KR101479134B1 - A novel NADH oxidase from Streptococcus pyogenes and its coupling with L-arabinitol dehydrogenase for L-xylulose production - Google Patents

Abstract

The present invention relates to a novel NADH oxidase producing Streptococcus pyogenes), relates to a method for producing NADH oxidase gene and the enzyme which generates a derivative from the strain, and more particularly include NADH oxidase to produce water, the nucleic acid molecule encoding it, the nucleic acid molecule A transformant containing the vector, and regeneration of the coenzyme using the NADH oxidase producing the water. In addition, L-arabinitol dehydrogenase, which generates L-rare saccharide, is coupled with NADH oxidase that generates new water, and the expensive coenzyme is regenerated and repeatedly used to economically produce L-rare saccharide And by using NADH oxidizing enzyme that generates water, it is possible to simplify the purification process because no byproducts other than water are produced.

Description

Production of L-xylulose by Coupling with New NADH Oxidase and L-arabinitol Oxidase Derived from Pseudomonas aeruginosa Streptococcus pyogenes and its coupling with L-arabinitol dehydrogenase for L-xylulose production }

The present invention relates to a method for producing L-xylulose by the coupling reaction of an NADH oxidizing enzyme and a L-arabinitol oxidase which produce new water derived from a pneumococcal strain. More specifically, the present invention relates to a method for producing L- A nucleic acid molecule encoding the NADH oxidase, a vector containing the nucleic acid molecule, a transformant containing the vector, and a couple of NADH oxidase and L-arabinitol oxidase using the same. And a method for producing L-xylulose by a ring reaction.

Biochemically, redox reactions are not only important in asymmetric reactions, but are also a field of interest in chemical substances or medicine that have optical purity. However, such an oxidation - reduction reaction requires an expensive coenzyme such as NAD (P) ⁺ or NAD (P) H. Therefore, it is necessary to economically ensure that these expensive coenzymes are regenerated and used continuously. Regeneration of coenzymes is usually accomplished by adding chemical, electrical, or photocatalytic substances or by adding enzymes during enzymatic reactions. There are two ways to regenerate coenzyme using enzymes. The first method is to regenerate enzymes and chemical additives, and the second method is to recover the coenzyme by coupling two enzymes. Formate dehydrogenase and glucose dehydrogenase are well known methods for regeneration of NAD (P) ⁺ and NAD (P) H. However, byproducts are generated during the reaction to lower the purity of the product and increase the purification cost . However, the use of NADH oxidase, which produces water, can produce a high economic efficiency because only water is produced as a byproduct.

In the present invention, NADH oxidase producing new water is proposed and coupled with L-arabinitol dehydrogenase to regenerate coenzyme to produce L-xylulose from L-arabinitol in high yield and continuously Xylulose by mass production of L-xylulose at a high yield by proposing optimum reaction conditions for the L-xylulose production.

Korean Patent Publication No. 10-2012-0083908 (July 26, 2012) Korean Patent Publication No. 10-2012-0034713 (2012.04.12)

A first object of the present invention is to provide a gene of NADH oxidase which produces water derived from a colony-forming strain.

A second object of the present invention is to provide an NADH oxidase which produces water expressed from the gene.

A third object of the present invention is to provide a recombinant expression vector containing the gene of NADH oxidase which produces the water.

A fourth object of the present invention is to provide all the transformed strains including the transformed recombinant E. coli.

A fifth object of the present invention is to provide an NADH oxidizing enzyme that produces a recombinant product of transformed recombinant E. coli.

A sixth object of the present invention is to provide an optimum condition for producing L-xylulose by coupling the enzyme with L-arabinitol dehydrogenase to regenerate coenzyme.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The present invention relates to a method for producing L-xylulose, NADH oxidase that produces water of the strain and Hypocrea < RTI ID = 0.0 > arabinitol dehydrogenase derived from L-arabinitol dehydrogenase derived from Lactobacillus jecorina is coupled to regenerate coenzyme. Also, the present invention is characterized by cloning an NADH oxidase gene that generates water from a pneumococcal strain through a horse hybridization and a colony hybridization.

In order to achieve the above object, the present invention provides an NADH oxidase which produces water having an amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof.

In one embodiment of the present invention, the NADH oxidizing enzyme producing the water is preferably but not limited to the one derived from a pneumococcal strain.

