Abstract:
After identifying the genes of a novel lipoxygenase and a novel allene oxide synthase derived from Lemna paucicostata SH strain, a plant growth regulating agent (KODA) was produced at high yield by using a Lemna paucicostata strain that expresses the lipoxygenase and the allene oxide synthase.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a method of preparing a plant hormone having a structure represented by the following Formula (I) (common name: 9-hydroxy-10-oxo-cis-12(Z),15(Z)-octadecadienoic acid, herein after referred to as KODA): 
         [0000]    
       
                 
         
             
             
         
       
     
       BACKGROUND ART 
       [0002]    KODA is known to be a plant hormone having a plant floral bud-formation promoting activity, a plant activating activity, and a plant growth regulating activity incorporating these activities (Japanese Unexamined Patent Publication (Kokai) No. 9-295908, Japanese Unexamined Patent Publication (Kokai) No. 11-29410, Japanese Unexamined Patent Publication (Kokai) No. 2001-131006, Japanese Unexamined Patent Publication (Kokai) No. 2009-17829). KODA is known to be present in various plant species, and Lemna paucicostata that was subjected to stress is known to release KODA at a very high level (a few hundred-fold higher) compared to other plants. Utilizing this property, KODA can be prepared using an extraction method in which KODA is obtained by extracting from Lemna paucicostata, a species of the family Lemnaceae. As other production methods, KODA can be prepared by using an enzymatic method in which α-linolenic acid (common name: cis-9,12,15-octadecatrienoic acid), which is an unsaturated fatty acid, is subjected to enzymes such as position 9-product specific lipoxygenase (LOX), allene oxide synthase (AOS) in accordance with the fatty acid metabolic pathway in a plant, or by using a chemical synthetic method in which generally known chemical synthetic methods are used. These production methods are disclosed in Japanese Unexamined Patent Publication (Kokai) No. 11-29410. 
         [0003]    Since KODA is a plant hormone having a plant growth regulating activity, its application in the agricultural filed is promising. When used in agriculture, unlike pharmaceuticals, it cannot be put into practical use unless produced at low cost and in large quantities. 
         [0004]    In an enzymatic method that employs α-linolenic acid as a starting substance, as described below, KODA can be prepared by acting 9-specific lipoxygenase (LOX) on α-linolenic acid as the substrate, thereby introducing a hydroperoxy group (—OOH) at position 9, and then acting allene oxide synthase (AOS) thereon. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0005]    However, position 9-specific lipoxygenase is not commercially available, and even the extraction from a plant requires a lot of time and effort on obtaining and processing materials. Also, the activity of position 9-specific lipoxygenases obtained to date was low. Furthermore, when cDNA of position 9-specific lipoxygenase known to date was expressed in  Escherichia coli , most of them are turned out to be insoluble, and thus it was difficult to obtain active protein at large quantities. 
         [0006]    Allene oxide synthase is an enzyme having an activity of converting hydroperoxylated fatty acid to allene oxide, and since allene oxide is unstable, it is non-enzymatically converted to α-ketol form. AOS is an enzyme present in plants, animals, and yeast, and in plants, it is present in throughout the angiosperms. However, allene oxide synthase generally has a suicide substrate-like property, and thus when the substrate concentration is increased, the amount of product conversely decreases. Due to the drawbacks of the lipoxygenase and allene oxide synthase, the enzymatic method was not suitable for a large scale production. On the other hand, in chemical synthesis, it was difficult to attain the low cost that was desired at the agricultural filed. 
         [0007]    On the other hand, while the conventional extraction method was carried out by culturing Lemna paucicostata 441 strain that are known to produce a floral bud-inducing substance at high efficiency, even the use of such strains could not produce a sufficient amount of KODA. Thus, there has been a need for a method of preparing KODA at low cost and in large quantities. 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0008]    An object of the present invention is to provide a method of preparing KODA in which the yield of KODA has been improved. 
       Solution to Problem 
       [0009]    In intensive research to solve the above problem, the present inventors conducted the screening of various strains of Lemna paucicostata. As a result, the present inventors have found that compared to other Lemna paucicostata strains, Lemna paucicostata SH strain produces an extremely high level of KODA. 
       Advantageous Effects of Invention 
       [0010]    Based on the above finding, the present inventors provide a method of preparing KODA at a high yield by using Lemna paucicostata SH strain as the starting substance in a method of preparing KODA based on the extraction method. 
