WO2016136508A1

WO2016136508A1 - Method for producing 2-aza-8oxohypoxanthine

Info

Publication number: WO2016136508A1
Application number: PCT/JP2016/054182
Authority: WO
Inventors: 真治徳山; 宰熏崔; 河岸　洋和
Original assignee: 国立大学法人静岡大学
Priority date: 2015-02-23
Filing date: 2016-02-12
Publication date: 2016-09-01
Also published as: JP6494738B2; JPWO2016136508A1

Abstract

The present invention provides a method for producing 2-aza-8oxohypoxanthine, comprising the steps of: reacting 2-azahypoxanthine with a microorganism to produce 2-aza-8oxohypoxanthine; and isolating 2-aza-8oxohypoxanthine from a reaction solution.

Description

Process for producing 2-aza-8 oxohypoxanthine

The present invention relates to a method for producing 2-aza-8 oxohypoxanthine.

2-Aza-8 oxohypoxanthine (hereinafter sometimes referred to as “AOH”) is a compound known to exhibit plant growth promoting activity. Patent Document 1 discloses a method for obtaining AOH by allowing xanthine oxidase (hereinafter, sometimes referred to as “XOD”) to act on 2-azahypoxanthine (hereinafter, sometimes referred to as “AHX”). Yes.

International Publication No. 2012/147750

A method for mass production of AOH by chemical synthesis has not been established. Further, the method of obtaining AOH by causing XOD to act on AHX is not suitable for producing a large amount of AOH because the production efficiency is low and the XOD is very expensive.

The object of the present invention is to provide a novel method capable of producing AOH.

The present invention is a method for producing AOH, which includes a step of reacting AHX with a microorganism to thereby generate AOH, and a step of isolating AOH from the reaction solution.

In the above method, the microorganism is a Burkholderia genus, Butiauxella genus, Pseudomonas genus, Escherichia coli, Bacillus genus, Saccharomyces genus, Kriveromyces genus, Candida genus, Chizosaccharomyces genus, Pichia genus, It is preferably at least one selected from the group consisting of Aspergillus and Trichoderma. The above microorganisms include Burkholderia Contaminance, Burkholideria cepacia, Burkholideria hungorum, Butiauxera Gabiniae, Butiauxera Aggressis, Shuudomonas petitda, Shudomonas synxanta, Shuudomonas bancoubenensis More preferably, it is at least one selected from the group consisting of Pseudomonas plecogrossida. By using these microorganisms, AOH can be generated with higher efficiency.

According to the present invention, a novel method capable of producing AOH can be provided.

It is a graph which shows AHX and AOH density | concentration in the reaction liquid which used Burkholderia contaminens CH-1. It is a graph which shows the AHX and AOH density | concentration in the reaction liquid using Burkholderia cepacia. It is a graph which shows the AHX and AOH density | concentration in the reaction liquid using Burkholderia fungorum. It is a graph which shows AHX and AOH density | concentration in the reaction liquid which used Burkholderia contaminens CH-1. It is a graph which shows the AHX and AOH density | concentration in the reaction liquid using XOD. It is a graph which shows the AOH density | concentration in the reaction liquid which used Burkholderia contaminens CH-1. It is a graph which shows AHX and AOH density | concentration in the reaction liquid which used Burkholderia contaminens CH-1. It is a graph which shows the AHX and AOH density | concentration in the reaction liquid using Pseudomonas synxantha A13. It is a graph which shows the AHX and AOH density | concentration in the reaction liquid using Pseudomonas putida A82. It is a graph which shows the AHX and AOH density | concentration in the reaction liquid using Buttiauxella gaviniae A111. It is a graph which shows the AHX and AOH density | concentration in the reaction liquid using Pseudomonas vancouverensis A112. It is a graph which shows the AHX residual rate in the reaction liquid using Buttiauxella gaviniae A111.

Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments.

The method for producing AOH according to the present embodiment includes a step of reacting AHX with a microorganism to thereby generate AOH, and a step of isolating AOH from the reaction solution.

2-Aza-8 oxohypoxanthine (AOH) is also represented by 3H-imidazo [4,5-d] [1,2,3] triazine-4,6 (5H, 7H) -dione and has the following formula (I) It is a compound represented by these.

