JPH10117800A - Production of monosaccharide, oligosaccharide and solubilized polysaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain a monosaccharide, an oligosaccharide or a soluble polysaccharide useful for a low-calorie sweetener, a health food, etc., by blasting or steaming or boiling a ligneous polysaccharide, a structural polysaccharide or an agricultural or marine product containing them. SOLUTION: A ligneous polysaccharide, a structural polysaccharide (e.g. xylan) or an agricultural or marine product (e.g. chaff) containing them is blasted, steamed or boiled with an acid solution having 10<-1> to 4×10<-6> M hydrogen ion concentration or a buffer solution to give an oligosaccharide or a soluble polysaccharide. The oligosaccharide or the soluble polysaccharide is treated with a glycolytic enzyme such as Streptomyces olivaceoviridis E-86 or Streptomyces olivaceoviridis E-86-N1-35 to efficiently give a monosaccharide or an oligosaccharide useful as a low-calorie sweetener and other health foods, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for efficiently producing monosaccharides, oligosaccharides and / or solubilized polysaccharides from agricultural and marine products. The present invention includes a method for efficiently producing a xylan solubilized solution from rice hulls, and a method for efficiently producing a xylo-oligosaccharide containing xylobiose as a main component from the xylan solubilized solution.

[0002]

2. Description of the Related Art Xylooligosaccharides are used as low-calorie sweeteners and other health foods. As a method for producing xylo-oligosaccharides, using corn cobs as a raw material,
A method has been proposed in which xylan is extracted therefrom and treated with an enzyme to produce xylo-oligosaccharides. In addition, Japanese Patent Application Laid-Open No. 1-25
No. 4692 discloses a method for producing a xylooligosaccharide containing xylobiose as a main component by enzymatically treating a hemicellulose solution obtained by steaming wood. Japanese Patent Application Laid-Open No. 2-117400 discloses a method for producing xylose by blasting cottonseed shells.
These methods have some difficulty in availability of raw materials.

[0003] In recent years, attempts have been made to produce xylooligosaccharides using rice husk, which is an agricultural waste, as a raw material. Rice husks have no major use at present and are incinerated as waste. However, there is a problem of smoke pollution and the like, and the disposal thereof is a major problem. The use of rice husk as a raw material not only makes it possible to reduce the production cost of xylo-oligosaccharides, but also leads to solving the problem of agricultural waste disposal.

As a method for producing xylo-oligosaccharides using rice husk as a raw material, there is known a method for producing xylo-oligosaccharides by treating rice husks with an alkali and saccharifying the obtained xylan with an enzyme. This method has a low yield, and requires alkali extraction of the rice husks as a chemical treatment. Therefore, the purity of xylan is low because inorganic components derived from silica in the rice husks are also entrained. In addition, the method requires a large amount of alkali as a pretreatment for the enzyme reaction raw material and an acid for neutralizing the alkali, which is a very costly method. Therefore, it is difficult to use it for food and drink, and it has not been put to practical use.

[0005] In addition to rice husks, agricultural and marine products, particularly agricultural and marine wastes that are currently disposed of without effective use methods, such as copra oiled cake, beet oiled cake, etc., are monosaccharides, oligosaccharides and / or There is a demand for the development of a technology for producing solubilized polysaccharides and effectively utilizing these.

[0006]

An object of the present invention is to provide a method for efficiently producing monosaccharides, oligosaccharides and / or solubilized polysaccharides from these agricultural and marine products. Another object of the present invention is to provide a method for efficiently producing monosaccharides or oligosaccharides from oligosaccharides and / or solubilized polysaccharides.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventor has found that when woody polysaccharides or structural polysaccharides or agricultural and marine products containing them are exploded or steamed, monosaccharides, oligosaccharides or solubilized polysaccharides can be obtained. Also, they have found that a monosaccharide or an oligosaccharide can be obtained by treating an oligosaccharide or a solubilized polysaccharide with a specific enzyme, and arrived at the present invention. That is, the first present invention comprises a step of exploding or steaming a wood-based polysaccharide or a structural polysaccharide or an agricultural or marine product containing the same, the monosaccharide, the oligosaccharide and / or the solubilized polysaccharide (hereinafter referred to as , Solubilized saccharides). The second invention is a method for producing a monosaccharide or oligosaccharide, comprising treating the oligosaccharide or the solubilized polysaccharide thus obtained with its glycolytic enzyme. The third invention is most preferred as a glycolytic enzyme
It is a mutant strain of Streptomyces olivaceoviridis E-86 Stre
ptomyces olivaceoviridis E-86-N1-35, a xylanase derived from this strain.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The agricultural and marine products containing a woody polysaccharide or a structural polysaccharide used in the present invention include, for example, rice hulls, rice straw, straw, buckwheat hulls, bagasse, apple juice cake, copra oil cake, beet juice. Oil cake, defatted rice bran, corn seed coat, seaweed and the like. These agricultural and marine products do not require any pretreatment such as grinding. However, there is no particular problem even if such a pre-processed one is used.

