight at 37°C and 160 rpm. This was used as a pre-culture solution.
400 mL of 2xYT medium (1.6% (w/v) high polypeptone, 1.0% (w/v) dry yeast extract, 0.5% (w/v) containing 100 μg/mL ampicillin in a 1 L baffled Erlenmeyer flask. ) NaCl) were prepared. 8 mL of a sterilized 50% (w/v) glucose solution and 400 μL of the pre-culture medium were added to each, and shaking culture was performed at 37° C. and 160 rpm by a rotation method. When the turbidity (OD600) of the culture reached 0.5-0.6, the culture was ice-cooled. After that, IPTG was added to the medium to a concentration of 0.1 mM, and cultured with shaking for 40 hours. After culturing, the culture solution was centrifuged at 4° C. and 6,800×g for 15 minutes, and the supernatant was removed to obtain wet cells.
3 mL of 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl and 20 mM imidazole was added to 3.0 g of wet cells and suspended. A small amount of DNase was added, and the mixture was allowed to stand at room temperature for 1 hour. After that, 27 mL of the same buffer solution as above was added to make a total of 30 mL, and suspended. The suspension was sonicated on ice for 3 minutes (OUTPUT: 4, DUTY: 40, TOMMY ULTRASONIC DISRUPTOR UD-201) and allowed to stand for 2 minutes to cool. By performing this operation five times, the cells were disrupted.
10 μL of the cell lysate was taken as a cell fraction for SDS-PAGE, and the remaining cell lysate was centrifuged at 4° C. and 9,700×g for 15 minutes. The supernatant was collected and centrifuged again under the same conditions, and the supernatant was used as the soluble fraction. In addition, a small amount of the precipitate obtained by the first centrifugation was collected and suspended in the buffer solution used for cell disruption to obtain an insoluble fraction.
2×SDS sample buffer was added to each of the cell fraction, soluble fraction, and insoluble fraction, and boiled for 3 minutes. The sample after boiling was subjected to SDS-PAGE prepared with 15% (w/v) polyacrylamide gel to examine the presence or absence of the target protein.

(5) Purification of SDR1 As a carrier for nickel affinity chromatography, about 3 mL of Ni Sepharose™ 6 Fast Flow (GE Healthcare) was packed in a PD-10 empty column (GE Healthcare). After washing the column, the carrier was equilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl and 20 mM imidazole. The equilibrated carrier was added to the soluble fraction obtained by sonication, and the mixture was stirred with a stirrer at 4°C for 1 hour to allow the target protein, SDR1, to be adsorbed onto the carrier. A solution containing this carrier was packed in a PD-10 empty column, and the solution coming out of the column was collected as a flow-through fraction. 20 mL of the same buffer as used for column equilibration was flowed to wash the column. This fraction was collected as the wash fraction. After that, 20 mL of 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl and 500 mM imidazole was flowed, 1 mL each was recovered, and these fractions were recovered as eluted fractions.
The eluted fraction was subjected to SDS-PAGE prepared on a 15% (w/v) polyacrylamide gel to examine fractions containing SDR1. Fractions in which SDR1 was detected were combined and placed in a dialysis membrane, size 36 (Wako), which is a dialysis membrane. This dialysis membrane was immersed in 10 mM sodium phosphate buffer (pH 7.4) containing 100 mM NaCl and dialyzed overnight at 4°C. After dialysis, the protein aggregated, so centrifugation was performed at 9,700 xg at 4°C for 15 minutes to obtain a supernatant. This supernatant was diluted 10-fold and subjected to SDS-PAGE prepared on a 15% (w/v) polyacrylamide gel to confirm the purification of SDR1.
The protein concentration of SDR1 was calculated by measuring the absorbance at a wavelength of 280 nm and using the molar extinction coefficient ε obtained from the following formula (1).
Equation (1) ε = (#Trp) x 5,500 + (#Tyr) x 1,490 + (#cyctine) x 125

