JPS61242592A - Production of decomposed xylan - Google Patents

Abstract

PURPOSE:To produce plural kinds of xylooligosaccharides at a desired ratios, by controlling the concentration of xylanase in a hydrolysis medium in the hydrolysis of xylan with a specific xylanase. CONSTITUTION:The ratio of produced xylobiose, xylotriose and xylotetraose is controlled to a desired ratio by controlling the concentration of xylanase in a hydrolysis medium in the hydrolysis of xylan with xylanase originated from bacterium belonging to Bacillus genus, preferably Bacillus pumilus SAM 0044 (FERM-P 8175).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field] The present invention relates to a method for producing a mixture of xylan decomposition products containing xylan decomposition products in a desired proportion.

[Conventional technology]

Conventionally, xylose has been produced from xylan by a hydrolysis method.

Recently, various oligosaccharides have been used as sweeteners, bulking agents, excipients,
It is thought that it can be widely used in the food and pharmaceutical industries as an inclusion agent, humectant, or thickener, and there is a demand for the development of various oligosaccharide production techniques suitable for these uses.

The method for producing xylo-oligosaccharides by partial hydrolysis requires complicated purification steps and has a low yield, making it unsuitable for industrial production. Therefore, xylobiose (X2), xylotriose (X3)
There is a need for the development of xylanases that can specifically produce xylo-oligo glues such as , xylotetraose (X4), or xylo-oligosaccharide mixtures containing them in desired ratios.

As xylanase, the enzyme produced by Bacillus subtilis (Fermentation Engineering Magazine ↓, 181-186.1963
), Streptomyces
sp,) enzyme produced by the strain (Journal of the Society of Agricultural Chemistry ↓3.
t45-1s3. t9e9) - an enzyme produced by Aspergillus niger [Biotech.

Letter) 3, 345-350.1981)
However, there is little research on methods for specifically producing xylo-oligosaccharides using xylanase. An example of the production of xylooligosaccharides is Ilumicola lanuginosa.
Production of xylobiose and xylotriose by xylanase derived from [Journal of Fermentation Technology (J, Ferment, Tech.
62415-420, 1984) and the production of xylose and xylobiose using xylanase derived from the genus Streptomyces (Journal of the Agricultural Chemistry Society, 14)
5-153.1969). however,
There is no known method for primarily producing xylotriose and xylotetraose.

[Problem that the invention seeks to solve]

Therefore, the present invention seeks to provide a method for producing a xylan decomposition product containing xylobiose, xylotriose, xylotetraose, etc. in a desired ratio.

[Failure to solve the problem]

The above object is achieved by a method in which xylan is hydrolyzed by xylanase, which acts on xylan under specific conditions to produce xylobiose, xylotriose, xylotetraose, etc. in desired ratios.

The present inventors conducted a search for microorganisms in soil in search of microorganisms with a strong ability to produce oligosaccharides from xylan, and as a result, the microorganism S A M2 was identified as Bacillus pumilus.
It was confirmed that the O2S strain produces a significant amount of xylanase, which efficiently produces xylo-oligosaccharides such as xylobiose, xylotriose, and xylotetraose, outside the bacterial cells.

Furthermore, as a result of examining the conditions for the decomposition of xylan by the xylanase produced by the S A M2O2S strain, we recognized that it is possible to create different xylooligosaccharides with different sugar compositions by appropriately setting the conditions. completed.

The properties of the xylanase produced by Pacillus pumilus and used in the present invention are described below.

(1) Action: This enzyme acts on xylan and mainly produces xylo-oligosaccharides such as xylobiose, xylotriose, and xylothy-1-laose. Furthermore, by changing the action conditions, the composition ratio of the sugar produced can be changed.

For example, xylobiose is 10-80%, xylotriose is 10-60%, and xylotetraose is 0-40%.
It is possible to change the composition ratio within the range of .

(2) Working pH range and optimal working pH: It works in the pH range of 5 to 9, and the optimal pH is about 6 (1% xylan,
Reaction at 50°C for 10 minutes) (Figure 1).

(3) pH stability: Stable in the pH range of 5.5 to 8.0 (
After standing in a 0.1M buffer for 3 hours (at room temperature), the remaining activity was measured (Figure 3).

(4) Working temperature range and optimum working temperature: works up to about 70°C, and the optimum working temperature is 45-55°C (1% xylan, p
H6, 0, 10 minute reaction) (Figure 2).

(5) Thermal stability: Residual activity was measured after heat treatment at pH 6 and 0 for 10 minutes. As a result, 30% at 50℃, 6
90% at 0°C and almost completely inactivated at 70°C (Figure 4)
.