In one embodiment of the present invention, the enzyme of the present invention has a molecular weight of 50 kDa.

The present invention also provides an NADH oxidase gene that produces water that encodes the enzyme of the present invention.

The gene of the present invention preferably has the nucleotide sequence of SEQ ID NO: 3.

The present invention also provides a method for producing NADH oxidizing enzyme producing water by culturing a strain transformed with a recombinant expression vector comprising an NADH oxidase gene which produces the water of the present invention.

The present invention also relates to a method for producing the water-containing NADH oxidase of the present invention by using Hypocrea arabinitol dehydrogenase and a method for producing L-xylulose from L-arabinitol at low cost and high yield by regenerating coenzyme through coupling reaction with L-arabinitol dehydrogenase.

The present invention also provides a composition for producing L-xylulose, which comprises the enzyme of the present invention as an active ingredient.

Hereinafter, the present invention will be described.

The NADH oxidase producing water of the present invention is characterized by having the amino acid sequence shown in SEQ ID NO: 4. In addition, the amino acid sequence of SEQ ID NO: 4 may contain at least one deletion, substitution, and addition of at least one amino acid within the range in which the activity of the NADH oxidase enzyme producing water represented by the protein having these amino acid sequences is not impaired The mutant NADH oxidase to which the mutation has been introduced is also included in the NADH oxidizing enzyme producing the water according to the present invention.

The present invention also includes an NADH oxidase gene encoding NADH oxidase which produces water having the amino acid sequence of SEQ ID NO: 4, and the gene sequence thereof is represented by SEQ ID NO: 3. Also, a mutant gene coding for an NADH oxidase which produces the aforementioned mutant obtained by mutating the nucleotide sequence of SEQ ID NO: 3 is also included in the NADH oxidase gene of the present invention.

In addition, the present invention includes a recombinant vector containing the NADH oxidase gene that generates the water, and a transformant transformed with the recombinant vector. In addition, the present invention includes a method for producing NADH oxidizing enzyme producing water, which comprises separating NADH oxidizing enzyme producing water from a culture obtained by culturing the transformant.

Hereinafter, the present invention will be described in more detail.

The water-producing NADH oxidase gene of the present invention is isolated from the cells of the pneumococcal bacteria. First, chromosomal DNA is obtained from a strain having an NADH oxidase gene that generates water.

Next, a polymerase chain reaction (PCR) is performed using the designed oligonucleotide as a primer and the chromosomal DNA of a strain of Streptococcus pneumoniae as a template to partially amplify the NADH oxidase gene that generates water. The thus-obtained PCR-amplified fragment is a fragment having 100% homology to the NADH oxidase gene that produces water of a pneumococcal strain. As a probe for performing colony hybridization, a high S / N ratio can be expected, Facilitates stringency control of hybridization. The above PCR-amplified fragment is labeled with an appropriate reagent, and the chromosomal DNA library is subjected to colony hybridization to select an NADH oxidase gene that produces water (Current Protocols in Molecular Biology, vol. 1, 603 , 1994).

A plasmid is recovered from E. coli selected by the above method using the alkali method (Current Protocols in Molecular Biology, Vol. 1, p.161, 1994) to obtain a DNA fragment containing an NADH oxidase gene that generates water . After the nucleotide sequence is determined by the above method, it is possible to obtain the entire gene of the present invention by hybridizing with a DNA fragment prepared by restriction enzyme digestion of the DNA fragment having the nucleotide sequence as a probe. SEQ ID NO: 3 shows the nucleotide sequence of the NADH oxidase gene of the present invention and SEQ ID NO: 4 shows the amino acid sequence encoded by the gene.

The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used in the production of the recombinant vector. For example, when a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention can be autonomously replicated in the host itself, and can also contain a DNA containing an NADH oxidase gene that produces a promoter and water, And the like. As the expression vector used in the present invention, pET28a was used, but any expression vector satisfying the above requirements can be used.

The production of water-producing NADH oxidase according to the present invention can be carried out by culturing a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same and culturing the transformant in a culture (culture cells or culture supernatant) Producing and accumulating NADH oxidizing enzyme producing phosphorus, and obtaining the enzyme from the culture.