(6) Measurement of SDR1 Activity The activity of purified SDR1 was measured. DEH used as a substrate was prepared in the laboratory to which the present inventor belongs. When SDR1 acts as a DEH reductase, it is thought that it requires reduced NADH or NADPH as a coenzyme. Therefore, DEH and purified SDR1 were reacted in the presence of NADH or NADPH, and activity was evaluated by monitoring the absorption wavelength of 340 nm possessed by NADH or NADPH.
Place the reaction solution adjusted to 1.0 mM DEH, 0.20 mM NADH or NADPH, and 50 mM sodium phosphate buffer (pH 7.0) in a quartz cell, place it in the cell holder of the spectrophotometer, and incubate at 25°C for 3 minutes. bottom. SDR1, which had been incubated at 25°C for 3 minutes, was added to 5.2 µM in the reaction solution, and after pipetting, the lid of the spectrophotometer was quickly closed and the decrease in absorbance at 340 nm was monitored. As controls, a sample containing only DEH in the reaction solution and a sample containing only SDR1 were measured.

2. Experimental result
(1) Construction of expression plasmid
FIG. 6 shows the results of subjecting the sdr1 gene amplified by PCR using the F. haliotis MA1-derived genomic DNA as a template to agarose gel electrophoresis. From this result, a band was detected at a position corresponding to the theoretical base number (750 bp) of the sdr1 gene, confirming amplification of the sdr1 gene.
After confirming the amplification of the sdr1 gene, the PCR product and the pET-22b(+) plasmid vector were treated with restriction enzymes and ligated. The ligation product was transformed into Escherichia coli, and the sdr1 gene was examined by colony direct PCR in order to confirm whether the transformant, which was a colony, retained the plasmid containing the sdr1 gene. A colony retaining the sdr1 gene was selected and cultured to extract the plasmid. The results of subjecting the extracted plasmid to agarose gel electrophoresis are shown in FIG. This result confirmed the construction of the plasmid.

(2) SDR1 expression
SDR1 transformants obtained by transforming pET-22b(+)-SDR1 into BL21(DE3) were cultured, and the presence or absence of IPTG-induced SDR1 expression was confirmed by SDS-PAGE. The results of SDS-PAGE are shown in FIG. As a control, Escherichia coli transformed with a pET-22b(+) vector into which no gene had been inserted was used as a control, and those subjected to the same operation were electrophoresed simultaneously. The target protein, SDR1, has a molecular weight of 26 kDa. A band was detected at the position corresponding to the molecular weight of SDR1 at all IPTG concentrations of 0, 0.1 and 1 mM in the culture medium, and a thick band was detected at 0.1 and 1 mM. This band was not detected in the control pET-22b(+) vector transformant.
(3) Mass culture and solubilization of SDR1
A total of 21.05 g of wet cells were obtained as a result of 40 hours of culture in two 400 mL 2×YT media. The bacterial cell fraction and the insoluble and soluble fractions after sonication were subjected to SDS-PAGE to confirm the expression of SDR1 and solubilization by sonication (Fig. 9). The molecular weight of SDR1 is 26 kDa, and a band was detected at a position corresponding to the molecular weight of SDR1 in all fractions. The soluble fraction had a thicker band than the insoluble fraction, and most of the expressed SDR1 was solubilized.

(4) Purification of SDR1 FIG. 10 shows SDS-PAGE after nickel affinity chromatography. The eluted fraction containing the largest amount of SDR1 in FIG. 10 was collected and dialyzed. FIG. 11 shows the results of subjecting the dialyzed solution diluted 10-fold to SDS-PAGE. The molecular weight of SDR1 is 26 kDa, and a single band was detected at the corresponding position. Therefore, this solution was used as the purified enzyme of SDR1. The yield of SDR1 was 295 mg/L medium.
(5) Measurement of SDR1 activity
FIG. 12 shows the results of reacting SDR1 with the substrate DEH in the presence of NADH or NADPH and monitoring the absorption wavelength of 340 nm possessed by NADH or NADPH. As controls, the results of a sample containing no DEH and a sample containing no SDR1 are shown. NADH reaction solution containing SDR1 and DEH was able to observe a decrease in NADH over time. In contrast, no such change was observed in the NADPH reaction solution.

