JP4308604B2 - Alliinase and method for producing allicin using the same - Google Patents

Description

  The present invention relates to an alliinase derived from a microorganism having an alliinase (Alliinase, EC 4.4.1.4) activity for producing allicin from alliin, and a method for producing allicin using the same.

  Allicin has been attracting attention in recent years because it is found in allium plants such as garlic, onion, and onion and is included as an aromatic component and has various physiological activities such as blood flow improvement, lipid metabolism improvement, and antibacterial activity. . Allicin is a by-product of pyrubin by condensation reaction and elimination reaction, as shown in the reaction formula of Fig. 1, where alliinase present in bulbs and stems and leaves acts on the precursor alliin present in mesophyll storage cells of the genus Allium. Produced with acid and ammonia. Accordingly, if allicin can be produced using alliinase, allicin can be put to practical use as a pharmaceutical having various physiological activities.

However, plant-derived alliinase is extremely unstable and unsuitable for industrialization and commercial production of allicin (see Non-Patent Document 1). Therefore, there is a proposal of a technique for continuously producing immobilized alliinase and allicin using garlic alliinase immobilized (see Patent Document 1).
S. Schwimmer, M. Mazelis, Arch. Biochem. Biophys., 100, 66 (1963) Special Table 2000-508535

  However, in order to industrially use unstable plant-derived alliinase, immobilization is necessary as described above, and there are problems in the complexity of production equipment and production cost. There has been no report on alliinase produced by Fusarium or bacteria.

  The present invention provides an alliinase derived from a microorganism capable of producing allicin from alliin on an industrial scale in a simple and efficient manner and putting it into practical use as a pharmaceutical, and producing allicin using the same. The challenge is to provide a law.

In order to solve the above-mentioned problems, the present inventors have sought extensively in the natural world for microorganisms capable of producing alliinase, and as a result of diligent searches, the strain obtained from soil produced the target alliinase. As a result, the present invention has been completed.
That is, it is a crude enzyme contained in a cell disruption liquid after culturing Fusarium sp. Strain AMA9394 (FERM P-19485), and the gist of the alliinase is heat-resistant compared to garlic-derived alliinase .

  In addition, the gist of the present invention is a method for producing alliinase in which a microorganism belonging to the genus Fusarium having the ability to produce alliinase is cultured, alliinase is produced in the culture, and this is collected.

  In the above invention, at least one selected from L-cysteine, S-allyl L-cysteine, N-acetyl L-cysteine and alliin is added to the medium and cultured. Further, after culturing for a predetermined period in a medium to which no inducer is added, the culture is further performed in a medium to which only alliin is added as an inducer.

  Moreover, this invention makes the summary the manufacturing method of allicin which makes said alliinase act on the solution containing an allium plant extract or alliin, and produces | generates allicin.

Alliinase of the present invention, since the stability has heat resistance, Ru can produce allicin from easily and efficiently alliin on an industrial scale without immobilization.

  Since the alliinase production method of the present invention can produce alliinase having the above properties, it can contribute to the efficient production of allicin.

  Since the allicin production method of the present invention can produce allicin simply and efficiently on an industrial scale, it can provide allicin as a pharmaceutical having various physiological activities.

The alliinase of the present invention is produced by a microorganism belonging to the genus Fusarium. Examples of such microorganisms include a novel microorganism found in soil, Fusarium sp. AMA9394 strain belonging to the genus Fusarium.

The morphological characteristics of Fusarium sp. Strain AMA9394 were as follows.
Bacto Potate Dextrose Agar (PDA) (Becton Dickinson, NJ, USA: BD), Bacto
Oatmeal Agar (OA) (BD) and 2% Bacto Malt extract (BD) + 1.5% Agar (MEA) plates are inoculated and cultured at 25 ° C for up to 2 weeks to observe the macroscopic characteristics of the colonies. went.
The description of colony color was according to Korncrup & Wanscher (1978).
(Macroscopic observation results)
The aerial hyphae were veltinous to fluffy (cottony), the surface color was white-yellowish white (3A1-2), and the OA plate showed a brown-colored reverse coloration.
There was no change in color on the colony surface due to conidia growth. Production of clear exudate was observed from PDA and OA plates after long-term culture, and production of a brown soluble pigment was observed on OA plates.
(Microscopic observation)
Microconidia and macroconidia were observed. Small conidia are phialidic.
The conidial pattern is almost single, the length of the stipe is medium, and almost no branching is observed.
Small conidia are slimy from the tip of the handle, and the shape is fusiform or luniform. Oiko was observed mainly in the aerial mycelium and was crescent-shaped and had a foot cell. Oita Ikuko is slightly thick and relatively short in length.
In addition, no pressure wall spore (chlamydospore) or telemorph (telemorph) has been confirmed from long-term culture specimens.