Acquisition and purification of NADH oxidizing enzyme producing water can be carried out by collecting the cells of the obtained culture through centrifugation, and performing cell disruption, affinity chromatography, cation or anion exchange chromatography, etc., alone or in combination .

The present inventors have cloned a gene of NADH oxidase which generates water from a colony-forming strain to develop water-producing NADH oxidase. L-arabinitol can be produced in a high yield from L-arabinitol through a coupling reaction between a recombinant NADH oxidase inserted with an electric gene and Hypocrease corina L-arabinitol dehydrogenase , The production of by-products can be greatly reduced, and the present invention has been completed.

The present invention clones a gene coding for NADH oxidase from a gene of Pseudomonas aeruginosa to prepare industrially useful water-producing NADH oxidase, analyzes the nucleotide sequence of the gene and the amino acid sequence deduced therefrom.

NADH can be oxidized by a recombinant strain inserted with an electric gene and it can be used for regeneration of coenzyme NADH and NAD ⁺ for the purpose of not producing any byproduct except water.

The optimum conditions for the production of L-xylulose through the coupling reaction of water-producing NADH oxidase and hypoclase K-L-arabinitol dehydrogenase were 4.9 U / ml, 8.2 mM NAD ⁺ Reaction pH 8.0, and reaction temperature 30.9 占 폚.

The NADH oxidizing enzyme producing the pyrethrinsophilus strain isolated in the present invention can be used for regenerating the coenzyme NAD ⁺ , and in particular, the NADH oxidizing enzyme producing water and the L-arabinitol dehydrating enzyme L-xylulose can be efficiently produced by a coupling reaction of digestive enzymes.

Fig. 1 is a vector map of vector pUC-LOX, which is obtained by cloning a fragment having an NADH oxidase gene that produces water in the chromosome of S. pneumoniae into a vector used in Escherichia coli.
2 is a diagram showing a method for producing an expression vector comprising an NADH oxidase gene that produces water derived from a pneumococcal strain.
Fig. 3 is an SDS-PAGE gel photograph of NADH oxidase producing water derived from a pneumococcal strain. One; Size marker, 2; Insoluble protein of NADH oxidase producing water, 3; Water-soluble protein of NADH oxidase producing water, 4; NADH oxidase producing purified water.
Figure 4 shows the conversion of L-xylulose produced from L-arabinitol to the concentration of L-arabinitol.
5 is an analysis of the conversion of L-xylulose produced from L-arabinitol through reaction surface analysis. (a) the correlation between the concentration of L-arabinitol dehydrogenase (U / ml) and the concentration of NAD (mM), (b) the ratio between the concentration of L-arabinitol dehydrogenase (C) Correlation between L-arabinitol dehydrogenase concentration (U / ml) and production temperature (° C).
FIG. 6 is a graph showing the results of analysis of L-arabinitol produced from L-arabinitol by high pressure liquid chromatography (HPLC) using L-xylulose produced through coupling reaction. A. Before Coupling, B. After Coupling
FIG. 7 is a graph showing the yield of L-xylulose produced by time in an optimized condition through a coupling reaction. FIG.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example  1: Water producing NADH  Oxidase Production bacterium  Selection

In order to isolate highly active colony producing water-producing NADH oxidase, 10 μl of the culture broth was suspended in 10 ml of physiological saline, and 10 μl (1 × 10 ⁴ cfu ml ^-1 ) of the suspension was added to 3% malt (Malt extract peptone agar) supplemented with the extract, and then cultured at 37 ° C for 2 days. After cultivating colonies on solid peptone medium, colonies were picked up and colonies with NADH oxidase activity were selected to produce water. Thus, a highly activated colony - forming strain producing water - producing NADH oxidase was searched.

Example 2: Cloning of the NADH oxidase gene to generate new water from colostrum (KCTC 3208)

Bacteria (KCTC 3208) were used to obtain the nucleotide sequence of the NADH oxidase gene, which produces water, L-arabinitol degrading enzyme. In general, genes with similar functions are known to be somewhat similar in size and sequence to each other. Therefore, it is assumed that the gene of NADH oxidase, which produces water of Pseudomonas aeruginosa (KCTC 3208), may have a size of about 1.4 kb. Based on the nucleotide sequence of NADH oxidase, which is already known water of other bacteria, (KCTC 3208) were cloned into the water-producing NADH oxidase whole gene.