(6) Activity measurement method = 1% (W/V) xylan (larc
50mM phosphate buffer (pH 6) containing
,0) 0.48m [to, 0.02mj! Add an appropriately diluted enzyme solution and conduct the reaction at 50°C for 1\0 minutes.
The resulting reducing sugar is colorimetrically determined using the DNS method. One unit is the amount of enzyme that produces a reducing power equivalent to 1 μmol of xylose per minute.

Next, the mycological properties of the S A M2O2S strain, which is a representative xylanase producing bacterium used in the method of this invention, will be described.

(A) Morphology (1) The cell size is a rod with a size of approximately 0.5 μm x 2.0 μm.

(2) There is no cell pleomorphism.

(3) It is motile, and the flagellum is peritrichous.

(4) Spore pattern, 1-A $6 VI: There is.

(5) Durham staining is positive.

(B) Growth status of L- in the medium (1) Broth liquid medium The bacterial body is yellowish white and forms a film on the surface. Nurture.

(2) Meat juice agar plate medium Growth is vigorous and the bacterial cells are yellowish white.

The diameter of the colony is approximately 2 mm, and the surface is Uneven, lack luster, and wavy edges exhibits.

(3) Meat juice agar slant medium It shows the same growth condition as (2) and spreads. It grows in a shape.

(4) Meat juice gelatin puncture medium Gelatin? It doesn't get dark at night.

(5) Ridmus milk The 1-mass dye turns red and solidifies.

(C) Physiological properties (1) Nitrate reduction - (2) vP test specialist (3) MR test + (4) OF test 0 (5) Indole production - (6) Hydrogen sulfide production - (7) Starch - (8) Decomposition of casein → - (9) Decomposition of hippuric acid (10) Utilization of propionic acid - (11) Growth in naerobic Agar - (
12) Resistance to 7% NaC] →-(13)
Production of catalase (14) Production of oxidase (15) Growth at 50°C →= (16) Attitude towards oxygen Aerobic (D) Production of acid and gas from sugar Production of acid Production of gas (1) L- Arabinose 10-(2) r
l-xylose 10-(3) D-glyose 10-(4) D-mannose
+ - (5) D-fructose →-- (6) D-galactose ± - (7) Maltose ± - (8) Zasocalose
10 - (9) Raketose - (10) ) Levalose 10 - (1
1) n-sorbitol − (12) D-mannitol + −(13
) Inosit - (14) Glycerin + - (15) Starch - (E) GC content of DNA The GC content of the DNA of this bacterium was 41.7% [Measurements were carried out by J. Tamaoka and Komagata;
1984. DN by reverse phase high performance liquid chromatography
Determination of A base composition
DNA base composition b
y reversed-phase high-per
formance 1i-quid chromato
FBMS Microbiology Letters (Microbiology I, etters)
25:125-128].

(F) Identification of the molecular weight of intracellular quinone Wood fungi contain menaquinone 7 (MK-7) [Measurements were made by Yamada h1, Miyuki Kuraishi, 1982, Ubiquinone and Menaquinone Moki, edited by Kazuo Komagata, Experiments on Chemical Classification of Microorganisms. Law J
P 143-155. Gakkai Publishing Center, Tokyo) ±].

According to the above mycological properties, the strain SAM0045 of the present invention
The taxonomic status of Hershey's Manual of Terminative Hateriology (Bergey'
sManual of I) eterminative
According to the 8th edition of Bacteri-ology, this strain is a Durham-positive bacterium, forms spores, is motile, and has a GC content of 41.7%. ) was identified as a bacterium belonging to the genus.

In addition, Gor
don R, E, W, C, Haynes and C, I (,
-N, Pang.

1973, The Genus Hasils, United Nations Stake Department of Agriculture
Hand Hook (The Genus Bacillus
, Un-4ted 5tates Departmen
to of Agriculture Handb-oo
k) No. 427, this bacterial strain B, subtilis ([
1゜5ubtilis), B, pumillus (B, pum
i 1us), B, licheniformis (B, ]i, ch
It turned out to be a species closely related to S. eniformis.

As a result of further detailed investigation, we found that there was no ability to reduce nitrate, no ability to decompose starch, and there was no ability to reduce starch.
This strain is similar to Bacillus pumilus because it does not grow in robic Agar and decomposes hippuric acid.
It was identified as P. llus pumilus).

Next, the culture conditions for producing xylanase using this bacterium will be described. As the culture method, a normal bacterial culture method can be used.

First, as a carbon source, xylan, malt saccharide of beer or whiskey, wheat bran, or other W! Use either alone or in combination. As nitrogen sources, inorganic compounds such as ammonium sulfate, ammonium nitrate, urea, etc., and organic nitrogen-containing substances such as yeast extract,
Peptone, malt extract, corn steep liquor, skim milk, various amino acids, etc. are used alone or in combination.

A suitable culture temperature is 20 to 35°C. Also, the first p
H can be used in a wide range from 5 to 11, but in particular,
Alkaline conditions are suitable. The number of days for culturing is usually 1 to 2 days. Although either solid culture or liquid culture is possible, liquid culture under aerobic conditions is preferred.