2-Azahypoxanthine (AHX) is also represented by 7H-imidazo [4,5-d] [1,2,3] triazin-4 (3H) -one, and is a compound represented by the following formula (II). is there.

The microorganism used in the present embodiment may be any microorganism that has the ability to generate AOH from AHX. According to the production method according to the present embodiment, reaction inhibition by the product (AOH) is suppressed compared to the case of using XOD, and therefore AOH can be produced efficiently even if the AHX concentration in the reaction solution is high. Can do. Moreover, it is not necessary to use expensive XOD, and AOH can be manufactured at low cost.

Examples of microorganisms include bacteria and fungi. Examples of bacteria that can be used include Burkholderia bacteria, Butiauxella bacteria, Pseudomonas bacteria, Escherichia coli, Bacillus bacteria, and the like. Examples of fungi that can be used include Saccharomyces sp., Criveromyces sp., Candida sp., Tizosaccharomyces sp., Pichia sp., Aspergillus sp., Trichoderma sp. These microorganisms may be used individually by 1 type, and may use multiple types together. The microorganism is preferably a Burkholderia bacterium, a Butiauxella bacterium, or a Pseudomonas bacterium, and more preferably a Butiauxella bacterium or a Pseudomonas bacterium. When these microorganisms are used, AOH can be generated with higher efficiency.

Examples of bacteria belonging to the genus Burkholderia include Burkholderia contamination, Burkholderia cepacia, Burkholderia, and the like. Examples of the bacteria belonging to the genus Butiauxella include Butiauxella gaviniae, Butiauxella agretis, and the like. The Pseudomonas genus bacterium, Pseudomonas putida (Pseudomonas putida), Pseudomonas and new Santa (Pseudomonas synxantha), Pseudomonas & vandalism Belem cis (Pseudomonas vancouverensis), Pseudomonas & Jesusenyi (Pseudomonas jessenii), Pseudomonas & plecos Gross Shi fern (Pseudomonas plecoglossicida ) And the like.

Among these, microorganisms include Burkholderia Contaminance, Burkholideria cepacia, Burkholideria hungorum, Butiauxera Gabiniae, Butiauxera Aggressis, Pseudomonas petitda, Pseudomonas synxantha, Pseudomonas banchouberensis It is preferably Jesseni, more preferably Burkholderia Contaminance, Butiauxera Gabiniae, Butiauxera Aggressis, Pseudomonas petitda, Pseudomonas synxanta, Pseudomonas vancouverensis or Pseudomonas jesuseni Gabiniae, Pseudomonas petitda, Pseudomonas synxanta or Pseudomonas More preferably from vandalism Belem cis, particularly preferably Butiaukusera-Gabiniae or Pseudomonas putida. When the above microorganism is used, AOH can be generated from AHX with higher efficiency.

The step of reacting AHX with microorganisms and thereby generating AOH can be performed, for example, by mixing a suspension containing microorganisms prepared in advance and a solution containing AHX. The step of reacting AHX with a microorganism to thereby generate AOH includes, for example, contacting AHX with a microorganism to thereby generate AOH, or contacting AHX with a microorganism, thereby converting AHX to AOH. It can also be referred to as a process of converting into

Microorganisms can be cultured under general culture conditions for microorganisms. The culture can be performed, for example, under conditions of pH 6 to 8, temperature 25 to 42 ° C. for 6 to 24 hours. The culture can be performed, for example, by shaking with aeration. As a culture medium for culturing microorganisms, various media containing nutrient components such as carbon sources, nitrogen sources, inorganic salts, vitamins and minerals used for microorganism culture can be used. Examples of the medium suitable for bacteria such as Burkholderia include TSB medium, YM medium, and the like. It is preferable to use a microorganism in the late logarithmic growth phase. The cultured microorganisms can be collected from the medium by centrifugation or the like and used for the reaction with AHX. Microorganisms washed after collection may be used for the reaction with AHX.

The solvent for performing the reaction between AHX and the microorganism is preferably a buffer solution. As the buffer solution, for example, phosphate buffered saline (PBS) can be used.

The AHX concentration in the reaction solution can be arbitrarily set. In the manufacturing method according to this embodiment, unlike the case where AOH is generated from AHX using XOD, even if the AHX concentration in the reaction solution is high, the AOH generation efficiency can be kept high for a long time.