Escherichia coli XL1-Blue and pUC18 vectors were used for cloning. As the culture medium for Escherichia coli, LB medium of the general composition was used, and Malt extract peptone agar was used for culturing of Streptococcus pneumoniae (KCTC 3208). As the plate medium for E. coli, LB agar and 3-5% sugar, 0.3-0.5% beef extract, 0.9-1.1% bactopeptone and 1.3-1.7% agar plate were used, respectively. 50 / ml amipicillin was added as needed.

In the case of colostrum (KCTC 3208), the culture was inoculated in a 250 ml Erlenmeyer flask containing 50 ml of medium, and cultured at 37 ° C and 200 rpm for 1 day. For Escherichia coli, 37 ° C and 200 rpm And cultured for 16 hours.

Most of the DNA was identified by agarose gel (TAE buffer, 0.5%) electrophoresis. The DNA band on the gel was purified using QiaXII gel extraction kit (QIAGEN, USA) Linking enzyme (NEB) was used. In addition, Qiagen plant total RNA kit (QIAGEN) was used for RNA extraction of pneumococcal bacteria (KCTC 3208). Oligo-dT RT-mix (intron) was used for reverse transcriptase for cDNA synthesis.

Chromosomal isolates of KCTC 3208 were isolated in order to clone NADH oxidase gene producing water. DhLOX F-5'-TCH TTY (SEQ ID NO: 1) Based on the NADH oxidase base sequence which produces water already known from other bacteria to amplify a portion of the NADH oxidase gene in the pneumococcal strain (KCTC 3208) YTH TCW TGT GGD AT -3 '(SEQ ID NO: 1) and DhLOX R-5'-CGH AYV GCR TTV GTD GCY A -3' (SEQ ID NO: 2). Using this, a portion of the 784 bp NADH oxidase gene was amplified by Chain Polymerase Chromosome (KCTC 3208) chromosome.

The genomic DNA of Bacillus subtilis (KCTC 3208) was completely digested with restriction enzymes BamHI, EcoRI, HindIII, SalI, and XbaI in the absence of the cleavage site of the amplified partial sequence. Then, a radioactively labeled probe was prepared using DNA fragments obtained by polymerase chain reaction. Using this method, we searched for DNA fragments with the gene that they were looking for by hybridization. When the chromosomes were cut with BamHI, HindIII and XbaI, the size of the DNA of the NADH oxidase gene, which generates water as a result of the hybridization, was about 20 to 23 kb, The desired gene was searched using a fragment of about 2.5 kb EcoRI and a fragment of about 5.8 kb SalI. The chromosomes were digested with EcoRI, and a DNA fragment of about 2.5 kb size and a DNA fragment of about 5.8 kb digested with SalI were cloned into pUC18 and named pUC-LOX (FIG. 1).

The pUC-LOX library was subjected to colony hybridization using the 1 kb probe prepared above to determine the clone with the NADH oxidase gene that produces the desired water. The determined nucleotide sequence of the clone was analyzed to reveal the entire nucleotide sequence of NADH oxidase producing water at 1,371 bp (SEQ ID NO: 3). As expected, it was similar in size to the NADH oxidase gene that produced water revealed in many other bacteria. In addition, it was confirmed that NADH oxidase producing waterborne streptococci (KCTC 3208) water has a common nucleotide sequence in NADH oxidase producing other water.

Example  3: Preparation of Recombinant Expression Vector and Recombinant Strain

In order to mass-express NADH oxidase in Escherichia coli using the gene encoding the NADH oxidase producing water according to Example 2, the enzyme gene was inserted into the BamH and XhoI sites of the expression vector pET28a (Novagen, USA) And then transformed into E. coli BL21 (DE3) (NEB, UK) (Fig. 2).

Example  4: Produce recombinant NADH  Expression and purification of oxidase

The recombinant strain prepared in Example 3 was inoculated on LB medium and cultured at 37 ° C for 24 hours, and the protein expressed on SDS-PAGE gel was confirmed (FIG. 3).

