JPH0458881A - Arthrobacter sp-k-1 - Google Patents

Abstract

NEW MATERIAL:An Arthrobacter.sp K-1 (FERM P-10736) as a microorganism growing at 41 deg.C without hydrolyzing starch nor reducing nitrate with generating an acid from fructose and generating a slight amount of acid from glucose or maltose and further containing lysine as a principal diamino acid in cell wall and containing 73.2-74.9mol% GC. USE:Used in production of a new beta-fructofuranosidase having strong saccharide- transferring activity and wide breadth of receptor specificity in outside of strain. Heterooligosaccharide or glycoside containing fructose is efficiently produced by said enzyme in a low cost. PREPARATION:A small amount of soil is added to a medium composed of 5% cane sugar, 0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4.7H2O and 0.02% MnCl2.4H2O and cultured at 30 deg.C for 2-3 day.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to Arthrobacter sp.

The present invention relates to Arthrobacter sp. K-1, and more particularly to Arthrobacter sp. K-1, which has broad receptor specificity and the ability to produce β-fructofuranosidase with strong metastatic activity.

[Prior art and problems to be solved by the invention] In recent years, with the increase in health consciousness, research has been carried out on the synthesis of oligosaccharides and useful glycosides with various physiological activities using glucosyl groups or fructosyl transferases. It is widely practiced. Coupling Shugo 6. Fructooligosaccharide, palatinose.

α-glucosyl stevioside does not cause cavities,
This is also an example of a substance that has been put to practical use as having properties such as a bifidobacteria growth factor.

Currently, fructosyltransferases include levansucrase produced by Bacillus subtilis, Penicillium sp.
) K-25 and other β-fructofuranosidases produced by fungi are known. Among these, xylosylfructoside and isomaltosylfructoside, which are synthesized using the transglycosylation action of levansucrase, have anti-cariogenic properties, and lactosylfructoside has activity as a bifidobacteria growth factor. These oligosaccharides have the potential to be put to practical use as useful functional carbohydrates. However, since the levansucrase used to produce these oligosaccharides is induced by sucrose, it is essential to add sucrose to the culture medium, which creates levan in the culture solution and increases the viscosity. Difficult to handle. In addition, problems such as low enzyme productivity and low heat resistance have been pointed out. On the other hand, β produced by conventional molds
-Fructofuranosidase is an intracellular enzyme and has narrow receptor specificity.

These enzymes transfer well to sucrose, so even if other sugars are added as acceptors, they transfer to sucrose and produce quesedoses, reducing the production rate of the desired hetero-olicosaccharide. have.

[Means to solve the problem]

In order to solve the above problems, the present inventors have searched for microorganisms that extracellularly produce highly heat-resistant enzymes capable of synthesizing carbohydrates with higher added value from sucrose. As a result, we discovered that by culturing microorganisms belonging to the genus Arthrobacter in a nutrient medium, β-fructofuranosidase with a wide range of target receptor specificities and strong transglycosylation activity can be produced extracellularly. The invention was completed.

That is, the present invention is a major diamino acid in cell walls that grows at 41°C, does not hydrolyze starch, does not reduce nitrate, produces acid from fructose, and only weakly produces acid from glucose and maltose. It relates to Arthrobacter sp. K-1, which has a GC content of 73.2 to 74.9 mol%.

This bacterium with the above characteristics is 5% sucrose, o, 3% Na
NO3, 0,1% 2HPO, 0,05% MgSO
A small amount of soil was added to a medium consisting of 4-7H20,0,02% Mn Cl 2''4820 and cultured for 2 to 3 days at 30°C. This bacterium was isolated on an agar medium containing the above medium. did.

The mycological properties of Arthrobacter sp. K-1 are described below.