3. consideration
We subcloned SDR1 and established a method for constructing and purifying an expression plasmid for Escherichia coli. The prepared plasmid was designed to add a base sequence encoding His-tag to the 3' end of the sdr1 gene. After sonicating the cells, most of SDR1 was present in the soluble fraction and could be purified by subjecting the solution to nickel affinity chromatography. Yield was 295 mg of SDR1 per liter of medium. It has been reported that the E. coli recombinant of FlRed, which is the same DEH reductase as SDR1, was 40 mg per 1 L of medium (Patent Document 1). In addition, it is reported that 82 mg of DEH reductase A1-R E. coli recombinant was obtained per 1 L of medium, and 91 mg of A1-R' E. coli recombinant was obtained per 1 L of medium (non-patent Reference 7). On the other hand, SDR1 yielded three times more purified enzyme than other DEH reductases.
As a result of reacting SDR1 with the substrate DEH in the presence of NADH or NADPH, the absorbance at 340 nm (concentration of NADH) decreased over time, indicating a decrease in NADH. In contrast, no decrease in absorbance was observed with NADPH. These results indicate that SDR1 is an NADH-dependent reductase. In addition, SDR1 was suggested to be a DEH reductase from the observation of a decrease in NADH in the reaction solution containing DEH. Therefore, an attempt was made to identify the product of this reaction solution.

<Identification of reaction products of SDR1>
FIG. 2 shows the reaction formula from DEH to KDG. The aldehyde group of DEH is reduced by DEH reductase using NADH or NADPH and a proton to form a hydroxymethyl group, thereby generating KDG. At this time, NADH is oxidized to NAD ⁺ and NADPH to NAPD ⁺ . Therefore, DEH has a molecular weight 2 higher than KDG. From the results of <Formosa haliotis-derived SDR1 expression/purification system construction>, SDR1 was found to be an NADH-dependent reductase. In order to investigate whether SDR1 works as a DEH reductase, a test was performed to confirm that the reaction product was KDG. That is, a reaction solution containing SDR1, DEH and NADH was prepared, and the reaction product was evaluated by thin layer chromatography (TLC) analysis. Furthermore, the reaction product was identified by LC/MS analysis.

1. experimental method
(1) Evaluation of Reaction Products by Thin Layer Chromatography Analysis A reaction solution of 500 μL was prepared so that the initial reaction concentrations were 0.35 μM SDR1, 10 mM DEH and 10 mM NADH. The solution was reacted with rotation at 25° C. for 41 hours. The reaction was then terminated by freezing at -80°C. After freezing overnight, the thawed product was subjected to TLC. The developing solvent had a composition of 1-butanol:acetic acid:water=2:1:1 (v/v). It has been reported that in TLC analysis, KDG spots were detected at lower positions than DEH spots (Non-Patent Documents 3 and 4). In this study, we attempted to distinguish between DEH and reaction products by using a diphenylamine-aniline reagent (DPA reagent), which detects different colors depending on the sugar. The DPA reagent used was prepared as follows. 1.0 g of diphenylamine was dissolved in acetone, and the volume was made up to 25 mL with acetone. 1.0 mL of aniline was dissolved in acetone, and the solution was made up to 25 mL with acetone, and this was used as B solution. 85% phosphoric acid was used as solution C. A solution A: solution B: solution C was mixed at a ratio of 5:5:1 (v/v) to obtain a DPA reagent.
As markers, 1 μL of a 17.5 mg/mL DEH aqueous solution as a substrate, 10 μL of an NADH aqueous solution having the same concentration as the reaction solution, and 10 μL of the reaction solution were spotted on a TLC plate. A TLC plate was immersed in a developing layer containing a developing solvent, and developed once. After completion of development, the TLC plate was dried and sprayed with the DPA reagent. After drying the TLC plate again, it was heated on a hot plate and the reaction product was detected.