In order to purify the NADH oxidase producing the recombinant expressed by the method of Example 4, the culture of the recombinant strain was centrifuged (8000xg, 10 minutes) to collect only the cells, and the cell wall of E. coli was disrupted by ultrasonication, The precipitate (cells) was removed by centrifugation at 20,000xg for 20 minutes and supernatant was obtained. After obtaining, the Ni-NTA His-tag binding chromatography (Qiagen, Germany) was finally performed to purify the NADH oxidase producing the recombinant product.

Example  4: NADH  Oxidase and L- Arabinitol  Lt; RTI ID = 0.0 > L- Xylulose  Optimal production condition experiment

(1) coupling of L- Xylulose  The optimal production of L- Arabinitol  Effect of Concentration

Arabinitol dehydrogenase enzyme concentration of 4.9 U / ml, NAD ⁺ concentration of 8.2 mM, pH of 8, temperature of 30 ° C and L - Enzyme activities were compared by varying the production temperature of xylulose to 50 ~ 300 mM. As shown in FIG. 4, the conversion of L-arabinitol to L-arabinitol was 90% or more at an L-arabinitol concentration of 50 mM to 150 mM.

(2) L- Xylulose  Optimal production test conditions

The concentration of NADH oxidase to produce water is 6.25 U / ml, L- arabinitol concentrations are 250 mM, L- arabinitol concentration of the dehydrogenation enzyme is 1.37 ~ 6.86 U / ml, NAD + concentration of 2.5 to 12.5 , the pH was varied from 2.5 to 12.5 and the production temperature was varied from 20 to 40 (Table 1).

Encrypted number -α -One 0 +1 + alpha Enzyme concentration (U / ml) 1.37 2.74 4.12 5.49 6.86 NAD ⁺ concentration (mM) 2.5 5.0 7.5 10.0 12.5 pH 6 7 8 9 10 Temperature (℃) 20 25 30 35 40

Table 1 is a conditional experiment table for enzyme activity optimization.

(3) L- Xylulose  Optimized for conversion efficiency

Based on the conditions of Example 4 (2), in order to optimize the conversion efficiency of L-xylulose from L-arabinitol through coupling reaction, NAD concentration, pH, reaction temperature, L-arabinitol dehydrogenation Enzyme concentration was varied. Thirty experiments were designed through Reaction Surface Methodology to optimize the production of L-xylulose. The L-xylulose conversion rate through each experiment was as shown in Table 2, and the correlation of each experimental variable was as shown in Fig.

number HjLAD
(U / mL) NAD +
(mM) pH Temperature ( ^o C) Conversion Rate (%) Estimated conversion rate (%) One 4.12 7.5 8 30 76.94 76.94 2 5.49 5 9 25 63.71 65.87 3 4.12 12.5 8 30 70.74 73.07 4 2.75 5 7 25 61.39 61.01 5 2.75 10 7 25 70.36 71.04 6 2.75 5 9 35 50.00 50.52 7 5.49 10 9 25 72.04 69.33 8 5.49 10 7 35 68.87 68.53 9 4.12 7.5 8 30 76.94 76.94 10 2.75 5 7 35 63.33 65.03 11 5.49 10 9 35 69.07 70.02 12 5.49 10 7 25 62.16 62.22 13 2.75 10 9 25 64.29 64.07 14 4.12 7.5 8 30 76.94 76.94 15 4.12 7.5 8 30 76.94 76.94 16 4.12 7.5 6 30 67.52 66.37 17 2.75 10 7 35 73.62 72.04 18 2.75 5 9 25 52.80 52.12 19 4.12 7.5 8 30 76.94 76.94 20 5.49 5 9 35 71.26 69.57 21 2.75 10 9 35 61.39 59.46 22 4.12 7.5 8 30 76.94 76.94 23 4.12 2.5 8 30 64.48 62.59 24 4.12 7.5 8 40 60.00 60.57 25 5.49 5 7 35 69.19 70.01 26 4.12 7.5 8 20 56.00 55.87 27 1.37 7.5 8 30 63.32 64.04 28 6.86 7.5 8 30 74.56 74.27 29 5.49 5 7 25 59.77 60.68 30 4.12 7.5 10 30 57.38 58.97

Table 2 shows L-xylulose production optimization table through RSM.