■ Morphological properties a Cell morphology During 24-hour culture on broth agar medium, young cells are rod-shaped with a size of 0.3 to 0.5 Xo, 6 to 6 μm, and older cells are spherical (0. ~0.5 μm), which varies depending on the growth stage.

b T-shaped and Y-shaped cells can be observed on polymorphic broth agar medium.

C motility, flagella They have no flagella and are not motile.

d Spore Does not form spores.

e Gram staining is positive in young cells and negative in old cells, changing depending on the growth stage.

f Acid-fast negative The above morphology was observed in the growth state on Lab M broth agar medium.

■Cultural properties The following results are obtained by culturing at 25°C using meat juice containing 0.02% yeast extract as the basic medium.

a. Cultivated on meat juice agar plate The growth is good, the colony is circular with a diameter of 2 mm, the circumference is gentle and opaque, but the colony has a glossy low color.
It becomes a competition class shape (low convex). The color of the colony is yellow and no diffusible pigment is produced.

b Meat juice agar slant culture It has good growth, grows linearly, and is glossy.

The color of the colony is yellow and no diffusible pigment is produced.

C. When growing on the surface of the broth liquid medium, a fungal film is not formed, or even if it is formed, it is very thin. The culture solution uniformly produces a precipitate after suspension.

d Meat juice gelatin puncture culture It grows only on the surface and its growth is good.

g Lidmus milk grows slowly and becomes peptonized in about 2 weeks. There may be soft solidification or alkalization. Ridmass does not give back.

■ Physiological properties Nitrate reduction Denitrification reaction MR test VP exam Generation of indole Production of hydrogen sulfide Decomposition of starch Use of citric acid Utilization of inorganic nitrogen sources ammonium salt.

production of pigment urease catalase oxidase cellulase Na5e β-galactosidase cytochrome oxidase arginine dehydrolase Both nitrates can be used.

Produces yellow pigment.

negative positive negative negative positive positive negative.

Growth in pH 5-9.5, pH 5-9.5 of negative positive negative negative negative negative negative negative negative negative negative negative negative sycercents, and optimal use of growth is around 7. Also, it grows at 10-41℃, and the optimum temperature for growth is 3.
The temperature is around 7℃.

t Attitude towards oxygen Aerobic uo-F test When glucose is used, it is weak or produces acid by fermentation.

■ Production of acid and gas from sugars Acid is produced from fructose, xylose, and sucrose, and is weak from glucose 2 maltose.

Not produced by starch or lactose. Furthermore, no gas generation was observed from any of the saccharides.

■ Chemical analysis a Cell wall component Mycolic acid Negative diamino acid Lysine (main constituent) b Fatty acid composition IsoC15:0 1.85% AntiisoC15:0 60.68 IsoC1
6:0 4.21isoC17:0
0.47 Anteiso C17:0
31.54c GC content 73.2-7
This strain, which exhibits properties of 4.9 ma1% (average 73.7 mo1%) or more, is classified by Bergey's Manual Determinative Bacteriology (Bergey's Manual Determinative Bacteriology).
Manual of Determinative B
Acteriology) 8th Edition (1974) and The Journal of General and Applied
Microbiology (The Journal
of General and Applied
According to Microbiology, Vol. 18, p. 417 (1972), it is classified into the genus Arthrobacter. A comparison of this bacterium with known related bacteria belonging to the genus Arthrobacter is as follows.

Table 1 Table 1 (Continued) Table 1 (Continued) 12 In the case of a positive reaction, a sugar reaction that produces more acid than sugar, it indicates a positive result within one week.

(+): Weakly positive reaction; in the case of sugar reaction, indicates positive after 1 week or more.

1: Negative reaction S-R: Rod-like, spherical, T-shaped, and Y-shaped objects are observed.

d: The reaction varies depending on the strain.