[0009] The explosion treatment in the present invention means to grind the rice hulls by immediately returning the agricultural and marine products under pressure for a certain period of time to normal pressure. The pressurizing pressure is usually 5 to 30 kg / cm ² in gauge pressure. Pressure is 5kg / c
If it is less than m ² or more than 30 kg / cm ² , the yield of solubilized saccharide will decrease. Preferably 5 to 25 kg / c
m ² , more preferably 5 to 20 kg / cm ² , most preferably 10 to 20 kg / cm ² . Pressing time is usually 1
Minutes to 2 hours. If the pressurization time is less than 1 minute or more than 2 hours, the yield of solubilized saccharide will decrease. Preferably it is 1 to 60 minutes, more preferably 2 to 60 minutes, most preferably 5 to 60 minutes. Although the temperature at the time of pressurization is not particularly limited, it is usually 158 to 225 ° C. (5 to 25 k in saturated vapor pressure).
g / cm ² ).

In the explosion treatment, agricultural and marine products are put in a pressure-resistant container,
This is performed by maintaining the pressure at a predetermined pressure for a predetermined time in the presence of steam, and then rapidly releasing the pressure to the atmospheric pressure. The pressurization includes a method of pressurizing the inside of the container with a compressor, a method of pressurizing the closed container by heating, and a method of introducing high-temperature and high-pressure steam into the container and pressurizing the container. A method of pressurizing with high-temperature and high-pressure steam is preferred.

The steaming treatment in the present invention is to treat agricultural and marine products in a liquid at high temperature and high pressure. The processing pressure is usually 5 to 30 kg / cm ² in gauge pressure. Pressure 5
It exceeds kg / cm ² less than a is either 30kg / cm ^2, of solubilized sugar yield is reduced. Preferably 5 to 25
kg / cm ² , more preferably 5 to 20 kg / cm ² ,
Most preferably, it is 10 to 20 kg / cm ² . The temperature is usually 158 to 225 ° C (5 to 25 kg / c at saturated vapor pressure)
m ² ). If the temperature is lower than 158 ° C., the amount of solubilized saccharides extracted decreases. When the temperature exceeds 225 ° C., the solubilized saccharide is further decomposed, and the amount of the solubilized saccharide decreases. Processing time is usually 1 minute to 2 minutes
Time. If the treatment time is less than 1 minute or more than 2 hours, the yield of solubilized saccharide decreases. Preferably 1 to 6
0 minutes, more preferably 5 minutes to 60 minutes.

When the explosion or the steaming treatment is performed by adding an acid or a buffer solution, the solubilized amount of the solubilized saccharide further increases. As the acid, for example, a mineral acid such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, and an organic acid such as acetic acid, citric acid and oxalic acid are used, and acetic acid is preferable. As the buffer, for example, an acetate buffer or a phosphate buffer is used, and an acetate buffer is preferred. Usually, an acid solution or a buffer solution having a hydrogen ion concentration of 10 ^-1 to 4 × 10 ^-6 M is added.

[0013] Explosive or steamed agricultural and marine products are usually obtained as a slurry. This slurry is subjected to solid-liquid separation. For solid-liquid separation, any of known separation methods such as squeeze separation, centrifugation, and filtration may be used. Further, either a batch type or a continuous type may be used. Preferably, a continuous screw press by squeezing separation or the like is used.

In this way, the solubilized saccharide can be easily recovered from agricultural and marine products at a high yield. For example, when rice husks are used as agricultural and marine products, xylan solubilized liquid is mainly obtained as sugars. Mannose and mannobiose are obtained from copra oiled cake, and arabinose, glucose and galactose are obtained from beet oiled cake.

To produce monosaccharides or oligosaccharides from oligosaccharides or solubilized polysaccharides, treatment with these glycolytic enzymes is carried out. The oligosaccharide or solubilized polysaccharide solution obtained by solid-liquid separation may be directly treated with a glycolytic enzyme, or may be concentrated and solidified as necessary. Known methods such as evaporation concentration, membrane concentration, and freeze concentration can be used as the concentration method used for this. Drying methods include ventilation drying, spray drying, freeze drying and the like.

Hereinafter, a technique for obtaining a xylo-oligosaccharide from a xylan solubilized solution will be specifically described. Xylanase is used as the glycolytic enzyme. As the xylanase, not only purified xylanase but also a cell culture solution containing xylanase or a cell itself producing xylanase can be used in some cases. Further, a known xylanase or a commercially available xylanase may be used. These include xynA (MW 43,000; pI 5.2) and xynB (MW 3), which are xylanases produced by Streptomyces lividans.
1,000; pI 8.4) and xynC (MW 22,000; pI> 10.25).