(4) Production of L-xylulose from L-arabinitol via coupling under optimal conditions

A coupling experiment using NADH oxidase which produces water derived from the strain of the present invention strain of the present invention and L-arabinitol dehydrogenase derived from Hippocase-Corina was carried out. The concentration of NADH oxidase to produce water in the coupling reaction mixture is 6.25 U / ml, L- arabinitol concentrations are 250 mM, L- Ara beanie concentration is 4.9 U / ml, NAD ⁺ concentration in the toll dehydrogenation enzyme 8.2 mM, pH 8.0, and temperature 30.9 ° C. The L-xylulose produced in the optimized coupling reaction was confirmed by HPLC (Fig. 6). FIG. 7 shows the conversion yield according to the reaction time under the optimized conditions. When the reaction was carried out for 8 hours, the maximum conversion rate was 78.4% (FIG. 7).

<110> Konkuk University Industrial Cooperation Corp. <120> A novel NADH oxidase from Streptococcus pyogenes and its coupling          with L-arabinitol dehydrogenase for L-xylulose production <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 tchttyytht cwtgtggdat 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 cghayvgcrt tvgtdgcya 19 <210> 3 <211> 1371 <212> DNA <213> Streptococcus pyogenes <400> 3 atgtctaaaa ttgttgttgt tggtgctaac cacgctggta ctgctgctat taaaactatg 60 ttaactaact atggtgatac taacgaaatt gttgttttcg atcaaaactc taacatttct 120 ttcttaggtt gtggtatggc tttatggatt ggtgaacaaa ttgctggtcc agaaggttta 180 ttctattctg ataaagaaga attagaatct ttaggtgcta aagtttatat ggaatctcca 240 gttcaatcta ttgattatga tgctaaaact gttactgctt tagttgatgg taaaaaccac 300 gttgaaactt atgataaatt aattttcgct actggttctc aaccaatttt accaccaatt 360 aaaggtgctg aaattaaaga aggttcttta gaattcgaag ctactttaga aaacttacaa 420 ttcgttaaat tatatcaaaa ctctgctgat gttattgcta aattagaaaa caaagatatt 480 aaacgtgttg ctgttgttgg tgctggttat attggtgttg aattagctga agctttccaa 540 cgtaaaggta aagaagttgt tttaattgat gttgttgata cttgtttagc tggttattat 600 gatcgtgatt taactgattt aatggctaaa aacatggaag aacacggtat tcaattagct 660 ttcggtgaaa ctgttaaaga agttgctggt aacggtaaag ttgaaaaaat tattactgat 720 aaaaacgaat atgatgttga tatggttatt ttagctgttg gtttccgtcc aaacactact 780 ttaggtaacg gtaaaattga tttattccgt aacggtgctt tcttagttaa caaacgtcaa 840 gaaacttcta ttccaggtgt ttatgctatt ggtgattgtg ctactattta tgataacgct 900 actcgtgata ctaactatat tgctttagct tctaacgctg ttcgtactgg tattgttgct 960 gctcacaacg cttgtggtac tgatttagaa ggtattggtg ttcaaggttc taacggtatt 1020 tctatttatg gtttacacat ggtttctact ggtttaactt tagaaaaagc taaacgttta 1080 ggtttcgatg ctgctgttac tgaatatact gataaccaaa aaccagaatt cattgaacac 1140 ggtaacttcc cagttactat taaaattgtt tatgataaag attctcgtcg tattttaggt 1200 gctcaaatgg ctgctcgtga agatatgtct atgggtattc acatgttctc tttagctatt 1260 caagaaggtg ttactattga aaaattagct ttaactgata ttttcttctt accacacttc 1320 aaccgtccat ataactatat tactatggct gctttaggtg ctaaagatta g 1371 <210> 4 <211> 456 <212> PRT <213> Streptococcus pyogenes <400> 4 Met Ser Lys Ile Val Val Gly Ala Asn His Ala Gly Thr Ala Ala   1 5 10 15 Ile Lys Thr Met Leu Thr Asn Tyr Gly Asp Thr Asn Glu Ile Val Val              20 25 30 Phe Asp Gln Asn Ser Asn Ile Ser Phe Leu Gly Cys Gly Met Ala Leu          