・：Unknown (1) This bacterium (2) Arthrobacter simplex (A, s
imp 1ex) (3) Arthrobacter globiformis (A, globiformis) (4) Arthrobacter citereus (A, citereus) (5
) Arthrobacter lamellacens (A, tumes
cens) (6) Arthrobacter telegens (A,
(7) Arthrobacter duodecadis (A, duodecadis) (8) Arthrobacter flavescens (A, f
1 avescens) As is clear from the table above, this bacterium has different properties from related bacteria. Therefore, this bacterium is considered to be a new bacterium, and the present inventors identified this bacterium as Arthrobacter sp.
rthrobacter sp.) K-1. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its deposit number is FERM P-10736.

The microorganism of the present invention has the ability to produce a novel β-fructofuranosidase with strong transposition activity. We investigated the culture conditions of microorganisms for producing this enzyme. first,
A basal medium containing 1.0% polypeptone, 0.2% ammonium nitrate, 0.1% magnesium sulfate, and a carbon source was used, and as the carbon source, various substances shown in Table 2 were used at a concentration of 1%. .

This medium was inoculated with Arthrobacter sp.-1 and cultured with shaking at 30°C for 2 days. The activity ratios (values when soluble starch is taken as 100) at this time are shown in Table 2.

As is clear from the table, this enzyme is not induced by sucrose, and anything other than highly fermentable substances such as glucose can be used, and soluble starch is the best carbon source.

Table 2 Activity (u/mj) Activity ratio Sucrose 0.18 39 Glucose
00 Maltose 0.19 41 Lactose 0.33. 72 bran
0.14 30 Soluble starch 0.46
100 powdered candy 0.16
35 Next, to consider the organic nitrogen source, 1.0%
Arthrobacter sp.-1 was inoculated into a medium containing soluble starch, 0.2% ammonium nitrate, 0.1% magnesium sulfate, and 1% organic nitrogen source, and cultured with shaking at 30°C for 2 days. The activity ratio at this time (value when 0.5% polypeptone + 0.5% yeast extract is taken as 100) is shown in Table 3.

As is clear from the table, when yeast extract is used, It shows high activity.

Table 3: Additive-free polypeptone corn staple extract activity (u/mj') 0.11 0.35 2.29 Meat extract 0.32 7 Defatted soybean powder OO Fish meat extract 0.42 10 Furthermore, the results of examining the medium composition , the optimal medium composition is 1.2
% yeast extract, 0.8% polypeptone, 4% soluble starch, 0.4% (NH4)2HPO.

and O, 1% M g SO4.7 H2O (pH 7
,0). Therefore, with reference to the above findings, the medium used for culture should be selected to have a composition that allows the test strain to produce a large amount of the desired enzyme.

In order to produce the β-fructofuranosidase of the present invention, the above-mentioned microorganisms are inoculated into a selected medium, and the pH is kept neutral to slightly alkaline and the temperature is kept at 20 to 45 degrees Celsius, preferably 30 to 40 degrees Celsius. The culture may be carried out with shaking or aeration under stirring for 10 hours to 5 days.

Enzymes can be collected and purified by conventional methods from the culture obtained as described above. For example, the supernatant obtained by centrifuging the culture and removing the bacterial cells can be used as a crude enzyme solution. moreover,
If necessary, known methods can be appropriately combined for purification and use.

Enzymes can be purified by various methods, one example of which is as follows.

Add solid ammonium sulfate to the crude enzyme solution at a low temperature of 5°C, collect the precipitated fraction at 0.6 saturation, dissolve in 10mM phosphate buffer (pH 7,0), and dialyze against the same buffer overnight. Perform the following operations on the selected item. The recovery rate in this ammonium sulfate salting out is about 90%. Next, DEAE-)
Ion exchange chromatography with Yopar 650,
Two specimens (fractions A and B) were purified by hydrophobic chromatography using Butyl Toyovar 650, gel filtration using Ultrogel AcA44, adsorption chromatography using hydroxyapatite, etc., and showed a single band on disk gel electrophoresis. Obtainable. Note that the recovery rates of this sample were 15% and 14%, respectively.