Preferably, Streptomyces olivaceoviridis is used for the production of xylo- oligosaccharides, which is the object of the present invention.
E-86 xylanase is preferred. The bacterium hardly produces sugar hydrolases such as cellulase and xylosidase other than xylanase. Therefore, even if the culture solution of the present bacterium is used directly, only xylo-oligosaccharide is preferentially produced without producing much glucose or xylose, and there is no need to use purified xylanase. Streptomyces olivaceoviridis E-8
6, and its culture method is disclosed in the literature (I. Kusakabe, T. Yasui and T Kobayashi; Agric . Bio
l . Chem ., vol.39, pp1355 (1975)).

Xylanase produced by Streptomyces olivaceoviridis E-86 has a molecular weight of 40,500 and a pI of 7.3.
(Kusukabe et al., Journal of the Japanese Society of Agricultural Chemistry, vol. 51, p429-437, 1)
977), with a molecular weight of 25,000. In the former, the optimum pH and temperature of the reaction are pH 5.5-6.2 and 55-60, respectively.
℃, stable below 60 ℃, stable at pH 4.5-10.5, and the specific activity of the produced enzyme is reported to be 253 units / mg. The latter was announced by Kuno et al. (Kusakabe's group) at the Japanese Society of Agricultural Chemistry in the spring of 1997.

The most preferred xylanase is S
treptomyces olivaceoviridis is a mutant strain of E-86 Stre
It is a xylanase derived from ptomyces olivaceoviridis E-86-N1-35. E-86-N1-35 is a mutant strain obtained by treating Streptomyces olivaceoviridis E-86 with nitrosoguanidine, as described in detail in Examples below, and deposited with the Biotechnology Research Institute as FERM P-15724. Have been. This xylanase is different from the one released by Kuno et al. Produced by the parent strain E-86 described above because the N-terminal sequence is different as described below and the molecular weight is 25,500, although there is a slight difference. It is.

A xylan solubilized solution containing a xylanase-containing solution,
Preferably Streptomyces olivaceoviridis E-86 culture solution , particularly preferably Streptomyces olivaceoviridis
Saccharification is performed by adding a culture solution of E-86-N1-35.
The unit amount of xylanase to be added is 5 to 20,000, preferably 50 to 1,000, for 50 mg of xylose in the xylan solubilized solution. The enzyme reaction temperature is in the range of 30 ° C to 80 ° C, preferably 40 ° C to 70 ° C. The enzyme reaction is carried out at a pH of 2 to 9, preferably 4
From 6.

In addition, Streptomyces olivaceoviridis E-8
6-N1-35 is a strain first provided by the present invention,
Extremely high xylanase activity. Therefore, not only xylan solubilizing solution obtained from rice hulls, but also bamboo,
Xylo-oligosaccharides can be obtained in high yield by treating all xylan-containing substances such as cottonseed and corn cob with this strain or xylanase derived therefrom, and these are all within the scope of the present invention.

The obtained enzyme reaction solution is a xylooligosaccharide containing xylobiose as a main component. Since this enzyme reaction solution contains various impurities, it is preferable to carry out a purification treatment for food use. Usually, first, activated carbon is added to decolorize. Next, organic acids such as acetic acid, lignin and its decomposition products, enzymes, inorganic salts, coloring substances, and the like generated by explosion or steaming are removed using an ion exchange resin or a synthetic adsorbent. As the ion exchange resin, a mixed bed of a strongly acidic cation exchange resin and a strongly basic anion exchange resin may be used, or they may be used separately. The purpose can be achieved by using an electrodialysis membrane instead of the ion exchange resin. The activated carbon treatment of the enzyme reaction solution may be performed after an ion exchange resin, a synthetic adsorbent, or the like or an electrodialysis treatment.

[0023]

The present invention will be described in detail with reference to the following examples.
The present invention is not limited in any way by these examples.

Example 1 Rice hulls were blasted using a blasting device (Tsukishima Kikai). 100 g of rice husk was sealed in a pressure vessel (withstand pressure 40 kg / cm ² , capacity ² L). Next, high-temperature and high-pressure steam is introduced into the pressure vessel, and the processing pressure is 10, 15, and 20 kg / cm ^2.
For a predetermined time. Immediately thereafter, it was rapidly discharged into a receiving tank equipped with a silencer at atmospheric pressure. The obtained slurry was added to 0.5
The powder was sieved with a stainless steel mesh having a thickness of 2 mm, and an explosion solubilized solution containing xylan was recovered. The xylan in the centrifugal supernatant of the filtrate was analyzed by the orcin-ferric chloride-hydrochloric acid method (WRFernell and HK).
King; Analyst, vol. 78, pp80, 1953). The yield was calculated based on the weight of the raw rice hulls. Table 1 shows the time-dependent formation of the solubilized xylan at each processing pressure. Further, the oligosaccharide composition of the obtained crushed supernatant was measured using DX-500.
FIG. 1 shows the results of analysis using a sugar analysis system (manufactured by Dionex). In FIG. 1, the unit of the vertical axis is nC (nano
(Coulomb), which is a value corresponding to the amount of sugar in the sample. Ara is arabinose, Glc is glucose, X1
Is xylose, X2 is a dimer of xylose (xylobiose), X3 is a trimer of xylose, and so on. At a processing pressure of 10 kg / cm ² , a processing time of 30 minutes, 1
In 5kg / cm ² 15 to 45 minutes, 20kg / cm
^In No. ² , the xylan solubilization rate was the maximum at 10 minutes at each treatment pressure. Up to about 12 g of xylan could be solubilized from 100 g of rice husk.