(2) Identification of Reaction Products by LC/MS Analysis In mass spectrometry, the accurate mass “monoisotopic mass” from only the main isotope of each element composing the sample molecule is used. The KDG monoisotopic mass is 178.0. LC/MS negative ion analysis detects deprotonated ions. The mass of the deprotonated ion of KDG is 177.0, in which case the m/z is the same as the mass of the ion. Therefore, if the reaction product is KDG, an ion should be detected at m/z 177, not at m/z 175 for DEH. Therefore, selective ion monitoring (SIM) was set to m/z 177 for LC/MS analysis. A total of five reaction solutions of the above (1) were prepared. Five were collectively ultrafiltered using an Amicon Ultra-4 3000 MWCO as the enzyme had to be removed for LC/MS. Negative ion analysis was performed using LC/MS on the solution from which the enzyme was removed after passing through the filter. A Shodex IC NI-424 column was used, and a mobile phase of 40 mM ammonium formate buffer containing 0.1% formic acid was used. For selective ion monitoring (SIM), we chose m/z 177, which corresponds to the mass of the ideal reaction product, the deprotonated ion [MH]- of KDG.

2. Experimental result
(1) Evaluation of reaction products by thin-layer chromatography analysis
FIG. 13 shows the results of subjecting the SDR1 reaction solution to thin layer chromatography (TLC). In Lane M, DEH (symbol D) spots with a color close to reddish brown were seen in a tailing pattern. In Lane 1, NADH (code N) had an indigo tail. In the reaction solution of Lane 2, the upper spot that seems to be DEH became faint, and below that, a pink spot (marked K) that was not seen in Lane 1 appeared. Tailing was seen. From this result, it was considered that a substance different from DEH was produced by the action of SDR1.
(2) Identification of reaction products by LC/MS analysis Figure 14 shows the LC/MS total ion chromatogram of the reaction products, which corresponds to the mass of the deprotonated ion [MH]- of KDG, which is the ideal reaction product. SIM chromatograms with m/z 177 selected are shown in FIG. 15, respectively. One prominent peak was detected at a retention time of 3.178 minutes on the total ion chromatogram. In addition, one prominent peak was detected at almost the same retention time of 3.166 minutes on the SIM chromatogram. As shown in FIG. 16, in the mass spectrum of the peak, a peak corresponding to [MH]-of KDG (relative intensity 100%) was observed at m / z 177.0, and the DEH peak (m / z 175) disappeared. bottom. In other words, SDR1 was found to produce KDG by reducing its substrate DEH. These results indicate that sdr1 acts as a DEH reductase. Moreover, when the reaction yield of DEH was determined from the peak of the mass spectrum, it was found that 90% or more of DEH was reduced to KDG.

3. consideration
DEH is uronic acid and KDG is gluconic acid. With the DPA reagent, uronic acid appears as bright reddish-brown spots. Gluconic acid, 2-keto-D-gluconate, is reported to appear as a reddish-brown spot and 5-keto-D-gluconate as a dark purple spot. As shown in FIG. 13, the spot of the reaction product in TLC was nearly pink, but it was considered to be a spot of KDG, which is gluconic acid, also from the results of LC/MS.
In the reaction solution of Lane 2, the DEH spot became lighter, but did not disappear completely, indicating that SDR1 could not completely reduce DEH. In the reaction solution used this time, there is a possibility that the activity was low and the reaction could not be completed because the buffer solution was not added in the LC/MS. In addition, since the enzymatic chemical properties of SDR1 have not been clarified, the reaction conditions such as temperature were set with reference to the previous paper on DEH reductase, so it is possible that the reaction conditions were outside the optimal range. There is We thought that it was important to investigate the enzymatic properties of SDR1 in our research.
The LC/MS results (Fig. 16) showed a small peak at m/z 175, which was thought to be due to the KDG isotope rather than the rest of the DEH substrate. Since the ion corresponding to KDG was detected, it was found that SDR1 works as a DEH reductase. In addition, almost no residue of DEH was detected, suggesting that the reaction yield of SRD1 was 90% or more. Thus, the F. haliotis strain MA1 was the first to demonstrate the presence of DEH reductase and to produce KDG in the presence of NADH. Next, various enzymatic properties of this enzyme were investigated.

