JPH02227074A - Production of new exo-type hydrolase and inulotriose and/or inulotetraose - Google Patents

Abstract

PURPOSE:To advantageously produce inulotriose and/or inulotetraose by reacting fructan with new enzyme hydrolyzing in every 3-4 saccharide units from fructose end side of said fructan. CONSTITUTION:Streptomyces sp.MCI 2423 strain (FERM P-2678) is cultured in medium containing fructan such as inulin as substrate and supernatant solution separated from culturing solution is purified. Then, fructose of said fructan is hydrolyzed in every 3-4 units from fructose end side to obtain purified substance of new exo-type hydrolase having properties of generating inulotriose and/or inulotetraose. Next, said purified enzyme is added to phosphoric acid buffer solution containing inulin and affected at 20-50 deg.C for 5min-100 hour, then inulotrigse and/or inulotetraose is specifically generated since initial stage of the reaction. Thus, the both are able to be advantageously produced.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention provides a method for extracting fructan from the fructose terminal side.
It is said that it specifically hydrolyzes each ~4A sugar unit.
This invention relates to a novel exo-type hydrolase [exo-inulotrioandtetraohydrolyase (tentative name)] that was completely unknown in the past, and a method for producing inulotriose and/or inulotetraose using the enzyme. be.

(Prior art and problems to be solved by the invention) Inulotriose and inulotetraose, which have 3 to 3 fructose bonds, have high functions (bifidus factor, anti-cariogenic properties,
It is an inulooligosaccharide with low calorie, etc.), and can be used as food,
Or it is useful in the pharmaceutical field.

BACKGROUND ART Conventionally, as a method for producing inulotriose and inulotetraose from fructans, a method of hydrolyzing fructans, such as inulin, with an acid is known.

However, according to this method, most of the products are fructose (Fl), inulooligosaccharide, inulotriose, and inulotetraose.The production yield is extremely poor, and colored substances are also produced. However, by-products such as difructose anhydride were produced, making it difficult to purify inulotriose and inulotetraose.

On the other hand, a method has been proposed for producing inulotriose and inulotetraose by decomposing fructan using enzymes. However, the only known enzyme is endo-type inulinase, which randomly hydrolyzes fructans. bios (Fri inulotetraose (F,)
Various types of fructose-linked inulooligosaccharides such as inulohexaose (F6) are produced, and the production yield is low as by-products such as fructose are produced, making it difficult to produce the desired inulotriose and inulotetraose. It was inappropriate.

(Means for Solving the Problems) The present inventors have discovered that canine rotitose and human
Or, as a result of intensive studies on the method for producing canine lotetraose, we discovered a novel exo type that was completely unknown until now and has the property of specifically hydrolyzing fructan into sugar units that have 3 to 1000 fructose bonded. He discovered for the first time a hydrolytic enzyme [echin-canine rotoriotriandtetrahydrolyase (tentative name)] and completed the present invention.

That is, the gist of the present invention is that it has the property of acting on fructan and hydrolyzing every 3 to μ sugar units from the fructose terminal side to produce inulotriose and/or inulotetraose. A novel exo-type hydrolase and fructan that can be extracted from the fructose end of fructan by 3 to 4! IJ ose and/or inulotetraose are produced by reacting with a novel exo-type hydrolase that has the property of hydrolyzing each sugar unit to produce inulotriose and/or inulotetraose. The present invention relates to a method for producing inulotriose and/or inulotetraose, which comprises obtaining the inulotriose and/or inulotetraose.

The present invention will be explained in detail below.

The enzyme conventionally known as the enzyme that produces inulooligosaccharides (inulinase) is an endo-type enzyme that randomly hydrolyzes inulin etc. to produce i- and nullooligosaccharides in which various numbers of fructose are bonded, and an exo-type enzyme. The enzyme that hydrolyzes each sugar unit to produce only fructose and the enzyme that produces difructose heptahendride is a completely new enzyme that has not been previously known. It is a type of exo-type hydrolase.

The enzyme of the present invention is obtained, for example, from a microorganism belonging to the genus Streptomyces.

The microorganisms used in the present invention may be those having the ability to produce exo-hydrolytic enzymes having the above-mentioned properties, including Streptomyces and its variants;
It is not limited to mutant species, etc.

Specific examples of such microorganisms belonging to the genus Streptomyces include, for example, Streptomyces sp, MCI J≠
Bacteria No. 23 is mentioned. Such strains [Feikokenbacterium IF No.
o51>3 (FERM P-7θ, flB)
It has been deposited as.