The properties of the purified enzyme of fraction A were investigated. The results are shown below.

(1) Action (a) Acts on 5% sucrose and breaks it down into glucose and fructose. Transfer reactions between sucrose molecules are unlikely to occur, and the production of transferred oligosaccharides is extremely rare.

As shown in FIG. 1, even if the reaction is carried out at a high concentration of 50%, the production rate of l-quesdose is only 20%.

(b) For neutral monosaccharides and oligosaccharides, when they take a chair conformation ('C+), the OH of C2°C3
When this enzyme is allowed to act on sucrose in the presence of a carbohydrate at the equitoria 1 position, a fructosyl group is transferred to the hydroxyl group at the C1 position of the reducing end, producing aldosyl fructoside. For example, D-xylose, D-galactose,
D= and L-fucose, L-sorbose, malto-oligosaccharide, isomalto-oligosaccharide, xylooligosaccharide, lactose, etc. are often transferred with fructosyl groups, and xylosylfructoside, galactosylfructoside, fucosylfructoside, sorbosylfructoside, maltooligosaccharide, etc. Produces silfructoside, isomaltosylfructoside, xylooligosylfructoside, and lactosylfructoside.

Figure 2 shows 50% of sucrose in the presence of xylose.
2 shows the change over time in the production rate of xylosylfructoside when the present enzyme was allowed to act on % substrate at 40°C. As is clear from FIG. 2, the production rate corresponds to 70% of the sucrose used.

(c) Methyl-α-glucoside, methyl-β-glucoside, methyl-α-galactoside, phenyl-α-glucoside, phenyl-α-galactoside, p-nitrophenyl-α-glucoside, p-nitrophenyl-β-galactoside When acting on sucrose in the presence of glycosides such as glycosides, fructosyl groups are transferred to these glycosides.

(d) D-xylitol, D-ribitol, D-galactitol, D-sorbitol, D-maltitol, D
- When acted on sucrose in the presence of a sugar alcohol such as erythritol, fructosyl groups are transferred to the sugar alcohol.

(e) In the presence of a primary alkyl alcohol having up to 10 carbon atoms such as methanol, ethanol, propatool,
When it acts on sucrose, it transfers the fructosyl group to an alkyl alcohol, producing an alkyl fructoside. Also, even in the case of secondary alcohols, the hydroxyl group
, 2-propanediol, 1,2-butanediol, 2
, 3-butanediol, and 1,2-cyclohexanediol.

(f) When it acts on sucrose in the presence of glycoside vitamins such as riboflavin, rutin, and esculin, and vitamins with hydroxyl groups such as pyridoxine, fructosyl groups are transferred to the vitamin.

(g) When acting on sucrose in the presence of nucleosides such as adenosine and inosine, fructosyl groups are transferred to the nucleosides.

(h) When acting on sucrose in the presence of glycosides such as stevioside and rubusoside, fructosyl groups are transferred to the glycoside.

(2) Substrate specificity In addition to sucrose, this enzyme supports eerulose, xylosylfructoside, raffinose, and neokesdose.

It decomposes lactosylfructoside and stachyose well, but has little effect on 1-kesdose, nisdose, canine bioses, and lebambiose.

(3) Activity measurement method 20% sucrose solution containing 40% xylose (50mM
Phosphate buffer pH 6,5) 500ul! Add 200 μl of an appropriately diluted enzyme solution to the solution and incubate at 40°C.

After reacting for a minute, a portion of the reaction solution is poured into boiling water to heat inactivate the enzyme. Next, the amount of glucose and fructose released is determined using the F kit, the amount of fructose is subtracted from the amount of glucose, and the amount of fructose transferred is determined. Calculate.

One unit was defined as the amount of enzyme that transferred 1 μmol of fructosyl group per minute.