The AOH produced by the reaction can be recovered, for example, by centrifuging the reaction solution to separate the cells, and collecting and concentrating the supernatant. The recovered supernatant may be subjected to a heat treatment or the like. The recovered AOH may be further purified and crystallized.

Hereinafter, embodiments of the present invention will be specifically described by way of examples, but the present invention is not limited to the following examples.

(Test Example 1: AOH production activity evaluation)
AOH production activity was examined using the following microorganisms.
Burkholderia contaminens CH-1
Burkholderia carryophylli NBRC 13591
Burkholderia cepacia NBRC 14595
Burkholderia caledonica NBRC 102488
Burkholderia fungorum NBRC 1024889
Burkholderia ferrariae NBRC 106233
Burkholderia ginsengisoli NBRC 100965
Burkholderia mimosarum NBRC 106338
Among the above-mentioned microorganisms, Burkholderia contaminens CH-1 (hereinafter also referred to as “CH-1 strain”) was isolated from the collected airborne fungus, and a standard strain of Burkholderia contaminens (strain J2956) was analyzed by 16S rDNA system analysis. ) Was 100%, confirming that it was the species. All other microorganisms were sold by NBRC (Biotechnology Center, National Institute of Product Evaluation Technology).

The medium used has the composition shown in Table 1. As phosphate buffered saline (PBS), a 10 × PBS solution having the composition shown in Table 2 was diluted 10-fold with distilled water.

Each platinum strain was inoculated in 5 ml of TSB medium dispensed into a test tube, and cultured with shaking at 30 ° C. and 180 rpm, and this was used as a preculture solution. 1% of the preculture was inoculated into a 500 ml Erlenmeyer flask into which 100 ml of TSB medium was dispensed, and cultured at 30 ° C. for 6 hours at 180 rpm. 1 ml of the obtained culture broth was centrifuged at 11000 rpm for 5 minutes to collect bacteria. To the collected cells, 1 ml of PBS was added and washed by centrifugation at 11000 rpm for 5 minutes.

A certain amount of the washed cells was suspended in 900 μl of PBS, and 100 μl of 0.7 mg / ml AHX in PBS was added to the suspension to prepare a reaction solution having an AHX concentration of 70 μg / ml. The reaction solution was transferred to a round bottom Spitz and reacted at 30 ° C. for 24 hours. After a predetermined time, the reaction solution was centrifuged to separate the cells, and the supernatant was collected and heat-treated at 98 ° C. for 5 minutes. The AHX and AOH concentrations in the reaction supernatant were analyzed by HPLC. HPLC analysis conditions were as follows: Column: Develosil C30-UG-5 (Nomura Chemical Co., Ltd.), mobile phase: water + 0.02% TFA, temperature: room temperature, flow rate: 0.8 ml / min, wavelength: 254 nm, injection: 20 μl It was. The retention times of AHX and AOH were 25.2 minutes and 26.6 minutes, respectively. The results are shown in Table 3. It should be noted that, under the HPLC analysis conditions of this example, separation from impurities may be insufficient, and therefore the AOH concentration may be apparently measured higher than it actually is.

It was confirmed that any bacteria used had the ability to produce AOH using AHX as a substrate. Among them, the CH-1 strain, Burkholderia cepacia and Burkholderia fungorum all converted AHX in the medium into AOH and had a conversion efficiency of 100%.

(Test Example 2: Comparison of AOH production activity)
Using three kinds of strains CH-1, Burkholderia cepacia, and Burkholderia fungorum, AOH generation activity up to 6 hours after the start of the reaction was examined. The reaction conditions were the same as in Test Example 1 except that the reaction time was a predetermined time. The AHX and AOH concentrations in the reaction solution were analyzed by HPLC in the same manner as in Test Example 1 at a predetermined time. The results are shown in FIG. 1 (CH-1 strain), FIG. 2 (Burkholderia cepacia), and FIG. 3 (Burkholderia fungorum). Among the above three species, the CHOH strain had the highest AOH production activity.