Streptomyces sp, Mc1.2!23 (hereinafter referred to as “
Actinobacterium MCI,? Morphological characteristics, properties on various media, and physiological
The biochemical properties and quality are as follows.

l) Morphological characteristics Pennett's agar culture and hiculcose-asparagine agar culture were used as the sporulation medium, and the cells were cultured at 27°C for one week.

On Pennett's agar medium, colonies initially appear yellowish-white, then turn light olive-gray as aerial mycelia form. Forms relatively short segmented spore chains on aerial hyphae. The spore chains occur in a 2- to 2-way radial pattern, stand upright, and do not coil in a spiral shape. Spore surface is smooth.

The spore has a short cylindrical shape, and its size is i-, at, iμm×
It is 2 to 8 μm.

Colonies appear yellowish white on a glucose-asparagine agar medium. Aerial hyphae and spore formation are poor. The basal hyphae develop well, and after 3 to 1/- days of culture, the basal hyphae segment at some places to form long spore chains.

2) Characteristics in various liquid media (1 week of shaking culture at 17°C) 3) Culture properties on various agar media The properties on various agar media after culturing at 27°C for 1 day are as shown below. .

In addition, the meanings of the symbols in the item section in the above table are as follows.

G: Growth, AM: Aerial mycelium, So: Spore formation, SP:
Soluble pigments 4/-) Physiological properties Production of melanin-like pigments (peptone, yeast extract, iron agar),

Hydrolysis of starch: Liquefaction of gelatin 0 Coagulation of skim milk Negative Positive Positive Negative Pebutosylation of skim milk: Positive Assimilation of carbon sources D-glucose + arabinose D-xylose + inositol D-mannitol D-7 lactose Shishi-rhamnose + Shelfrose Shi-raffinose +: Possible to assimilate, ±: Doubtful, 1: No assimilation ability Biochemical properties Amino acid composition of cell wall: L L -At ptns Glycine (Cell wall type ■ type) Main menaquinone : MKri (H6), MK
(H8) GC content of DNA Niao, /% Bacterial fatty acids Contains biantennary fatty acids B) Taxonomic considerations Identification at the genus level The actinomycete MCIJ4cJj that produces canine oligosaccharides
The fungus has the following characteristics: 1) It has an upright spore chain, the colony is bright olive gray, 3) The formation of aerial hyphae is poor, and most of the aerial hyphae develop into spores. The basal hyphae segment and form spores. The hyphae are seen to be fragmented during liquid culture. Cell wall composition:
L mountain - Contains diaminopimelic acid and glycine (cell wall type I) 6) Major menaquinones: MKP (H@), MKri (H8)
7) Contains branched fatty acids.

Based on these morphological and biochemical properties, this strain is Streptomyces.
) belongs to the genus. As an actinomycete with segmented basal hyphae,
Elythrosporangium (Elytrosporangium)
iun) genus “Miku a x o Bosboria (Micro
ellobosporia) genus "Chainia"
ia) genus Actinobacteria were known, but good-fellows and others (System
, Appl, Microbiol, lr: u
(Hiro-At, 1916) found that these temperature ranges are Streptococcal.
Emphasizing that it has biochemical properties in common with the genus Streptomyces, it is treated as a synonym for a foreign substance in the genus Streptomyces.

Identification at the species level This strain, MCI2112J, l) forms an upright spore chain, and c) the colony exhibits a light olive gray color.The ISP species search table for the genus Streptomyces (Hideo Nonomura, Fermentation Engineering Journal Vol. 12, Vol. 2) When I searched for No. 7 by 1 octopus, 127 years), Rec tu
It was suggested that it belongs to the s-white clause.

As a peripheral bacterium, Streptomyces Candithreptomyces settonii (S, 5etonii).

Streptomyces syndenensis (S.

can be lost. When we compared the properties of this species and the various species described in ISP, these ISP-described species did not contain any information regarding division of basal hyphae, and were distinguished from this strain.

In addition, conventional “Elytrosporangium”
” , “Microe-11 obosporia A
ctinosporangium II and” Act
When we compared the morphological characteristics and physiological properties of this strain with the original descriptions of the bacterial species of each genus included in the genus inopycnidium''', this strain was clearly distinguished from each of these bacterial species. This strain was identified as Streptomyces sp. MCI No. 4!23.

Cultivation of Streptomyces MCIJ≠23 for producing the novel exo-type hydrolase of the present invention can be carried out in a conventional medium containing nutrients that can be utilized by microorganisms.