         [0011]    Furthermore, the present inventors have focused on the metabolic pathway of Lemna paucicostata SH strain which is a high KODA-producing strain, and have succeeded for the first time in identifying the gene sequence (SEQ ID NO: 1) of position 9-specific lipoxygenase and the gene sequence (SEQ ID NO: 2) of allene oxide synthase in the Lemna paucicostata The base sequences of said SEQ ID NO: 1 and SEQ ID NO: 2 are sequences both derived from Lemna paucicostata SH strain. 
         [0012]    Thus, the present inventors provide a method of preparing KODA based on the extraction method wherein KODA can be produced at a high yield by using a Lemna paucicostata strain having a gene consisting of a DNA consisting of a sequence represented by SEQ ID NO: 1 and/or SEQ ID NO: 2, or a DNA substantially identical to the above DNA, 
         [0013]    The present invention also provides KODA produced by the production method using a high KODA-producing Lemna paucicostata strain described above. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0014]      FIG. 1  is a graph showing the amount of KODA produced for 62 strains of Lemna paucicostata. 
           [0015]      FIG. 2A  shows the gene sequence of the LOX gene of Lemna paucicostata SH strain. 
           [0016]      FIG. 2B  shows the continuation of gene sequence of  FIG. 2A . 
           [0017]      FIG. 2C  shows the continuation of gene sequence of  FIG. 2B . 
           [0018]      FIG. 3A  shows the gene sequence of the AOS gene of Lemna paucicostata SH strain. 
           [0019]      FIG. 3B  shows the continuation of gene sequence of  FIG. 3A . 
           [0020]      FIG. 3C  shows the continuation of gene sequence of  FIG. 3B . 
           [0021]      FIG. 4  is a graph showing the comparison of the activity of a rice plant LOX (r9-LOX) and LOX of Lemna paucicostata SH strain expressed in  Escherichia coli.    
           [0022]      FIG. 5  is a a graph showing the comparison of the activity of AOS of Arabidopsis thaliana and AOS of Lemna paucicostata SH strain expressed in  Escherichia coli.    
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0023]    The method of preparing KODA of the present invention comprises applying stress to a specific high KODA-producing Lemna paucicostata strain, extracting KODA with a solvent from said stressed Lemna paucicostata strain, and purifying KODA. 
         [0024]    In an embodiment of the present invention, the specific high KODA-producing Lemna paucicostata strain to be used in the present invention is a strain that expresses a protein encoded by a DNA identical with or substantially identical with a DNA consisting of a base sequence represented by SEQ ID NO: 1 and/or a DNA that is identical with or substantially identical with a DNA comprising a base sequence represented by SEQ ID NO: 2. Furthermore, the specific high KODA-producing Lemna paucicostata strain of the present invention to be used in the present invention is a strain that expresses a protein identical with or substantially identical with a protein consisting of an amino acid sequence represented by SEQ ID NO: 3, and/or a protein identical with or substantially identical with a protein consisting of an amino acid sequence represented by SEQ ID NO: 4. SEQ ID NO: 3 is the amino acid sequence of position 9-product specific lipoxygenase derived from Lemna paucicostata SH strain, and SEQ ID NO: 4 is the amino acid sequence of allene oxide synthase derived from Lemna paucicostata SH strain. 
         [0025]    Since KODA is prepared from linolenic acid by the acting of LOX and AOS in the plant, a strain that expresses LOX and/or AOS identical to or substantially identical to LOX and/or AOS of the high KODA-producing Lemna paucicostata SH strain is considered to have a high KODA productivity similarly to the Lemna paucicostata SH strain. As used herein the term “substantially identical DNA” refers to a DNA that has a 70% identity with the reference DNA, and that encodes a protein, when transcribed and translated, having the same enzyme activity as that of a protein produced by the transcription and translation of the reference DNA (the LOX activity in the case of a DNA consisting of a base sequence represented by SEQ ID NO: 1, and the AOS activity in the case of a DNA consisting of a base sequence represented by SEQ ID NO: 2). The identity may preferably be at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. 
         [0026]    Also, “substantially identical DNA” refers to a DNA that can hybridize to a DNA consisting of a base sequence complementary to the reference DNA under a high stringent condition, and that encodes a protein having the same enzyme activity as that of a protein encoded by the reference DNA (the LOX activity in the case of a DNA comprising a base sequence represented by SEQ ID NO: 1, and the AOS activity in the case of a DNA comprising a base sequence represented by SEQ ID NO: 2). Hybridization can be carried out by a known method or a method based on said method, such as a method described in J. Sambrook et al., Molecular Cloning, 2nd., Cold Spring Harbor Laboratory Press, 1989, and a high stringent hybridization condition refers to, for example, a NaCl concentration of about 10-40 mM, preferably about 20 mM, and a temperature of about 50-70° C., preferably about 60-65° C. Furthermore, the present invention also relates to a DNA fragment of said DNA sequence, said fragment encoding a protein having the enzyme activity of the protein encoded by the original DNA. 