(4) Optimal pH and stable pH The optimal pH for action is 0.1 ml of enzyme mixed with 0.05 M buffer (p
H3-8. Add 0.9 mj' of Matskiben buffer, 8.0 to 11 Korsoff buffer, and add 0.2 mρ of this to 20
% sucrose and 0.2771j' of a 40% xylose solution, and the activity was measured. pH stability was determined by adding 0.4 ml of the same 0.1 M buffer to 0.1 ml of the enzyme, keeping it at 40°C for 2 hours, then adding 1.0 ml of 0.1 M phosphate buffer, pH 6.5. Residual activity was measured using 200 μl.

As shown in FIG. 3, the optimum pH is around 6.5 to 6.8, and this treatment is stable in the pH range of 5 to 10.

(5) Optimal temperature and temperature stability The optimal temperature was determined by reacting for 10 minutes at each temperature and measuring the activity.

Temperature stability was determined by holding the enzyme solution at each temperature for 30 minutes, then cooling it for 10 minutes, and then measuring the residual activity. As shown in FIG. 4, the optimum temperature of this enzyme is around 55°C, and it has a residual activity of 72% even at 60°C.

(6) Inhibition and activation This enzyme is inhibited by 90% or more in the presence of 1 mM silver, mercury, or copper, and 40 to 50% inhibited in the presence of 1 mM zinc or tin. Other lomM calcium.

barium, magnesium, manganese, strontium,
There was little inhibition in the presence of ImM nickel, cadmium, and iron. Furthermore, it is inhibited by 90% or more with 10mM sodium lauryl sulfate and 200mM ribose. It is not inhibited in the presence of 0 and 1 mM p-chloromercuribenzoic acid, which acts specifically on cysteine residues.

This enzyme is activated by the presence of xylose and galactose, which are effective receptors for this enzyme.

Dithiothreitol and reduced glutathione, which are SH reducing reagents, had no effect on stabilizing this enzyme. Further, no effect on increasing stability is observed for calcium and magnesium.

(7) Molecular Weight 5DS - The molecular weight was 52,000±2.500 as measured by disk gel electrophoresis and gel filtration.

(8) Isoelectric point As a result of investigation by ampholine electrophoresis, the isoelectric point is 4
．． It was 3.

(9) Crystal structure and elemental analysis No crystal preparations have yet been obtained for this enzyme, or preparations showing a single band when electrophoresed have been obtained.

Table 4 shows the substrate specificity of fungal β-fructofuranosidase that has been reported so far.
It is expressed as the relative speed when Table 5 shows the receptor specificity of levansucrase and fungal β-fructofuranosidase.

As shown in Table 4, this enzyme decomposes raffinose well, but it is difficult to decompose ■-quesdose. It is difficult to catalyze, and as shown in Table 5, it transfers fructosyl groups to galactose, lactose, etc. with a wide range of receptor specificity.

From the above, this enzyme is a novel β-fructofuranosidase that is clearly different from the conventional β-fructofuranosidases.

Furthermore, as a result of examining the enzyme stability, influence of metal salts, substrate specificity, receptor specificity, etc. of the partially purified product of fraction B, almost the same results as fraction A were obtained.

Sucrose raffinose stachyose neokestose 1-kestose nystose turanose Table 4 Main enzyme 67.7 15.6 53, 5D O1 pull N, D N, D N, D N, D 0.02 N, D N, D 0.01 B: Penicillium sp, enzyme derived from K-25, C: Aspergills niger ATC
Transfer activity of the enzyme lactose derived from C20611) N, D: No data available / Sucrose l-Quesdose Raffinose D-Glucose Lactose Galactose D-Xylose L-Arabinose-Sorbose-Rhamnose D-Sorbitol D-Mannitol Kano xylitol maltose isomaltose lactose methanol ethanol 10+ 10+, produced a transfer product, -2 produced no transfer product, N,
D: No data A: Pencilliun+ frequentan
Enzyme B derived from s T-1: Penicillium s
This enzyme is an extracellular enzyme and accumulates in the culture solution. Furthermore, instead of sucrose-induced enzymes, it is possible to culture using other carbon sources without adding sucrose to the medium. Furthermore, unlike levansucrase, levan is not produced in the culture solution, so the enzyme can be easily recovered and is also useful for practical purposes.