In addition, the gene sequence of about 320 bp (SEQ ID NO: 1 in the sequence listing) of the D2 region of 28SrDNA (rRNA gene) is determined, and the classification of which taxon is closely related by molecular genetic techniques It was. A blast search of the nucleotide sequence based on the DDBJ / GENE BANK database suggested that the nucleotide sequence was most closely related to the Fusarium genus or Fusarium solani complex with a high homology of 98% or more. In addition, in order to derive phylogenetic relationships, arbitrary strains belonging to the genus Fusarium and AMA9394 strains were selected that were closely related by molecular genetics, and a phylogenetic tree was created (FIG. 3).
As a result, several groups of Fusarium sp. Strains were systematically recognized, but AMA9394 strain belongs to a group, and Fusarium sp. 1 strain, Fusarium sp. 2 strain (AF513980, AY234907) due to homology of 99% or more. ). It was also suggested that it is closely related to the third strain of Fusarium solani. The microorganism that produces this novel alliinase active substance is judged to be Fusarium, an imperfect fungus, based on the above morphological characteristics and molecular genetic analysis. (IPOD, 〒305-8566 Tsukuba City, Ibaraki Pref., 1st Higashi 1-chome, 1st Central, 6th) The deposit number is FERM P-19485. The strain producing the alliinase of the present invention is not limited to the above-deposited strain, and is preferably 95% or more, more preferably 98% or more, and more preferably, the D2 region of 28S rDNA (rRNA gene) of SEQ ID NO: 1. A filamentous fungus having a D2 region showing 99% or more, most preferably 100% homology, and producing alliinase can be used.

Using the Strain this, in order to produce the alliinase crude enzyme, a carbon source necessary for the strain satisfactorily growing is produced steadily enzymes, nitrogen sources, nutrients such as inorganic salts This is cultured in a synthetic or natural medium containing The medium for culturing does not need to be special, and a normal medium can be used.

  As a carbon source, carbohydrates, such as starch or its composition fraction, glucose, sucrose, can be used, for example.

  Examples of the nitrogen source include polypeptone, casein, meat extract, yeast extract, corn steep liquor, organic nitrogen source substances such as extracts such as soybeans or soybean meal, inorganic salt nitrogen compounds such as ammonium sulfate and ammonium phosphate, glutamic acid, etc. The amino acids can be used.

  In order to increase the productivity of alliinase, cysteine derivatives such as L-cysteine, S-allyl-L-cysteine, N-acetyl-L-cysteine and alliin can be used. In particular, after culturing for a predetermined period in a medium to which no inducer is added, it is preferable to further culture in a medium to which only alliin is added as an inducer. By this culture method, alliinase can be produced at high yield.

  Examples of inorganic salts include phosphates such as monopotassium phosphate and dipotassium phosphate, magnesium salts such as magnesium sulfate, potassium salts such as potassium chloride, iron salts such as iron sulfate, and zinc salts such as zinc sulfate. Copper salts such as copper sulfate can be used.

  The culture is adjusted to a medium pH range of 5 to 10, preferably pH 6 to 8, and a temperature range of 10 to 40 ° C., preferably 25 under aerobic conditions such as shaking culture or aeration and agitation culture. Although it is desirable to carry out at ˜37 ° C., there is no particular limitation as long as it is a condition that allows microorganisms to grow and produce the target enzyme even under other conditions.

  When culturing is carried out in this manner, alliinase is usually produced in the cells in 2 to 7 days after the start of the culturing.

  Next, the microbial cells are collected from the culture solution, washed with a phosphate buffer or the like, crushed by glass beads or the like, and the enzyme is recovered.

  The crude enzyme alliinase thus obtained can be used as it is for the allicin production reaction, but if necessary, known purification operations such as cation exchange resin, anion exchange resin, hydrophobic chromatographic resin, fractionation by gel filtration, etc. It can also be used as a purified enzyme.

Since the alliinase produced by the microorganisms of the genus Fusarium described above has heat resistance and stability, it is useful for producing allicin from alliin by an industrial scale enzymatic reaction .

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

[Reference Example 1] (Production of alliin)
Alliin used in the following examples was produced as follows.
H ₂ O ₂ solution was mixed with L-allyl-cystine (manufactured by Tokyo Chemical Industry Co., Ltd.) so that the molar concentration was 10 times. After reacting at room temperature for 30 minutes, acetone was mixed to 90%, stirred gently, and left in an ice bath for 30 minutes. After confirming the precipitate, it was centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. 90% acetone was newly added to the precipitate, and the same operation was repeated a few times. The washed precipitate was dried overnight in a desiccator. Thereby, (±) alliin (S (±) -L-cysteine sulfoxide) containing an isomer was synthesized. L-allyl-cystine and (±) alliin were separated by TLC (1-butanol: acetic acid: H ₂ O = 4: 1: 1) and detected by ninhydrin reaction.