         [0027]    As used herein “a substantially identical protein” refers to a protein that comprises an amino acid sequence in which one or a few amino acids have been deleted, substituted or added in the amino acid sequence of the reference protein, and that has the activity of the reference protein. Furthermore, “a substantially identical protein” refers to a protein that comprises a sequence having an identity of 98% with the reference amino acid sequence, and that has the activity of the reference protein. The identity may preferably be at least 99%, at least 99.5%, or at least 99.9%. 
         [0028]    The method of preparing KODA of the present invention utilizes an extraction method. Specifically, a homogenate of Lemna paucicostata is centrifuged (8000×g, for about 10 minutes), and among the supernatant and the precipitate obtained, the supernatant is removed, and the rest is used in the next step as a KODA-containing fraction. Using such a fraction as the starting substance, KODA can be isolated and purified. In order to promote the production of KODA from Lemna paucicostata, it may be preferred to apply a specific stress described below to Lemna paucicostata before centrifugation. 
         [0029]    As a preferred starting substance in terms of preparation efficiency, there can be mentioned an aqueous solution obtained after Lemna paucicostata was allowed to float or immersed. This aqueous solution may not be specifically limited as long as Lemna paucicostata can grow therein. Specific examples of the aqueous solution will be described in the Examples described below. 
         [0030]    The immersing time may be about 2-3 hours at room temperature, but should not be limited. When KODA is prepared by this method, it may be preferred in terms of production efficiency to apply to Lemna paucicostata a specific stress that can induce KODA. 
         [0031]    Specifically, as a specific stress, there can be mentioned a drying stress, a heat stress, an osmotic stress and the like. The drying stress can be applied by, for example, allowing Lemna paucicostata to be left spreaded on a dry filter paper at a low humidity (preferably a relative humidity of 50% or less) and at room temperature, preferably about 24-25° C. The drying time in this case is roughly 20 seconds or more, preferably 5 minutes or more, and more preferably 15 minutes or more. 
         [0032]    The heat stress may be applied by, for example, immersing Lemna paucicostata in a warm water. The temperature of the warm water may be 40-65° C., preferably 45-60° C., and more preferably 50-55° C. As the time of immersing in the warm water, roughly 5 minutes may be sufficient, and when the temperature is relatively low, for example when Lemna paucicostata is treated in a warm water of about 40° C., it is preferable to treated for 2 hours or more. After the above heat stress treatment, Lemna paucicostata may preferably be quickly returned to cold water. 
         [0033]    The osmotic stress may be applied by, for example, contacting Lemna paucicostata with a high osmotic solution such as a high-concentration sugar solution. The sugar concentration at this time, in the case of a mannitol solution, may preferably be 0.3 M or more, and preferably 0.5 M or more. When, for example, a 0.5 M mannitol solution is used, the treating time may preferably be one minute or more, and preferably 3 minutes or more. Thus, the desired starting substance containing KODA of the present invention can be prepared. 
         [0034]    Then, the starting substance prepared as described above may be subjected to the separation and purification treatment as described below to prepare the desired KODA. The separation means shown in this specification is only for illustrative purposes, and a separation means for producing KODA from the above starting substance should not be limited to the separation means. 
         [0035]    First, it may be preferred that the above starting substance is subjected to solvent extraction to extract a component containing KODA of the present invention. The solvent to be used in such solvent extraction should not be specifically limited, and for example chloroform, ethyl acetate, butanol etc., may be used. Among these solvents, chloroform is preferred since it can remove impurities relatively easily. 
         [0036]    The oil phase obtained by this solvent extraction may be washed and concentrated by a commonly known method, and subjected to high performance liquid chromatography (HPLC) using a reverse phase partition chromatography with an ODS (octadecylsilane) column for identification and isolation of a fraction having a floral bud-inducing activity to isolate KODA of the present invention. Depending on the properties of the starting substance, other generally known separation means such as ultrafiltration, gel filtration chromatography etc., may be used in combination. 
       EXAMPLES 
     Example 1 
     Screening of a High KODA-Producing Lemna Paucicostata Strain 
       [0037]    62 types of Lemna paucicostata harvested from different places were prepared and were subcultured in the ½-diluted Hutner&#39;s medium under continuous illumination of daylight fluorescent light at 24-25° C. The ½-diluted Hutner&#39;s medium comprises the following ingredients: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Sucrose 
                 10 
                 g/l 
               