Specifically, fructans such as 7-lacto-oligosaccharides, inulooligosaccharides, and inulin containing β-col' fructoside bonds are used as substrates, and components necessary for the growth of the bacteria, such as soybean flour, wheat germ, etc., are used as nitrogen sources. corn staple
Liquor Use cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. to generate sodium, potassium, calcium, magnesium, cobalt, chlorine, sulfuric acid, and other ions as necessary. Culture in a medium containing inorganic salts, vitamins, etc. In this case, the effective amount of fructan added to the medium is, for example, in the case of inulin, in the range of 0.15 to 20%.

Furthermore, the culture temperature is 20-37℃, and the culture time is 20-37℃.
26 hours is appropriate.

After completion of the culture, the novel exo-hydrolase of the present invention can be purified from the culture supernatant by appropriately combining various known purification methods. One example is as follows. It is common knowledge.

Remove bacteria from the culture solution by appropriate means such as filtration and centrifugation to obtain a supernatant, which is then dialyzed against distilled water. Add ammonium sulfate to the obtained dialysate to make it 30% saturated,
aO% saturated ammonium sulfate-containing phosphate buffer (0,
Toyopearl HW equilibrated with 02M1pH7,0)
Adsorb to 4tF (manufactured by Tosoh Corporation) and perform column chromatography. Elution is carried out using a linear concentration gradient of an aqueous solution of ammonium sulfate that is saturated at go-o%. Next, fractions with inulinase activity are collected and dialyzed against 20rrM phosphate buffer (pH 7.0). The obtained crude enzyme solution is DE
A'E-) When passed through a Yopar column (manufactured by Tosoh Corporation), inulinase activity was observed in the non-adsorbed fraction, and the enzyme solution was further separated by molecular sieving using Wotoyopar HWljF (manufactured by Tosoh Corporation), and then / It is adsorbed onto QAE-Toyopearl (manufactured by Tosoh Corporation) equilibrated with OmM Tris-HCl buffer (pHr, 7). A purified product of the novel exo-type hydrolase can be obtained by performing elution with a linear concentration gradient of O-0.1M saline, collecting the active fraction, and dialyzing it.

For example, the purified enzyme of the present invention obtained as in Example 1 below has the following properties.

b) From the fructose terminal side of fructan, fructose is hydrolyzed into every 3 to 4 c units to produce inulotriose and/or inulotetraose.

This enzyme acts on pentaose and higher sugars in l-kesdose oligosaccharides, producing shakerose and inulotriose from pentaose, and both inulotriose and inulotetraose O from heptaose and higher.

Inulotriose (Fl) and Inulotetraose (F4)
) is determined by the chain length of inulin; when the chain length is long, Fa > Fs, and when the chain length is short, F.

〉F4　.

(b) The optimum pH is t~7, and it is stable at pHLj~.

(c) The optimum temperature at pHJ~7 is around 10°C, and good activity is shown at 4tO-0°C.

) pH 4-7, heat treatment conditions for 30 minutes, bottom: C! Q
Stable up to ’°C.

After standing at various temperatures at pH 1j for 30 minutes, the reaction was carried out at 50°C and the residual activity was measured. As a result, there was almost no decrease in activity up to 10°C, and the activity was rapidly lost at temperatures higher than that.

(e) The molecular weight determined by 5DS-polyacrylamide gel electrophoresis is approximately 7♂0.00.

The method for producing inulotriose and/or inulotetraose using the enzyme of the present invention uses a fructan containing at least 100% of fructose, particularly inulin, as a substrate,
Inulotriose and/or
Alternatively, canine lotetraose can be produced. As the enzyme to be acted on, a purified enzyme purified from the culture supernatant may be used, or a culture supernatant obtained by culturing the bacterial cells in a medium containing the above-mentioned substrate may be used. Alternatively, enzymes or microbial cells may be adsorbed, bound, and encapsulated on an immobilization carrier by a known method.

For example, when treating purified enzymes, inulin o, i N
1 oz6 containing 0. / M phosphate buffer (pH 6, t) with enzyme o, ot-io units, 20~! O℃! minutes to 10
When allowed to act under conditions of 0 hours, inulotriose and/or inulotetraose are specifically produced from the initial stage of the reaction.