<Effect of pH and temperature on SDR1>
We have shown that F. haliotis SDR1 is a DEH reductase. However, its enzymatic properties have not been elucidated. The optimal pH and optimal temperature for all DEH reductases currently reported are shown as [enzyme name, optimal pH, optimal temperature]: [PDEHR, pH7.0-7.5, -], [A1- R, pH6.5-7.5, 50°C], [A1-R′, pH6.5, 51°C], [FlRed, pH7.2, 25°C], [HdRed, pH7.0, 50°C]. Among these, FlRed and A1-R' are NADH-dependent SDR enzymes like SDR1. There were considerable differences in temperature optima and pH optima among the enzymes. Therefore, we conducted experiments to clarify the optimum pH and temperature of SDR1.
1. experimental method
(1) Effect of pH The buffer solutions were 250mM MES buffer (pH 5.5, 6.0, 6.5, 7.0) to which NaCl was added so that the ionic strength was 0.5M, 250mM HEPES buffer (pH 7.0, 7.5, 8.0), 250 mM Tricine buffers (pH 8.0, 8.5, 9.0) and 250 mM Glycine-NaOH buffers (pH 9.0, 9.5, 10.0) were prepared. DEH used as a substrate was prepared in the laboratory to which the present inventor belongs.
A mixed solution adjusted to 1.0 mM DEH, 0.20 mM NADH, and 50 mM each buffer solution (ionic strength 0.1 M) was placed in a quartz cell, placed in a constant temperature cell holder of a spectrophotometer (Simadzu UV-1800), and 15 Preincubated for 4 min 30 sec until reaching °C. After adding SDR1 preincubated at 15° C. for 3 minutes to 5.2 μM, pipetting was performed, the lid of the spectrophotometer was quickly closed, and the decrease in absorbance at 340 nm was monitored. Time was plotted on the horizontal axis and the absorbance at 340 nm was plotted on the vertical axis, and the slope was defined as the reaction rate. This operation was performed while changing the pH, and the reaction rate at each pH was determined. Then, pH was plotted on the horizontal axis and the relative activity (%) of the reaction rate was plotted on the vertical axis to investigate the effect of pH on SDR1.

(2) Effects of temperature
Put the mixed solution adjusted to 1.0mM DEH, 0.20mM NADH, and 50mM Tricine buffer (pH 8.0, ionic strength 0.1M) into a quartz cell and place it in the constant temperature cell holder of the spectrophotometer (Simadzu UV-1800). bottom. The temperature of the cell holder was adjusted to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C and 45°C, and the mixed solution was preincubated until each temperature was reached. After adding 5.2 μM of SDR1 preincubated to each temperature, pipetting was performed, the lid of the spectrophotometer was quickly closed, and the decrease in absorbance at 340 nm was monitored. Time was plotted on the horizontal axis and the absorbance at 340 nm was plotted on the vertical axis, and the slope was defined as the reaction rate. Plotting the temperature on the horizontal axis and the relative activity (%) of the reaction rate on the vertical axis, the effect of temperature on SDR was investigated.

2. Experimental result
(1) Effect of pH A graph prepared by the method described in 1(1) above is shown in FIG. From this result, it was found that the optimum pH of DehR is pH 5.5-10.5 (preferably pH 6.0-10.0, more preferably pH 7.0-9.0, most preferably pH 8.0).
(2) Influence of Temperature A graph prepared by the method described in Top 1 (2) is shown in FIG. From these results, the optimum temperature for DehR is 10°C to 44°C (preferably 10°C to 35°C, more preferably 15°C to 30°C, most preferably 20°C to 25°C), and almost disappears at 45°C. found to live.
(3) Effect of time The reaction time can be varied between 5 minutes and 72 hours for the reaction. It was found that KDG production began about 5 minutes after the start of the reaction within the optimum pH and optimum temperature ranges described above. In order to obtain a sufficient reaction yield, it is preferable to carry out the reaction for several tens of minutes (20 minutes, 30 minutes, 40 minutes) to several hours (1 hour, 2 hours, 3 hours, 6 hours). Therefore, preferred reaction times are 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 48 hours and 72 hours. You can choose a suitable time from
3. consideration
It was found that SDR1 has a slightly higher pH optimum and the lowest optimum temperature than the known DEH reductases. The reason for the low optimum temperature is that F. haliotis, the origin of this enzyme, is a bacterium isolated from the digestive tract of abalone that lives in the sea. It was thought that the enzyme may not be very heat resistant. It has been reported that 19°C is the optimum water temperature for aquaculture of giant abalone, from which F. haliotis was isolated, which is very close to the optimum temperature for SDR1.
However, HdRed, a DEH reductase present in Ezo abalone liver and pancreas extracts, had an optimum temperature of 50°C. is.