               
                   
                 K 2 HPO 4   
                 200 
                 mg/l 
               
               
                   
                 NH 4 NO 3   
                 100 
                 mg/l 
               
               
                   
                 EDTA free acid 
                 250 
                 mg/l 
               
               
                   
                 Ca(NO 3 )•4H 2 O 
                 176 
                 mg/l 
               
               
                   
                 MgSO 4 •7H 2 O 
                 250 
                 mg/l 
               
               
                   
                 FeSO 4 •7H 2 O 
                 12.4 
                 mg/l 
               
               
                   
                 MnCl 2 •4H 2 O 
                 8.92 
                 mg/l 
               
               
                   
                 ZnSO 4 •7H 2 O 
                 32.8 
                 mg/l 
               
               
                   
                 Na 2 MoO 4 •2H 2 O 
                 12.6 
                 mg/l 
               
               
                   
                 H 3 BO 3   
                 7.1 
                 mg/l 
               
               
                   
                 Co(NO 3 )•6H 2 O 
                 0.1 
                 mg/l 
               
               
                   
                 CuSO 4 •5H 2 O 
                 1.97 
                 mg/l 
               
               
                   
                   
               
               
                   
                 and pH was adjusted to 6.2-6.5 with KOH (50%). 
               
             
          
         
       
     
         [0038]    The grown Lemna paucicostata was spreaded on a filter paper, and after incubating for 2 hours, it was immersed in water for 1 hour. The water was subjected to high performance liquid chromatography (HPLC; column: TYPE UG120 5 μm, SIZE: 4.6 mm I.D.×250 mm; guard filter: INERTSTL 4.6 mm×50 mm; eluent: 50% acetonitrile+0.1% trifluoroacetic acid; condition: absorption wavelength 210 λ(nm), flow rate: 1 ml/min, column temperature: 40° C.) to analyze the concentration of KODA. The mean of the production amount of KODA in all Lemna paucicostata strains was 4.97 μM. Among them, Lemna paucicostata SH strain produced 60.2 μM of KODA, which is about 12-times higher than the mean amount of KODA produced, giving a very high production amount ( FIG. 1 ). 
       Example 2 
     Cloning of Lipoxygenase Derived from Lemna Paucicostata SH Strain and Measurement of the Activity Thereof 
       [0039]    From Lemna paucicostata (SH strain), total RNA was extracted by using the RNeasy Plant Mini Kit (QIAGEN), and then cDNA was synthesized by using 1.8 μg of total RNA as the template in LongRange 2 Step RT-PCR Kit (QIAGEN). 
         [0040]    Then, degenerate PCR (PCR condition: initial denaturation at 94° C. for 3 minutes; a cycle comprising 94° C. for 0.5 minute, 47° C. for 0.5 minute, and 72° C. for 1.3 minute is carried out for 39 times) was carried out by using the cDNA as the template, and using the following degenerate primers (LpDPf, LpDPr) to obtain a partial sequence of 9-lipoxygenase of interest. 
         [0000]    
       
         
               
               
             
           
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 LpDPf: 5′-GCITGGMGIAGIGAYGARGARTTY-3′ 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 LpDPr: 5′-GCRTAIGGRTAYTGICCRAARTT-3′ 
               
             
          
         
       
     
         [0041]    wherein, I represents inosine. 
         [0042]    After the base sequence of said partial sequence was determined, BLAST search was carried out based on the sequence information obtained. As a result, the sequence obtained exhibited a high homology with LOX derived from a plurality of known plants (75% with  Corylus avellana,  74% with  Actinidia deliciosa,  75% with  Solanum tuberosum,  76% with  Oryza sativa,  74% with  Nicotiana tabacum,  75% with  Cucumis sativus,  73% with  Arabidopsis thaliana , etc.). 
         [0043]    Based on this sequence information, primers for the following 3′ or 5′ RACE method (Rapid Amplification of cDNA end) were constructed, and the full-length sequence was determined by the 3′ or 5′ RACE method ( FIG. 2 ). 
         [0000]    
       