Furthermore, when the bacterial cells are cultured in the above substrate-containing medium, inulotriose and/or inulotetraose are produced in the culture solution. This culture is sterilized, and the resulting culture supernatant is treated with activated carbon and desalted to remove impurity salts and the like. Next, the obtained product is subjected to activated carbon column chromatography. When gradient elution with 0-10% ethanol is performed, inulotriose and/or inulotetraose are eluted in the j-io% fraction, and by removing ethanol, inulotriose and/or inulotetraose in the culture supernatant can be removed.
Alternatively, canine lotetraose can be purified.

(Effects of the Invention) The enzyme of the present invention is a novel echin-type hydrolase that specifically hydrolyzes fructan from the fructose terminal side to each sugar unit to which 3 to ≠ fructose is bonded, /or Canine rotidolaose can be advantageously produced.

(Examples) Next, the present invention will be explained in detail using Examples, but the present invention is not limited to the following unless it exceeds the gist thereof.

Example 1 Streptomyces sp, MCI-2≠J J No. , oz%, flp mother extract 0.02%, and a trace amount of iron chloride.
After culturing for days, a portion of the culture was taken and transferred to a liquid medium of the same composition (100 yd medium/100 ml Erlenmeyer flask).
Aerated shaking culture was performed at 30°C for 3 days.

Next, after dialyzing the crude enzyme solution against distilled water, 4! Ammonium sulfate was added and fractionated to achieve O saturation. Then, 20mM phosphate buffer containing ≠Q% saturated ammonium sulfate (p
It was adsorbed onto a Toyopearl HW4 jF column chromatograph (φj 4

The active fraction was dialyzed against 20 mM phosphate buffer (pH 7,0) and then subjected to DEAE-) Jobar column chromatography (φj cm (φJ
, /X70cIn) for molecular separation and purification.

Furthermore, the active fraction was adsorbed onto a QAE Toyopearl column chromatograph (φJ, /cm Elution was performed by applying a concentration gradient.

Table 1 shows the purification results in each step. The enzyme of the present invention is isolated and purified by the above operations.

Table 1 Crude enzyme fraction lhiro 10/3/ 3/, ri Q, λ≠ 100 Note that the titer was measured as follows.

That is, 0. x-related dog squirrel aqueous solution Q, coyo d and p) li
t of 0.2 M + 7 phosphoric acid buffer 0. 0.1 jttl of the enzyme solution was added to the mixed substrate of /, 1 and reacted at 37°C for 1 hour, and the produced inulotriose and/or inulotetraose was quantified by HPLC (high performance liquid chromatography). The amount of enzyme that produced canine oligosaccharide/μmol per minute was defined as 1 unit (u).

Example 2 0.1% (w/v) inulin as substrate.
Example 1 in 01M phosphate buffer (pH 1j)/l
The purified hydrolytic enzyme obtained in 0. / A solution containing the unit was added and the reaction was carried out at 30°C for 10 minutes.

After heating the reaction solution to inactivate the enzyme, the production rate of inulooligosaccharide in the reaction solution was determined using HPLC, and it was found that inulotriose was 70. / %, dog lotetraose is one! ,,
r% of each, and no other inulo-oligosaccharides were observed to be produced. The amount of unreacted inulin was tj, 1%.

In this way, by using the enzyme of the present invention, inulotriose and inulotetraose can be specifically produced.

Example 3 The reaction was carried out in the same manner as in Example 2 for 2j hours, and the results were analyzed in the same manner as in Example 2. As a result, the production yields of inulotriose and inulotetraose (inulotriose: inulotetraose = group) :6) was 1r/(1) in total.

Example 1 Streptomyces sp, Mc
I 24AAl1 bacteria was cultured and the enzyme solution was sterilized after culturing + rO1! Inulin for Ll, 20? was added, and ≠ was allowed to react at 30°C for a period of time.

After the reaction, the reaction solution was analyzed by liquid chromatography, and the production rate of inulotriose and inulotetraose (inulotriose:inulotetraose=4c:6) in the reaction solution was 73 in total.

Claims (2)

[Claims] (1) A novel exo-type hydration characterized by having the property of acting on fructan and hydrolyzing every 3 to 4 sugar units from the fructose terminal side to generate inulotriose and/or inulotetraose. Degrading enzyme. (2) Reacting fructan with a novel exo-type hydrolase that has the property of hydrolyzing every 3 to 4 sugar units from the fructose terminal side of fructan to produce inulotriose and/or inulotetraose. A method for producing inulotriose and/or inulotetraose, which comprises producing inulotriose and/or inulotetraose and obtaining the inulotriose and/or inulotetraose.