<Substrate specificity of SDR1>
Since the substrate specificity of SDR enzymes shows considerable diversity, we investigated whether SDR1 reacts with DEH in a substrate-specific manner. DEH is a 6-carbon uronic acid. Among sugar derivatives, uronic acid is a carboxylic acid in which the hydroxymethyl group at the end of the main chain is changed to a carboxy group. Therefore, hexasaccharides and pentasaccharides with similar carbon numbers, and sugar derivatives were selected as substrates. Since DEH has various functional groups such as aldehyde group, carboxyl group and ketone group, we selected compounds such as aldehyde, ketone and carboxylic acid and attempted to elucidate the substrate specificity.
1. Experimental method In addition to DEH as a substrate, α-ketoglutaric acid and pyruvic acid as α-keto acids, D-glucose, D-mannose, D-galactose, D-ribose, L-arabinose and D-xylose as aldoses, and Ketose D-fructose as sugar alcohol, D-mannitol, D-sorbitol as sugar alcohol, 2-deoxy-D-ribose, D-fucose as deoxy sugar, D-glucuronic acid as uronic acid, maleic acid, citric acid as carboxylic acid Using DL-glyceraldehyde and benzaldehyde as acid, 4-methy-w-pentanone and 2,5-hexanedione as ketone, and ethyl pyruvate, ethyl benzoylacetate and methyl pyruvate as esters, we investigated the substrate specificity of SDR1. examined.
A mixed solution adjusted to 1.0 mM each substrate, 0.20 mM NADH, and 50 mM Tricine buffer (pH 8.0, ionic strength 0.1 M) was placed in a quartz cell, placed in the constant temperature cell holder of the spectrophotometer, and heated to 15°C. Pre-incubated for 4 min 30 sec until reached. Separately, SDR1 preincubated for 3 minutes at 15° C. was added to 5.2 μM, followed by pipetting, quickly closing the lid of the spectrophotometer, and monitoring the decrease in absorbance at 340 nm. Time was plotted on the horizontal axis and the absorbance at 340 nm was plotted on the vertical axis, and the slope was defined as the reaction rate. The relative activity was determined for each substrate, with the reaction rate for DEH set to 100%.

2. Experimental result
When the activity of DEH was defined as 100%, the relative activity of each substrate was ND (not determined). Thus, no enzymatic activity of SRD1 was observed for substrates other than DEH examined.
3. consideration
For SDR1, we selected compounds with structures similar to those of DEH and compounds with functional groups in common. Therefore, SDR1 was found to react specifically with DEH as a substrate.
In this study, in order to investigate whether the sdr1 gene acts as a DEH reductase, we constructed a recombinant SDR1, identified the reaction products, and investigated their enzymatic properties. A large-scale expression system was constructed by inserting the sdr1 gene into the pET-22b(+) plasmid vector as an expression plasmid. This was transformed into E. coli BL21(DE3), and the mass culture was sonicated, solubilized, and purified to homogeneity by nickel affinity chromatography. Recombinant SDR1 was able to be produced in a large amount of 295 mg per 1 L of medium, compared with previously reported DehR. When SDR1 was reacted with DEH in the presence of NADH, enzymatic activity was observed, suggesting that SDR1 is an NADH-dependent reductase.
Analysis of the SDR1 reaction product by TLC and LC/MS identified the reaction product as KDG and revealed that SDR1 was DehR. As a result of MS, the peak of DEH disappeared, so it was considered that almost all DEH was converted to KDG (reaction yield of 90% or more). As a result of examining the effect of pH, the optimum pH was 8, which was slightly larger than the previously reported DehR. As a result of investigating the effect of temperature, the optimum temperature was 20°C, which was the lowest among the reported DEH reductases. As a result of examining the substrate specificity of SDR1, it was found that only DEH reacts specifically.
SDR2 is expected to have similar results as SDR1.