〔Example〕

Next, the present invention will be described in detail with reference to Examples.

Example 1 Arthrobacter sp.-1 on ordinary agar slant medium
(FERM P-10736) and incubated at 37℃ for 2 hours.
After culturing for one day, take a loopful of it and add 1.2% yeast extract, 0.8% polypeptone, and 4% soluble starch.

0.4%(NH4)2HP04.0.1%M g S
The cells were inoculated into a liquid medium (C60m1 medium/500 ml shoulder flask) consisting of O4.7H20 (pf 47.0), and cultured with aeration and shaking at 37°C for 2 days. This was used as a seed culture, dispensed into a liquid medium having the same composition, and cultured with aeration and shaking at 37°C for 5 days. The culture solution was centrifuged to obtain about 1.1 f of supernatant (crude enzyme solution). This solution contains 30 units of β
-Contained fructofuranosidase.

Example 2 1.2% yeast extract, 0.8% polypeptone, 4% lactose, 0.4% (NH4)2HPO4, 0.1%M
g S 04.7 H2O (pH 7.0) was used as a medium,
Dispense 60 ml into a 500 ml Yosaka Lough flask, inoculate each with 4 ml of inoculum that has been precultured in the same medium for 2 days, and
Preculture was carried out at ℃ for 2 days. Use this as a starter, 5% of 121
Corn staple liquor, 3% sucrose, 0.4% (N
H4) 2HPO4,o, 1%MgS○, 7 H2O(
The cells were inoculated into a medium having a pH of 7.0, and cultured with aeration for 25 hours while adjusting the pH to 7. The culture solution was centrifuged to remove bacterial cells and crude enzyme solution 121 was obtained. The activity of this solution was 140 units/rnl.

Application example 1 Dissolve 5 kg of lactose and 5 kg of sucrose in water in advance, and
H was set to 6.5, 5 units of the crude enzyme solution of Example 2 was added per 1 g of sucrose, the solid content concentration was set to 40%, and the reaction was carried out at 50°C for 5 hours to obtain a reaction solution containing 30% of lactosyl fructoside. Ta.

〔Effect of the invention〕

According to the present invention, it is possible to efficiently obtain a novel β-fructofuranosidase with strong transfer activity.

Furthermore, by using this enzyme, fructose-containing hetero-oligosaccharides and glycosides can be produced efficiently and at low cost.

Xylosylfructoside and isomaltosylfructoside have anti-cariogenic properties, and lactosylfructoside has activity as a bifidobacteria growth factor, making these fructose-containing hetero-oligosaccharides useful. It is expected that it will be put to practical use as a functional carbohydrate.

Therefore, the present invention, which efficiently produces β-fructofuranosidase with strong transfer activity, is extremely useful industrially.

[Brief explanation of drawings]

Figure 1 shows the reaction progress of β-fructofuranosidase (50
% sucrose, IOU/g substrate), Figure 2 is a graph showing the reaction progress of this enzyme in the presence of 25% xylose as a receptor (25% sucrose, IOU/g substrate), Figure 3
The figure is a graph showing the optimum pH and pH stability of this enzyme, and FIG. 4 is a graph showing the optimum temperature and temperature stability of this enzyme. iI 鴫U exchange next IQ I! (I4lisu ■mi wise uncle) 1su J next size wise uncle four ■ next

Claims (1)

[Claims] It grows at 41℃, does not hydrolyze starch, does not reduce nitrate, produces acid from fructose, produces weak acid from glucose and maltose, has lysine as the main diamino acid in its cell wall, and has a low GC content. 73.2-74.
Arthrobacter sp. K-1 at 9 mol%.