(Test Example 3: Examination of reaction inhibition by products)
Using the CH-1 strain, the reaction was conducted for 96 hours under the same conditions as in Test Example 1, except that the initial concentration of AHX in the reaction solution was 2 mg / ml and the bacterial cell concentration was 3 times that of Test Example 1. It was. During the reaction, the AHX concentration and AOH concentration in the reaction solution were measured by HPLC in the same manner as in Test Example 1 at a predetermined time. The results are shown in FIG. In addition, the AHX concentration was similarly 2 mg / ml, and AOH was produced at 30 ° C. using 0.5 U of xanthine oxidase (from buttermilk, Oriental Yeast Co., Ltd.). Similarly, the AHX concentration and AOH concentration in the reaction solution Measurements were made. The results are shown in FIG.

In the reaction using xanthine oxidase, the AHX concentration did not change while maintaining a constant amount after a certain period of time, suggesting that reaction inhibition by the generated AOH occurred (FIG. 5). On the other hand, in the reaction using the CH-1 strain, the reaction resulted in an AHX concentration of 0 mg / ml in the reaction solution, indicating that all the AHX used was converted to AOH (FIG. 4).

(Test Example 4: AOH decomposition reaction evaluation)
Using the CH-1 strain, the reaction was carried out in the same manner as in Test Example 1 except that the bacterial cell concentration in the reaction solution was twice that of Test Example 1 and AOH was used as the substrate instead of AHX. The concentration of AOH in the reaction solution was measured by HPLC as in Test Example 1. The results are shown in FIG. Suspending AOH with the CH-1 strain did not reduce the AOH concentration. It was confirmed that the AOH degradation activity of the CH-1 strain was extremely weak.

(Test Example 5: AOH yield evaluation)
The same reaction conditions as in Test Example 1 except that the concentration of cells in the reaction solution was 3 times the amount of Test Example 1 and the initial concentration of AHX and the reaction temperature were as shown in Table 4. The reaction was carried out for 8 hours. After the reaction, the reaction solution was centrifuged to separate the cells, and a supernatant was obtained. The supernatant was heat treated at 98 ° C. for 5 minutes, concentrated to 1/7 volume with a rotary evaporator, and allowed to stand at 4 ° C. for 3 days to form a precipitate. Thereafter, the liquid containing the precipitate was filtered with a filter paper, the obtained product was dried to recover AOH, and the yield of AOH based on the amount of AHX used was calculated. Table 4 shows the initial amount of AHX used and the AOH yield after 30 hours of culture.

AOH can be produced with high efficiency by a method using microorganisms such as CH-1 strain (microbe method). In addition, compared to a method of carrying out an AOH production reaction using XOD (XOD method), in the microbial method, reaction inhibition due to the produced AOH is less likely to occur, so that the substrate concentration in the medium can be increased by about 60 times. It was possible. As a result, when compared with the amount of AOH produced, the amount of the reaction solution used can be reduced to about 1/45 of that in the case of the XOD method by the microbial method. Was able to be shortened. In addition, the microbial method could produce AOH at a significantly lower cost than the XOD method.

(Test Example 6: AOH production activity of various microorganisms)
AOH production activity was examined using various microorganisms and CH-1 strains shown in Table 5 below. Collect strains that have been cultured in advance according to the type of various microorganisms, suspend a certain amount of cells in PBS, add AHX solution to an AHX concentration of 70 μg / ml, and The reaction was performed for 24 hours. After a predetermined time, the reaction solution was centrifuged to separate the cells, and the supernatant was collected and heat-treated at 98 ° C. for 5 minutes. The AHX and AOH concentrations in the supernatant of the reaction solution were measured by the same method as in Test Example 1. The results are shown in Table 5. It was confirmed that all the microorganisms have an activity to generate AOH from AHX.

(Test Example 7: Search for AOH-producing microorganism)
Forest soil was collected, and 1 scoop of medicine was suspended in 3 ml of physiological saline. The suspension was allowed to stand and the supernatant was diluted and smeared on the medium. As the medium, TSB medium or YM medium having the composition shown in Tables 6 and 7 was diluted 5-fold. The colonies that appeared on the medium were streaked into the TSA medium to obtain each strain.