<Method for producing KDG from alginic acid>
Next, a method for easily producing KDG by mixing three types of enzymes with alginic acid is examined.
1. Test method
(1) Method of sequentially adding enzymes
2.0 mg/mL (20 mM Tris 1 mL of an aqueous solution of -HCl buffer pH 7.5) and 1 mL of an aqueous solution of 2.0 mg/mL (20 mM Tris-HCl buffer pH 7.5) of a polypeptide (endo-alginate lyase) having the amino acid sequence shown in SEQ ID NO: 6 are added. This reaction solution is incubated at 37° C. for 6 to 24 hours. Next, 0.5 mL of an aqueous solution of 4.0 mg/mL SDR1 (10 mM sodium phosphate buffer containing 100 mM NaCl, pH 7.4) and 0.5 mL of an aqueous solution of 2.0 mg/mL NADH dissolved in purified water were added. Incubate at °C for 2-24 hours. The solution after the reaction is analyzed by TLC according to the method described in <Identification of reaction products of SDR1> 1(1).
As a result, it was found that KDG was produced from alginic acid.
In the above method, the polypeptides having the amino acid sequences shown in SEQ ID NOs: 5 and 6 are added to alginic acid, reacted at 45°C or lower for 6 hours or longer, and then SDR1 is added without changing the temperature. good. Since the temperature can be kept constant, the reaction method can be simplified.
(2) Method of adding enzymes at the same time
2.0 mg/mL (20 mM Tris -HCl buffer pH 7.5) 1 mL of aqueous solution, 1 mL of aqueous solution of 2.0 mg / mL (20 mM Tris-HCl buffer pH 7.5) of polypeptide (endo-type alginate lyase) having the amino acid sequence shown in SEQ ID NO: 6, 4.0 mg 0.5 mL of an aqueous solution of SDR1 (10 mM sodium phosphate buffer containing 100 mM NaCl, pH 7.4) at 1/mL of the three enzymes and 0.5 mL of an aqueous solution of 2.0 mg/mL NADH dissolved in purified water were added. Incubate at 20-30°C for 6-72 hours. After that, the reaction solution is analyzed by TLC according to the method described in <Identification of reaction products of SDR1> 1(1).
As a result, it was found that KDG was produced from alginic acid.
SDR2 is expected to have similar results as SDR1.

In this study, we obtained a novel DEH reductase from F. haliotis MA1 strain. This is the first protein identified in F. haliotis strain MA1. Information on DEH reductase is very limited, and this study provided new knowledge about DEH reductase. It was found that KDG can be produced easily and efficiently from alginic acid by applying the DEH production method developed in this laboratory.
Thus, according to this embodiment, a method for efficiently producing KDG from DEH could be provided. In addition, KDG could be produced by contacting alginic acid with three kinds of enzymes. Therefore, a biorefinery (energy production and production of useful substances) can be easily constructed from brown algae.

Claims (4)

4-deoxy-L-erythro-5-hexoseuloseuronic acid (DEH) is contacted with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 and having DEH reducing activity to convert 2-keto-3-deoxy- A KDG manufacturing method for producing D-gluconate (KDG). 4-deoxy-L-erythro-5-hexoseuloseuronic acid (DEH ) plasmid for expression of reductase. The plasmid for DEH reductase expression according to claim 2 is for E. coli, and after incorporating the plasmid into E. coli, an enzyme induction step of expressing DEH reductase, an enzyme purification of purifying the induced DEH reductase A method for producing DEH reductase, comprising the steps of: (a) for alginic acid, (1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 5 and having exo-type alginate lyase activity, and (2) an endo-type alginate lyase having the amino acid sequence shown in SEQ ID NO: 6 Enzyme addition step of contacting a polypeptide having activity with (3) a polypeptide having an amino acid sequence set forth in SEQ ID NO: 2 and having 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) reducing activity and (b) an enzymatic action step of maintaining a mixture of the alginic acid and the three polypeptides at a temperature at which the exo-type alginate lyase activity, the endo-type alginate lyase activity, and the DEH reduction activity work. -Method for producing deoxy-D-gluconate (KDG).

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