         
               
               
             
           
               
                   
                 (SEQ ID NO: 7) 
               
               
                   
                 SH-3′-TP: 5′-AGCTCTTCATCTTGGACC-3′ 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 SH-5′-TP: 5′-TTTCATCCTTCTTGTCGC-3′ 
               
             
          
         
       
     
         [0044]    The sequence of SHLpLOSX obtained was introduced into a vector (pET23d, Novagen) for protein expression, and then transformed into  Escherichia coli  (BL21(DE3), Novagen), so as to allow the expression of SHLpLOX protein. Using this SHLpLOX protein, the activity test in KODA production was carried out. 
         [0045]    In the activity test in KODA production, to an aqueous solution comprising 5 mM linolenic acid solution (dissolved in 0.1% Tween 80 solution; 25 μl), 0.2 M sodium phosphate buffer (pH 7, 10 μl), and distilled water (5 μl), an enzyme solution (10 μl) was added, and allowed to react at room temperature for 30 minutes. After the reaction was over, the reaction mixture was subjected to HPLC to determine the site specificity and the amount of linolenic acid hydroperoxide to be formed. HPLC analysis was carried out with a column: Capcell pak C-18 UG120 (4.6×250 mm, Shiseido), by using column temperature: 40° C., mobile phase: 50% acetonitrile solution (0.02% TFA), flow rate: 1 ml/min, detection wavelength: 210 nm. In this case, as a position 9-specific control enzyme, r9-LOX1 which is a position 9-specific lipoxygenase derived from rice germ. It was revealed that novel LOX obtained from Lemna paucicostata SH strain is a position 9-product specific lipoxygenase having a much higher activity than r9-LOX1 that had been considered to be the most potent among the known 9-product specific lipoxygenases ( FIG. 4 ). 
         [0046]    Among the novel LOXs derived from Lemna paucicostata, SHLpLOX that had the highest production amount of linolenic acid-9-hydroperoxide was subjected to kinetic analysis. Using a reaction mixture comprising 40 mM phosphate buffer (pH 6.0) and 0.1% Tween 80, the analysis was carried out at a temperature of 25° C. The substrate α-linolenic acid was tested at the substrate concentration of 10-100 μM. 100 μl of the reaction mixture was added into a cuvette, and absorbance at 234 nm was scanned over time for 10 minutes at an interval of 15 seconds using the SmartSpec Plus Spectrophotometer (Bio-Rad). The amount of the reaction product was calculated from the A 234  determined (e=25,000). Kinetic parameters were determined using the Hanes-Woolf plot ([S]/v versus [S] plot). As shown in Table 1, the result shows that the K m  value which is an affinity parameter for substrate is lower in SHLpLOX than in r9-LOX1, thereby indicating that SHLpLOX had a high affinity for the substrate α-linolenic acid. The maximum reaction velocity V max  is almost comparable between r9-LOX and SHLpLOX, while the k cat  value which is the number of reactions per unit time was higher in SHLpLOX than in r9-LOX1. The k cat /K m  value which is an index of enzyme activity was about 1.6-fold higher in SHLpLOX than in r9-LOX1. This revealed that the novel 9-LOX derived from Lemna paucicostata SH strain is a very highly active 9-LOX. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Kinetic parameters of SHLpLOX and r9-LOX1 
               
             
          
           
               
                   
                 K m   
                 V max   
                 k cat   
                 K cat /K m   
               
               
                   
                 (μM) 
                 (μmol min −1 ) 
                 (min −1 ) 
                 (×10 5 M −1 min −1 ) 
               
               
                   
                   
               
             
          
           
               
                 LpLOX- 
                 19.2 ± 4.1 
                 3.0 ± 0.1 
                 768.2 ± 32.3  
                 41.4 ± 10.0 
               
               
                 SH 
               
               
                 r9-LOX1 
                 22.9 ± 1.6 
                 2.8 ± 0.6 
                 614.1 ± 124.6 
                 26.7 ± 4.2  
               
               
                   
               
             
          
         
       
     
       Example 3 
     Cloning of the AOS Gene Derived from Lemna Paucicostata SH Strain and the Activity Measurement Thereof 
       [0047]    Total RNA was extracted from Lemna paucicostata (SH strain), and cDNA was synthesized by the RT-PCR method. Then, using the synthesized cDNA as the template and using the primers derived from  Arabidopsis thaliana  as described below, a partial sequence information of allene oxide synthase derived from the SH strain (SHLpAOS) was obtained by setting the annealing temperature for PCR at a low temperature of 45° C. 
         [0000]    
       