Each of the above strains was inoculated into 5 mL of TSB medium dispensed into a test tube, cultured with shaking at 30 ° C. and 180 rpm, and this was used as a preculture solution. 1% of the preculture was inoculated into TSB medium and cultured at 30 ° C. for 8 hours at 180 rpm. The resulting culture solution (1 ml) was collected by centrifugation.

After washing the collected cells with PBS, 100 μl of 0.7 mg / ml AHX in PBS was added to the suspension, and a reaction solution with an AHX concentration of 70 μg / ml was added to the suspension. Produced. The reaction solution was transferred to a round bottom Spitz and reacted at 30 ° C. for 5 hours. After a predetermined time, the reaction solution was centrifuged to separate the cells, and the supernatant was collected and treated at 98 ° C. for 5 minutes. The AHX concentration in the supernatant of the reaction solution was analyzed by HPLC in the same manner as in Test Example 1, and the AHX residual rate was calculated. The results are shown in Table 8. Of the 192 strains isolated from the forest soil, 11 strains showing at least the same level of AOH production activity as the CH-1 strain were obtained.

Among the 11 strains with low AHX survival rate, 10 strains were subjected to 16S rDNA lineage analysis. The analysis confirmed that each strain was the same species as the standard strains shown in Table 9.

(Test Example 8)
Using the above 10 strains and the CH-1 strain, the reaction with AHX was carried out under the same conditions as in Test Example 7, except that the reaction time was 2.5 hours. The AHX concentration in the supernatant of the reaction solution was analyzed by HPLC in the same manner as in Test Example 1. The results are shown in Table 10. All strains had higher AOH production activity than the CH-1 strain.

(Test Example 9)
Among the strains used in Test Example 8, 7 strains having high AOH production activity were reacted with AHX under the same conditions as in Test Example 7 except that the reaction time was 0.5 hour or 1 hour. The AHX concentration in the supernatant of the reaction solution was analyzed by HPLC in the same manner as in Test Example 1. The results are shown in Table 11.

(Test Example 10)
Among the strains used in Test Example 9, four strains with higher AOH production activity were tested except that the AHX concentration in the reaction solution was 2 mg / ml and the reaction time was 1 hour, 2 hours or 4 hours. Reaction with AHX was carried out under the same conditions as in Example 7. AHX and AOH concentrations in the reaction solution were measured by HPLC in the same manner as in Test Example 1. The results are shown in Table 12 and FIGS.

(Test Example 11)
About A111 strain | stump | stock, the reaction liquid volume was enlarged as follows and the reaction experiment with AHX was conducted. As shown in Table 13, the reaction volume was 3.2 l, 3.4 l, or 6.0 l, the AHX concentration in the reaction liquid was 4 g / l or 5 g / l, and the reaction time was 24 hours. The reaction between A111 strain and AHX was carried out under the same conditions as in No.7. After the reaction, the obtained AOH was crystallized and recovered and quantified. The AOH yield was calculated based on the initial amount of AHX used and the amount of AOH recovered. The results are shown in Table 13. In Table 13, No. For 3, the AHX concentration in the reaction solution was measured at a predetermined time, and the AHX residual rate was calculated. The results are shown in FIG.

Even when the reaction solution volume was large, AOH could be generated with high efficiency by the microbial method.

Claims

A method for producing 2-aza-8 oxohypoxanthine, comprising:
Reacting 2-azahypoxanthine with a microorganism, thereby producing 2-aza-8 oxohypoxanthine;
Isolating 2-aza-8 oxohypoxanthine from the reaction solution;
Including a method.
Microorganisms include Burkholderia bacteria, Butiauxella bacteria, Pseudomonas bacteria, Escherichia coli, Bacillus bacteria, Saccharomyces bacteria, Kriveromyces bacteria, Candida bacteria, Tychosaccharomyces bacteria, Pichia bacteria, Aspergillus bacteria and The method according to claim 1, wherein the method is at least one selected from the group consisting of Trichoderma.
Microorganisms include Burkholderia Contaminance, Burkholideria cepacia, Burkholideria hungorum, Butiauxera Gabiniae, Butiauxera Aggretis, Shudomonas Petitda, Shudomonas Synxanta, Shudomonas vancouvenus, Shudomonas The method according to claim 1, wherein the method is at least one selected from the group consisting of plecogross ferns.