[0025]

[Table 1] Table 1 Solubilization of xylan by explosion treatment ──────────────────────────────────── Treatment Pressure 10 kg / cm ² Processing pressure 15 kg / cm ² Processing pressure 20 kg / cm ² ───────── ───────── 時間 Processing time (min) Xylan Yield (%) ────────────────────────────────────2 4.26 5 0.961 5 0.91 8.68 8 9.29 10 2.71 10.66 10.6 15 6.03 11.07 1.26 30 11.4 11.08 45 11.83 ──────────────────────────

Example 2 Rice hulls were blasted using a blasting apparatus. A pressure vessel (withstand pressure 40 kg / cm ² , capacity ² L) was filled with 100 g of rice husk and 0.01 to 0.5 mol of acetic acid. Next, high-temperature and high-pressure steam was introduced into the pressure vessel, and kept at a processing pressure of 15 kg / cm ² for 30 minutes. Immediately thereafter, it was rapidly discharged into a receiving tank equipped with a silencer at atmospheric pressure. 0.5 mm of the obtained slurry
And a blasting solubilized solution containing xylan was recovered. Xylan in the centrifuged supernatant of the filtrate was measured in the same manner as in Example 1. Table 2 shows the time-dependent formation of the solubilized xylan at each processing pressure. Acetic acid 0.05
At the time of the molar addition, up to about 14 g of xylan could be solubilized from 100 g of rice husk.

[0027]

[Table 2] Table 2 Solubilization of xylan by explosion treatment (in the presence of acetic acid) ──── N-acetic acid ────────────────────────── 0 0.01 0.05 0.2 0.2 0.5 ──── {Xylan yield (%) 11.1 14 13 12 12} ───────────────────────────────

Example 3 10 g of rice hulls and 200 ml of water were placed in a 500 ml pressure-resistant stainless steel container (manufactured by Pressure Glass Co., Ltd., portable reactor TVS-N2-500) and heated from outside the stainless steel container with a plate heater. After the steam was blown for about one minute after the steam started to come out of the container, pressurization was started. Processing pressure is 10, 15, 20 kg / cm ²
For a predetermined time. Thereafter, the heating was stopped and the mixture was allowed to cool. The reaction mixture was recovered, and a supernatant was obtained using a small centrifuge. Table 3 shows the solubilized xylan in the supernatant at each processing pressure. The yield was calculated based on the weight of the raw rice hulls. As a result, the amount of solubilized xylan was 15 kg / cm
² was 5 minutes, and 30 minutes at 10 kg / cm ² was the maximum. Up to about 1 g of xylan could be solubilized from 10 g of rice husk.

[0029]

[Table 3] Table 3 Solubilization of xylan by steaming treatment ────────────────────────── Treatment pressure 10 kg / cm ² Treatment pressure 15 kg / cm ² ───────── ───────── Processing time (min) Xylan yield (%) ──────────────────── {5 7.46 15 9.53 6.04 30 11.1 4.33 45 8.88 3.09} ─────────

Example 4 10 g of rice hulls and 200 ml of 0.01-0.5N acetic acid were added to 5
It was placed in a 00 ml pressure-resistant stainless steel container and heated with a plate heater from the outside of the stainless steel container. After the steam was blown for about one minute after the steam started to come out of the container, pressurization was started. Treatment pressure is 3 at 10 kg / cm ²
Treated for 0 minutes. Thereafter, the heating was stopped and the mixture was allowed to cool. The reaction mixture was recovered, and a supernatant was obtained using a small centrifuge. Table 4 shows the solubilized xylan in the supernatant at each treatment pressure. The yield was calculated based on the weight of the raw rice hulls. As a result, the amount of solubilized xylan was maximum in the 0.01 N acetic acid solution. Up to about 1.
2 g of xylan could be solubilized.