         
               
               
             
           
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 AOS-Forward: 5′-GGAACTAACCGGAGGCTACCG-3′ 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
                 AOS-Reverse: 5′-CCGTCTCCGGTCCATTCGACCACAA-3′ 
               
             
          
         
       
     
         [0048]    Based on this sequence information, the full-length sequence was determined by the 3′ or 5′ RACE (Rapid Amplification of cDNA end) method. As a result, a novel AOS homolog (nucleotide sequence: 1443 bp, amino acid sequence: 480 aa, deduced molecular weight: 53.3 KDa) of one sequence was obtained from the SH strain ( FIG. 3 ). 
         [0049]    The sequence of SHLpAOS obtained was introduced into a vector (pET41a, Novagen) for protein expression, and then transformed into  Escherichia coli  (BL21(DE3), Novagen), thereby allowing the SHLpAOS protein to be expressed. Using this SHLpAOS protein, the activity test in KODA production was carried out. 
         [0050]    In the activity testing in KODA production, 5 mM linolenic acid solution (dissolved in 0.1% Tween 80 solution) was prepared, and reacted with lipoxygenase extracted from rice germ at pH 7 at room temperature for 10 minutes to synthesize 9-hydroperoxylinolenic acid (9-HPOT) reaction mixture. To 20 μl of this 9-HPOT reaction mixture, 0.32 ng of the SHLpAOS protein or  A. thaliana -derived AOS (AtAOS) protein was added and reacted at room temperature for 10 minutes. After the reaction was over, the reaction was terminated by a heat treatment at 50° C. for 3 minutes. 10 μl of this solution was analyzed by HPLC. HPLC analysis was carried out with a column: Capcell pak C-18 UG120 (4.6×250 mm), and using mobile phase: 50% acetonitrile solution (0.02% TFA), flow rate: 1 ml/min, detection wavelength: 210 nm. 
         [0051]    As a result, the SHLpAOS protein had an activity nearly 7-fold higher than the AtAOS protein ( FIG. 5 ). 
         [0052]    SHLpAOS obtained from Lemna paucicostata SH strain was subjected to kinetic analysis. Using a reaction mixture comprising 40 mM phosphate buffer (pH 7.5) and 1% EtOH, the reaction was carried out at a temperature of 25° C. The substrate 9-HPOT was tested at the substrate concentration of 5-53 μM. The substrate 9-HPOT was added as an EtOH solution, and the final concentration of EtOH was adjusted to 1%. 100 μl of the reaction mixture was added into a cuvette, and a decrease in absorbance at 234 nm was scanned over time for 1 minute at an interval of 2 seconds using the SmartSpec Plus Spectrophotometer (Bio-Rad). From the A 234  determined, the amount of the reaction product was calculated (e=25,000). Kinetic parameters were determined using the Hanes-Woolf plot ([S]/v versus [S] plot). As shown in Table 2, the result indicates that the K m  value is significantly lower in SHLpAOS than in AtAOS, and had an about 5-fold higher affinity for 9-HPOT in SHLpAOS than in AtAOS. The V max  was about 2.8-fold higher in SHLpAOS than in AtAOS. The K cat  value was also about 2.8-fold higher in SHLpAOS than in AtAOS, indicating that reaction turnover is occurring very efficiently. The k cat /K m  value was about 14-fold higher in SHLpAOS than in AtAOS. It is believed that compared to AtAOS, SHLpAOS is a very useful AOS in the practical production of KODA. The above revealed that SHLpAOS cloned from Lemna paucicostata SH strain is an AOS having a very high activity that has not been reported before. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Kinetic parameters of SHLpLOX and AtAOS 
               
             
          
           
               
                   
                 K m   
                 V max   
                 k cat   
                 k cat /K m   
               
               
                   
                 (μM) 
                 (μmol sec −1 ) 
                 (sec −1 ) 
                 (×10 6 M −1 sec −1 ) 
               
               
                   
                   
               
             
          
           
               
                 LpLOX-SH 
                 7.1 ± 1.9 
                 4.7 ± 0.3  
                 709.2 ± 44.3 
                 99.0 ± 23.7 
               
               
                 AtAOS 
                 36.3 ± 1.3  
                 1.7 ± 0.03 
                 255.5 ± 4.8  
                 7.0 ± 3.8