[Reference Example 2] (Measurement method of alliinase activity)
The enzyme activity of alliinase in each of the following examples was measured as follows.
As a substrate, 20 μl of 200 mM (±) alliin, 20 μl of 100 mM pyridoxal 5 ′ phosphate, 100 μm of sodium phosphate buffer (pH 6.5) containing 20% glycerol, 40 μl, Fusarium sp. AMA9394 cell-free 20 μl of the extract was added, and 100 μl of the reaction solution with a total volume of 100 μl was incubated at 30 ° C. for 30 minutes. After the reaction, 100 μl of 1 mM 2,4-dinitrophenyl hydrazone (2,4-dinitrophenyl hydrazone) solution—2 N hydrochloric acid was added and left at room temperature for 20 minutes. Thereafter, 1.0 ml of 0.4 N NaOH was added and stirred gently, and then the absorbance at 505 nm derived from pyruvic acid was measured with a spectrophotometer to measure alliinase activity.
The measurement of pyruvic acid was performed as follows. Figure 2 shows the reaction formula.
Add 100 μl of the enzyme reaction solution to 100 μl of 1 mM 2,4-dinitrophenyl hydrazine (2,4-dinitorophenyl hydorazine) solution-2 N hydrochloric acid and let stand at room temperature for 20 minutes. Thereafter, 1.0 ml of 0.4 N sodium hydroxide is added and stirred, and then the absorption at 505 nm is measured. At that time, the reaction solution before the reaction is also measured, and the UV absorption value is the net increase. A calibration curve is drawn with sodium pyruvate purchased in advance, and the measured value is converted to a concentration.
Under this measurement condition, the amount of enzyme that produces 1 μM pyruvic acid per minute was defined as 1 unit.

Example 1
Medium containing 0.5% L-cysteine, S-allyl-L-cysteine, N-acetyl-L-cysteine and alliin in a medium (YPD medium) consisting of 1.0% yeast extract, 2.0% polypeptone and 2.0% glucose Fusarium sp. AMA9394 strain was cultured at 37 ° C. for 4 days, and dry weight and alliinase activity were measured. All cultures were performed in test tubes and 5 ml of medium was used.

After culturing, the cells are washed with a solution containing phosphate buffer (50 mM, pH 6.5) and 10% glycerol (add the solution, voltex for a few seconds, precipitate at 8000 rpm for 30 minutes, and discard the supernatant) several times. After that, 15 cycles (30 minutes) were performed with glass beads for 1 minute voltex and 1 minute ice bath. At this time, it was confirmed with an optical microscope whether the mycelium was destroyed. If it was not sufficiently destroyed, several cycles were performed to obtain a crude enzyme cell disruption solution.
The microbial cells after each culture were washed several times with distilled water, and the weight after drying for 2 days with a 60 ° C. air dryer was taken as the dry weight of the microbial cells. The final concentration of alliin for activity measurement was 100 mM. The results are shown in Table 1.

  From Table 1, alliinase activity of the cell disruption liquid was increased in the medium containing S-allyl-L-cysteine and N-acetyl-L-cysteine, and the dry weight of the cells was also increased compared to others. . However, when alliin was added, the dry weight was extremely reduced and the growth of the cells was not sufficient.

[Example 2]
5 ml of YPD medium was put in a test tube, sterilized by a conventional method, inoculated with Fusarium sp. AMA9394 strain, and cultured at 37 ° C. for 4 days.

After culturing in YPD medium, the cells were washed several times with phosphate buffer (50 mM, pH 6.5), then K ₂ HPO ₄ 0.1%, MgSO ₄ 7H ₂ O 0.05%, KCl 0.001%, FeSO ₄ Alliin was added to 7H ₂ O 0.001%, ZnSO ₄ · 7H ₂ O 0.0005%, CuSO ₄ · 5H ₂ O 0.0005% to a final concentration of 0.5%, and further adjusted to pH 7.0 (A medium). The cells were cultured at 37 ° C for 1 day.
The results were compared with those obtained when cultured for 5 days at 37 ° C. using only YPD medium.

After culturing in medium A, the cells are washed with a solution containing phosphate buffer (50 mM, pH 6.5) and 10% glycerol (add the solution, voltex for several seconds, precipitate at 8000 rpm for 30 minutes, and discard the supernatant) After several times, 15 cycles (30 minutes) were performed using glass beads for 1 minute voltex and 1 minute ice bath. At this time, it was confirmed with an optical microscope whether the mycelium was destroyed. If it was not sufficiently destroyed, several cycles were performed to obtain a crude enzyme cell disruption solution.
The weight after washing several times with distilled water and drying for 2 days with a 60 ° C. ventilator is taken as the dry weight of the cells. The final concentration of alliin for activity measurement was 100 mM. The results are shown in Table 2.