[0031]

Table 4 Table 4 Solubilization of xylan by steaming treatment (in the presence of acetic acid) ──── N-acetic acid ────────────────────────── 0 0.01 0.05 0.2 0.2 0.5 ──── {Xylan yield (%) 11.1 12.1 11.9 11 12} ───────────────────────────────────

Example 5 10 g of copra oiled cake (containing 3.0 g as mannose), 100 ml of 1N acetic acid were placed in a pressure-resistant stainless steel container and heated from the outside. After steaming at a processing pressure of 10 kg / cm ² for 30 minutes, the mixture was allowed to cool. The reaction mixture was recovered and a supernatant was obtained using a small centrifuge. 3.3 g of total sugar was solubilized with respect to the raw material. The lysate contained 243 mg of mannose and 72 mg of mannobiose.

Example 6 Apple pectin (5.0 g), 200 ml of 0.1 M acetate buffer (pH 4.0) was placed in a pressure-resistant stainless steel container, and heated from the outside. After steaming at a processing pressure of 5 kg / cm ² for 10 minutes, the mixture was allowed to cool. The reaction mixture was recovered and a supernatant was obtained using a small centrifuge. The lysate contained 0.11 g of galacturonic acid.

Example 7 5.0 g of citrus pectin, 200 ml of 0.1 M acetate buffer having a pH of 4.0 was placed in a pressure-resistant stainless steel container, and heated from the outside. After steaming at a processing pressure of 5 kg / cm ² for 10 minutes, the mixture was allowed to cool. The reaction mixture was recovered and a supernatant was obtained using a small centrifuge. The lysate contained 0.15 g of galacturonic acid.

Example 8 10 g of beet juice lees (including 2 g of arabinose)
100 ml of water was put in a pressure-resistant stainless steel container and heated from the outside. After steaming at a processing pressure of 10 kg / cm ² for 5 minutes, the mixture was allowed to cool. The reaction mixture was recovered and a supernatant was obtained using a small centrifuge. Arabinose, glucose, and galactose were solubilized in the supernatant, and 0.28 g of arabinose was obtained as a main product.

Example 9 10 g of defatted rice bran and 200 ml of water were placed in a pressure-resistant stainless steel container and heated from the outside. After steaming at a processing pressure of 10 kg / cm ² for 10 minutes, the mixture was allowed to cool. The supernatant contained sucrose, maltose, and maltotriose as main products, and maltotetraose, arabinose, and glucose monosaccharide as small products. 5 kg / cm ²
Under the treatment conditions of -5 minutes, arabinose, glucose and sucrose were the main products.

Example 10 10 g of corn seed coat and 200 ml of 0.1 M acetate buffer at pH 4.0 were placed in a pressure-resistant stainless steel container and heated from the outside. After steaming at a processing pressure of 10 kg / cm ² for 15 minutes, the mixture was allowed to cool. The supernatant contained xylooligosaccharides in addition to arabinose and xylose monosaccharides as main components.

Example 11 Streptomyces olivaceoviridis E-86 was inoculated in a storage medium having the composition shown in Table 5 and cultured at 30 ° C. for one week. The spores sufficiently settled as a parent strain. The spores were scraped from the slant and suspended in a sterile phosphate buffer (5 ml) at 1/15 M, pH 7.5. Nitrosoguanidine was added to this solution to a final concentration of 1500 to 2000 μg / ml, and the mixture was allowed to stand at room temperature for 30 minutes. The nitrosoguanidine-treated solution was filtered through filter paper (Advantech No. 2),
10 ^-1 with a sterile phosphate buffer of 1 / 15M, pH 7.0
The solution was diluted to 10 ^-5 (10 to 100,000 times) and spread on a plate storage medium prepared in advance. Incubate at 30 ° C for 7 days,
Colonies were formed (survival rate 5-10%).

Then, strains were selected by the agar-piece method and the plate activity assay method. The production medium shown in Table 6 plus 2% agar was dispensed in 30 ml portions into a 9 cm diameter petri dish and solidified on a plate. It is aseptically punched with a 6 mm diameter cork borer into an agar piece.
Arranged individually. The previously obtained mutagenized colonies were
Each plant was planted and cultured at 30 ° C. for 1 to 2 weeks. 1 g of birchwood xylan (Fluka Biochemical 95588) and 2 g of agar were added to 0.05 M sodium citrate buffer (pH 5.
3) After suspending in 100 ml and sterilizing at 121 ° C. for 20 minutes,
The mixture was poured horizontally into a sterile antibiotic test plate (made by Iwashiya) and solidified. The agar pieces in which the bacteria were cultured were arranged at equal intervals on the activity assay plate, and placed in an incubator at 37 ° C. for 24 to 48 days.
Time left. The size of xylanase production resulted in large and small dissolved circles on the white turbid birchwood xylan plates. Strains with a large lysis circle were selected.