35 40 45 Trp Ile Gly Glu Gln Ile Ala Gly Pro Glu Gly Leu Phe Tyr Ser Asp      50 55 60 Lys Glu Glu Leu Glu Ser Leu Gly Ala Lys Val Tyr Met Glu Ser Pro  65 70 75 80 Val Gln Ser Ile Asp Tyr Asp Ala Lys Thr Val Thr Ala Leu Val Asp                  85 90 95 Gly Lys Asn His Val Glu Thr Tyr Asp Lys Leu Ile Phe Ala Thr Gly             100 105 110 Ser Gln Pro Ile Leu Pro Pro Ile Lys Gly Ala Glu Ile Lys Glu Gly         115 120 125 Ser Leu Glu Phe Glu Ala Thr Leu Glu Asn Leu Gln Phe Val Lys Leu     130 135 140 Tyr Gln Asn Ser Ala Asp Val Ile Ala Lys Leu Glu Asn Lys Asp Ile 145 150 155 160 Lys Arg Val Ala Val Val Gly Ala Gly Tyr Ile Gly Val Glu Leu Ala                 165 170 175 Glu Ala Phe Gln Arg Lys Gly Lys Glu Val Val Leu Ile Asp Val Val             180 185 190 Asp Thr Cys Leu Ala Gly Tyr Tyr Asp Arg Asp Leu Thr Asp Leu Met         195 200 205 Ala Lys Asn Met Glu Glu Gly Gly Ile Gln Leu Ala Phe Gly Glu Thr     210 215 220 Val Lys Glu Val Ala Gly Asn Gly Lys Val Glu Lys Ile Ile Thr Asp 225 230 235 240 Lys Asn Glu Tyr Asp Val Asp Met Val Ile Leu Ala Val Gly Phe Arg                 245 250 255 Pro Asn Thr Thr Leu Gly Asn Gly Lys Ile Asp Leu Phe Arg Asn Gly             260 265 270 Ala Phe Leu Val Asn Lys Arg Gln Glu Thr Ser Ile Pro Gly Val Tyr         275 280 285 Ala Ile Gly Asp Cys Ala Thr Ile Tyr Asp Asn Ala Thr Arg Asp Thr     290 295 300 Asn Tyr Ile Ala Leu Ala Ser Asn Ala Val Arg Thr Gly Ile Val Ala 305 310 315 320 Ala His Asn Ala Cys Gly Thr Asp Leu Glu Gly Ile Gly Val Gln Gly                 325 330 335 Ser Asn Gly Ile Ser Ile Tyr Gly Leu His Met Val Ser Thr Gly Leu             340 345 350 Thr Leu Glu Lys Ala Lys Arg Leu Gly Phe Asp Ala Ala Val Thr Glu         355 360 365 Tyr Thr Asp Asn Gln Lys Pro Glu Phe Ile Glu His Gly Asn Phe Pro     370 375 380 Val Thr Ile Lys Ile Val Tyr Asp Lys Asp Ser Arg Arg Ile Leu Gly 385 390 395 400 Ala Gln Met Ala Ala Arg Glu Asp Met Ser Met Gly Ile His Met Phe                 405 410 415 Ser Leu Ala Ile Glu Glu Gly Val Thr Ile Glu Lys Leu Ala Leu Thr             420 425 430 Asp Ile Phe Leu Pro His Phe Asn Arg Pro Tyr Asn Tyr Ile Thr         435 440 445 Met Ala Ala Leu Gly Ala Lys Asp     450 455

Claims (10)

delete delete delete delete delete A method for regenerating NAD + from NADH using an NADH oxidase consisting of the amino acid sequence of SEQ ID NO: 4. NADH oxidase consisting of the amino acid sequence of SEQ ID NO: 4 is coupled with L-arabinitol dehydrogenase from Hypocrea jecorina to produce L-xylulose from L-arabinitol in the presence of NAD ⁺ Way. The method of claim 7, wherein in the method, the concentration of NADH oxidase in a concentration of 250 mM are, L- arabinitol dehydrogenation enzyme 6.25 U / ml, L- arabinitol concentrations of 4.9 U / ml, NAD ⁺ Wherein the concentration is 8.2 mM. 8. The method of claim 7, wherein the method is performed at a pH of 8.0 and a temperature of 30.9 &lt; 0 &gt; C. An NADH oxidase consisting of the amino acid sequence of SEQ ID NO: 4,
Composition for the production of L-xylulose from L-arabinitol containing L-arabinitol dehydrogenase derived from Hypocrea jecorina and NAD ⁺ as an active ingredient.

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