  After culturing in YPD medium for 4 days and culturing in A medium for 1 day, alliinase activity of the cell disruption liquid increased, and it was found that alliinase was induced and produced during that time. Further, the addition of alliin did not reduce the weight of the cells, and the cells were sufficiently grown and alliinase could be produced at a high yield.

Example 3
The physicochemical properties of alliinase were examined using the cell disruption solution obtained by culturing in the YPD medium and A medium of Example 2. As a control, a filtrate obtained by crushing garlic fragments and performing filtration (hereinafter, garlic crush solution) was used. In FIGS. 4 to 7 below, alliinase derived from Fusarium sp. Strain AMA9394 is indicated as AMA9394, and alliinase derived from garlic is indicated as garlic.
(1) optimum temperature various temperatures each reacted with bacterial cell lysate and garlic lysate to alliin in to measure the respective alliinase activity, and the results are shown in FIG.

As is clear from FIG. 4 , the alliinase (hereinafter also referred to as the present enzyme) in the cell disruption liquid had an optimum temperature at 37 ° C. like the alliinase in the garlic disruption liquid.

(2) Optimum pH
under pH conditions of pH 4 to 9, subjected to the enzymatic reaction respectively reacted with bacterial cell lysate and garlic lysate to alliin to measure the respective alliinase activity, and the results are shown in Figure 5. A 500 mM sodium phosphate buffer (pH 4-9) containing 10% glycerol was used as the buffer.

As is clear from FIG. 5, the optimum pH of this enzyme was around 7.5, and the alliinase in the garlic crushing solution was around 6.5.

(3) after 2 hours standing the temperature stability cell lysate and garlic lysate at a temperature of 0 to 55 ° C., to determine their residual activity, and the results are shown in Figure 6.

As is apparent from FIG. 6 , the present enzyme showed a residual activity of about 80% at 35 ° C., but the alliinase in the garlic crushing liquid was extremely poor at 10% or less.

(4) Specificity for optical isomers The final concentrations of the substrates (+) alliin and (−) alliin were 500 mM, and the effects of the bacterial cell disruption solution and garlic crushing solution on the optical isomers were examined. In addition, (+) alliin and (-) alliin were separated by HPLC (ODS column, flow rate 1.0 ml / min, 10 mM phosphate buffer (pH 7.5, 5 mM tetra-n-butylammonium dihydrogen phosphate), UV 220 nm detection).
The reaction solution was sampled every hour and subjected to HPLC analysis. And the peak area was converted into the density | concentration using the analytical curve created beforehand. The results are shown in FIG.

From FIG. 7 , this enzyme reacted with (+) alliin compared to (−) alliin, like the garlic crush solution. When both were compared, there was a slight difference in the effect on (-) alliin.

The reaction formula by which allicin is produced from alliin by alliinase is shown. The reaction formula in the measuring method of alliinase activity is shown. The phylogenetic tree created in order to identify Fusarium sp. AMA9394 strain is shown. It is a graph of the optimal temperature which compared the alliinase derived from a Fusarium sp. AMA9394 strain and the alliinase derived from a garlic. It is the graph of the optimal pH which compared the alliinase derived from Fusarium sp (Fusarium sp.) AMA9394 strain and the alliinase derived from a garlic. It is a graph of temperature stability comparing the alliinase derived from Fusarium sp. AMA9394 strain and the alliinase derived from garlic. It is a graph which shows the reactivity to the optical isomer which compared the alliinase derived from a Fusarium sp. AMA9394 strain and the alliinase derived from a garlic.

Claims (5)

An alliinase that is a crude enzyme contained in a cell disruption liquid after culturing Fusarium sp. AMA9394 strain (FERM P-19485), and has higher heat resistance than an alliinase derived from garlic .   A method for producing alliinase, comprising culturing a microorganism belonging to the genus Fusarium having the ability to produce alliinase, producing alliinase in the culture, and collecting this.   The method for producing alliinase according to claim 2, wherein at least one inducer selected from L-cysteine, S-allyl L-cysteine, N-acetyl L-cysteine and alliin is added to the medium and cultured.   The method for producing alliinase according to claim 3, further comprising culturing in a medium to which only alliin is added as an inducer after culturing in a medium to which no inducer is added. The solution containing the extract or alliin in Allium plant by the action of alliinase mounting serial to claim 1, method for producing allicin to produce allicin.

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