The productivity of several xylanase high-producing strains selected in this manner was examined by flask liquid culture. 60 ml of the production medium was dispensed into a 300 ml Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes. The above-mentioned bacteria were inoculated in this medium and cultured at 35 ° C. for 4 days in shaking culture (220 rpm, 20 cm). The culture was centrifuged, and the supernatant was examined for enzyme productivity by the following xylanase assay. 1% xylan (Fluka Biochemical 9558)
100 μl of the sample was added to 900 μl of the substrate solution of 8) 0.05 sodium citrate, pH 5.3, and reacted at 50 ° C. for 5 minutes. 1.5 ml of dinitrosalicylic acid (DN
S) The reagent was added to stop the reaction. Heated in boiling water for 5 minutes. After cooling in running water, the absorbance at 540 nm was measured, and the xylanase activity was calculated from the standard curve. By the above method, the mutant with the highest xylanase activity, St
reptomyces oli vaceoviridis strain E-86-N1-35 was selected. Streptomyces olivaceoviridis E-86-N1-35 has been deposited with the Institute of Biotechnology Industry as FERMP-15724.

[0041]

[Table 5] Table 5 Storage medium ──────────────────────────────────── Ingredient weight% ─── ───────────────────────────────── Sumicelco AA (crushed rice hulls) 2.0 Polypeptone 0.1 Yeast extract 0.05 KH ₂ PO ₄ 0.1 MgSO ₄ .7H ₂ O 0.05 agar 2.0 ─────────────────────────── ＰＨ pH 5.5

[0042]

[Table 6] Table 6 Production medium ──────────────────────────────────── Ingredient weight% ──ミ Sumicelco AA (crushed rice hulls) 6.0 Polypeptone 1.4 Yeast Extract 0.1 Corn steep liquor (powder) 0.5 KH ₂ PO ₄ 1.0 MgSO ₄ .7H ₂ O 0.05 ───────────────────── ＰＨ pH 5.5

Example 12 Streptomyces olivaceoviridis E-86-N1-35 was examined using a jar fermenter for culturing conditions, particularly pH. E-86-N1-35 was cultured in the flask for 4 days,
The seed mother was created. Put 1 L of production medium in a jar and add 121 L
Sterilized at ℃ for 20 minutes. Plant seeds and ventilate 1VV
M, stirring speed was 300 rpm for the first 24 hours, and then 5
The mixture was adjusted to 00 rpm, the culture temperature was set to 35 ° C., and the cells were cultured for 4 to 5 days, and the xylanase activity and the growth rate of the bacteria were measured over time. The pH of the culture was initially set at about 5.5,
Thereafter, from the time when the pH rose to a certain value, 1N hydrochloric acid was added dropwise to control the pH to a substantially constant value. The method for measuring xylanase activity is the same as for flask culture. The growth rate of the bacteria was determined by extracting and measuring the nucleic acid (NA) of the cells. The results are shown in FIG. Fig. 2 (a) shows the pH during culture.
It is a figure showing a change. As shown in FIG. 2 (a), the pH was kept almost constant from the time when it reached about 5.5, 6.0, 6.5, 7.0 and 7.5, respectively. FIG. 2 (b) shows the change in activity at each pH, and FIG. 2 (c) shows the growth rate of the bacteria with respect to the culture time. When the pH was adjusted to 6.5 to 7.0, a decrease in activity was suppressed. The activity of the mutant strain E-86-N1-35 was about 2 against the activity (10 U / ml) of the parent strain E-86.
A 0-fold increase in activity (200 U / ml) was obtained.

Example 13 A 30 L jar fermenter (manufactured by Marubishi Bio-Engineering Co.) was charged with 15 L of an enzyme production medium, and after sterilization,
Seed mother E-86N1-35 cultured at 35 ° C for 3 days (300ml)
Was inoculated. Temperature 35 ° C, ventilation rate 1 VVM, stirring speed 2
Culture was performed at 00 rpm. The pH was initially 5.5 and increased with the lapse of culture, but was adjusted so as not to rise above 6.5. After culturing for 60 hours, the culture solution was subjected to solid-liquid separation with a pressure filter (manufactured by Advantech) pre-coated with celite, and the crude enzyme solution (10 L, 18
0 U / ml). Elute the crude enzyme solution with an evaporator for about 1
It concentrated under reduced pressure to L. The concentrated solution was dialyzed against running water for 24 to 48 hours using a cellulose tube. At this time, the dialysis tube was replaced every four hours. Dialysate (2.7
L) was passed through a DEAE-cellulose column (500 mL) previously equilibrated with a 20 mM acetate buffer (pH 5.5) to remove the dye. The xylanase fraction (3100 ml) passed through the DEAE column was concentrated (610 ml) using an evaporator, and then dialyzed against distilled water for 24 hours. After dialysis, acetic acid was added to adjust the pH to 4.0 so that the final concentration became 20 mM, and then SP-Toyopearl 650M previously equilibrated with a 20 mM acetate buffer (pH 4.0).
The column (250 ml) was loaded. After the column was washed with the same buffer, xylanase was eluted by applying a concentration gradient of 0 to 0.5 M sodium chloride. The fraction having the xylanase activity was dialyzed and again subjected to SP-Toyopearl column chromatography to obtain a xylanase preparation (15%).
0 mL, 240,000 U).

This xylanase has the following characteristics. (1) Action and substrate specificity It acts on xylan and xylo-oligosaccharides, and their β-1,
Hydrolysis of the 4-xylosidic bond produces xylose and xylooligosaccharides. (2) Titer measurement method As a substrate, 1.0% Birchwood xylan (Fluka Bioch
emie) was suspended in 0.05M citrate buffer at pH 5.3 and uniformly dispersed by sonication.
to 0.1 l of a sample containing xylanase,
At 5 ° C. for 5 minutes, and the generated reducing sugar was quantified by a method using 3,5-dinitrosalicylic acid.
1 unit (unit) of enzyme that produces 1 xylose
And (3) Optimum pH and stable pH range The optimum pH range is 5.5-7.5, and the stable pH range is 4.5.
−9.5 (in the case of treatment at 50 ° C. for 60 minutes).

(4) Range of suitable temperature for operation In the case of treatment at pH 5.5 for 60 minutes, it is stable at 55 ° C or less. (5) Purification method Streptomyces olivaceoviridis strain E-86-N1-35 is removed from the culture broth cultured in a medium containing rice hulls by filtration to obtain a clear solution. Concentrate and dialyze this purified liquid,
Pass through DEAE-cellulose equilibrated with 0.02 M acetate buffer, further adsorb to SP-Toyopearl,
Elute and purify with a concentration gradient of 0.5M sodium chloride. (6) Molecular weight The molecular weight of the product obtained by the purification method of (5) above is S
It is 25,500 by DS-PAGE. (7) Isoelectric point The isoelectric point of the product obtained by the purification method of (5) is 9.2. (8) N-terminal The N-terminal has the sequence of SEQ ID NO: 1.

The present xylanase was prepared using St.
xylanase xynA, xyn of reptomyces lividans
Table 7 shows a comparison with B and xynC.

[0048]

Table 7 Table 7 Ｍ MW Km Vmax Km Vmax pI (Oat spelt xylan) (Birchwood xylan) ──────────────────────────────────── xynA 43,000 2.6 5570 1.1 616 5.2 xynB 31,000 3.7 1960 1.8 921 8.4 xynC 22,000 4.2 650 4.1 3022> 10.25 Example 13 25,500 4.21 4901 2.52 2264 9.2 ────────────────────────── ──────────

The comparison of the N-terminal amino acid sequences is as follows. E-86 (25Kd) and E-86
(45 kD) is a xylanase produced by the parent strain. Example 13 AVIITNQQGGNNGFYYY-E-86 (25 kD) ATVITTNQTGTNNGFYYSFW- xynC AATTITTNQTGTD * GMYYSFW- xynB * DTVVTTNQEGTNNNGIYYSFW-E-86 (45 kD) AESTLGAAAAQSGRYxTAAAGSGKLAGASGLAGSLGAAGGSGASGLGSGAGSGLAGSAGAAGGSGAAGGTG

Example 14 1 ml of xylan solubilizing solution (50 mg as xylose)
1 ml (0.5 to 5,000 units) of xylanase of Streptomyces olivaceoviridis E-86 was added to the mixture, the pH was adjusted to 5.5, and the mixture was reacted at 50 ° C. for 30 minutes. The oligosaccharide composition in the reaction solution was analyzed with a DX-500 sugar analysis system. Table 8 shows the ratio of the produced xylooligosaccharide to xylose. Increasing xylanase with respect to the xylan lysate increased xylo-oligosaccharides with a low degree of polymerization, and the ratio of xylobiose was maximized at 5,000 xylanase units. Thus, by changing the amount of xylanase added to the xylan solubilized solution, the composition ratio of xylo-oligosaccharide can be arbitrarily changed.

[0051]

[Table 8] Table 8 Enzymatic degradation of xylan soluble solution (investigation of enzyme concentration) ──────────────────────────────── ──── Oligosaccharide formation ratio ────────────────────────── Xylanase (unit amount) X1 X2 X3 X4 X5 ────── ０ 0 1 0.55 0.26 0.22 0.12 0.5 1 0. 56 0.26 0.22 0.15 1 0.58 0.37 0.34 0.18 50 1 0.84 0.84 0.39 0 500 1 1.54 0.6 00 5000 5000 1 1. 570 000 ──────────────────────────────────── Note) X1 to X5 are xylose polymerization degrees Represents That is, X1 is xylose, X2 is a dimer of xylose (xylobiose), X3 is a trimer, and so on.

Example 15 A xylan solubilized solution was concentrated under reduced pressure. After adjusting the pH of the concentrate to 5.5, the concentrate (2.4 as xylose).
9 g) 5 m of crude enzyme solution containing 25,600 units of xylanase of Streptomyces olivaceoviridis E-86 in 50 ml
1 was added and reacted at 50 ° C. for 4 hours. Reaction solution at 80 ° C
After heating, 1 g of activated carbon (Takecol) was added and stirred for 20 minutes. The activated carbon-treated solution was filtered through Nutsche precoated with Hyflo Super Cell. Next, in order to remove organic acids and phenolic compounds such as acetic acid generated by the explosion and enzymes and inorganic salts at the time of the enzyme reaction, the obtained filtrate is subjected to a strongly acidic cation exchange resin and a strongly basic anion exchange resin. Was passed through a mixed bed column (3 cm × 16 cm). The same operation 2
Batched. By concentrating two batches of the liquid passing through the column under reduced pressure, Brix35 xylo-oligosaccharide syrup 1
2.8 g were obtained. FIG. 3 shows the results of sugar analysis of this syrup. A xylooligosaccharide containing xylobiose as a main component was obtained.

Example 16 Xylane solubilized solution (10 ml, 90 ml as xylose)
To g), 5.8 ml (47,000 units) of a xylanase solution obtained from Streptomyces olivaceoviridis E-86-N1-35 was added, and the mixture was adjusted to pH 5.5 and reacted at 50 ° C. for 4 hours. As a result of analyzing the generated oligosaccharide composition in the reaction solution, X1: X2 = 1: 1.8, and a xylo-oligosaccharide having almost the same composition as that of Example 9 was obtained. From this result, E-8
The xylanase of 6-N1-35 can be presumed to be qualitatively the same as that of the parent strain.

[0054]

As is clear from the above examples, the method of the present invention makes it possible to easily obtain solubilized saccharides from agricultural and marine products in high yield. Furthermore, it has become possible to efficiently obtain monosaccharides and oligosaccharides from oligosaccharides and solubilized polysaccharides. The method of the present invention can be applied to woody or structural polysaccharides or all agricultural and marine products containing them. For example,
From rice straw, straw, buckwheat husk, bagasse, apple juice cake, seaweed, etc., neutral sugars such as glucose, arabinose, rhamnose, galactose, mannose, uronic acids such as glucuronic acid and galacturonic acid, and oligosaccharides containing these monosaccharides It can also be used for polysaccharide production.

[0055]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Ala Val Ile Ile Thr Asn Gln Gln Gly Gly Asn Asn Gly Phe Tyr 1 5 10 15 Tyr Tyr

[Brief description of the drawings]

FIG. 1 is a view showing the results of analyzing the oligosaccharide composition of a supernatant obtained by exploding rice hulls.

FIG. 2 is a diagram showing changes in pH, activity and growth rate of bacteria during culture of flasks of E-86-N1-35 with respect to culture time, (a) showing changes in pH, and (b) showing changes in pH. Changes in activity,
(C) Changes in the growth rate of bacteria.

FIG. 3 is a diagram showing a sugar analysis result of the sugar syrup obtained in Example 15.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 19/14 C12P 19/14 A // (C12N 1/20 C12R 1: 465) (72) Inventor Norio Shibamoto Akita, Akita 4-26 Sunaya, Shinyacho Inside Akita Prefectural Food Research Institute (72) Inventor Shunzo Inoue 25-1 Yamazaki, Yamagata, Akita-shi, Akita

Claims (9)

[Claims] 1. A method for producing a monosaccharide, oligosaccharide and / or solubilized polysaccharide, comprising a step of exploding or steaming a wood-based polysaccharide or a structural polysaccharide or an agricultural or marine product containing them. 2. 10 ^-1 to 4 × 1 during explosion or steaming treatment.
2. The method according to claim 1, wherein an acid solution or a buffer having a hydrogen ion concentration of 0 ^-6 M is added. 3. The method according to claim 1, wherein the agricultural or marine product is rice husk and the polysaccharide is xylan. 4. A method for producing a monosaccharide or oligosaccharide, comprising treating the oligosaccharide or the solubilized polysaccharide obtained by the production method according to claim 1, 2 or 3 with its glycolytic enzyme. 5. The method according to claim 5, wherein the glycolytic enzyme is Streptomyces olivaceovir.
idis E-86 or Streptomyces olivaceoviridis E-86-N
The production method according to claim 4, which is an enzyme derived from 1-35. (6) Streptomyces olivaceoviridis E-86-N1
-35. (7) Streptomyces olivaceoviridis E-86-N1
Xylanase from -35. 8. A xylanase having a molecular weight of about 25.5 kDa and an N-terminal having the sequence of SEQ ID NO: 1. 9. A xylan solubilized solution obtained by blasting or steaming a xylan-containing substance is used to prepare Streptomyces olivaceoviridis E
-86 or Streptomyces olivaceoviridis E-86-N1-35
Alternatively, a method for producing a xylo-oligosaccharide, comprising treating with a xylanase derived therefrom.