After completion of the culture, xylanase can be collected using known techniques. An example is shown below.

First, bacterial cells and solids in the culture solution are removed by centrifugation to obtain a supernatant. It is also possible to use this supernatant as crude enzyme. This supernatant is precipitated with about 40% ammonium sulfate. The precipitate is dialyzed overnight, passed through a LJ,:T)EAE cellulose column equilibrated with 20mM phosphate buffer (pH 7,0), and the flow-through fraction is collected. next,
This passage section was filled with 50mM phosphate buffer (pH 6.5).
After adsorption on CM cellulose equilibrated with , oxygen was eluted by changing the salt concentration from 0.1 to 0.7M,
Collect the xylanase activity fraction. Furthermore, in order to improve the enzyme purity, gel filtration is performed using a Sephadex G-100 column to obtain the final sample.

The enzyme reaction for producing the xylan hydrolyzate mixture by the method of this invention is carried out at pH 5.5-8.0.30°C.
It is carried out at a temperature of -60° C. for 3 to 48 hours, particularly preferably for 3 to 15 hours. The xylan concentration in the reaction solution is usually 1 to 40%, particularly preferably 1 to 20%. Preferably, a buffer, for example a phosphate buffer, is used as the reaction medium.

Even if the reaction pH, reaction temperature, and reaction time were changed within the above range, the ratio of xylooligosaccharide in the product hardly changed. Furthermore, as the initial concentration of xylan, the substrate, was increased, the yield of xylooligosaccharides tended to decrease, but in this case as well, the production ratio of xylooligosaccharides did not change.

On the other hand, changing the enzyme concentration significantly changed the composition of the sugar produced. That is, xylobiose (X
2) is 10-80%, xylotriose (X3) is 10%
~60%, and xylotetraose (X4) is 0-4
It showed a change of 0%. Xylose (X, ), xylobiose (X2), xylotriose (X3), and xylotetraose (X4) when the initial xylan concentration was 5% and various xylanase concentrations were used to hydrolyze
) is shown in Figure 6 and the table below.

The reaction can be stopped by heating the reaction solution to inactivate the xylanase. Next, the hydrolysis product is separated and collected from the reaction solution according to a conventional method. For example, a heat-treated reaction solution can be filtered to obtain a transparent reaction solution, and this can be concentrated to obtain a product concentrate or syrup.

Moreover, a powder product can be obtained by freeze-drying the reaction solution or its concentrate.

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

U-Kari 1-0 Gunpo version p-adapted xylan 1%: yeast extract 0.3%, polypeptone 0.
5%, K211PO4,0,1%, and Mg5O,l
・7■ Medium 27 consisting of 200.1%! into a 5p jar fermenter, and add Bacillus pumilus (Feikoken Bacteria No. 8175) to 1 Vvll, 300rp.
The cells were cultured for 48 hours at m. After culturing, the culture solution was centrifuged to obtain supernatant synovial fluid. This -superior synovial fluid is 585 units/ml
It contained xylanase.

” Ue 1. 0.01ml of the culture supernatant obtained in Anti-epidemic Example 1 was added to 10ml of 50mM phosphate buffer at pH 6.0 containing 4% xylan.
7! and reacted at 45°C for 10 hours.

The produced sugar was collected by liquid chromatography (column Co5m0-
sil ・5NH2, eluent acetonitrile 70%:
As a result of analysis using 30% water and a detector 5hodexRI-3E51), it was found that xylobiose was 17%, xylotriose was 43%, and xylotetraose was 37%, with xylotriose and xylotetraose as the main components (80% in total). A product was obtained (Figure 5a).

Example 3. 0.5g of 0.1rrl of the culture supernatant obtained in Soniwa Example 1
50mM phosphate buffer (pH 6.0) containing xylan
) and reacted at 45° C. for 12 hours. As a result of analysis of the produced sugars, a product containing 41% xylobiose and 53% xylotriose, with xylobiose and xylotriose as the main components (94% in total) was obtained (Figure 5b).

Example 4. The culture supernatant obtained in Pox Example 1 was precipitated with 40% ammonium sulfate. The crude enzyme solution obtained by dialyzing this precipitate was
Diluted with 50mM phosphate buffer (pH 6.5)
00 units/m7! Closed. To 10 ml of this enzyme solution, add 0.
5 g of xylan was added and the reaction was carried out at 45°C for 15 hours. As a result of analyzing the sugar produced, xylose was 13%.
, 76% xylobiose, 11% xylotriose, and a product containing xylobiose as the main component was obtained (Figure 5C). In addition, the yield of xylobiose at this time was 3
It was 4%.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between pH and activity of the xylanase used in the present invention, Figure 2 is a graph showing the relationship between temperature and activity of the xylanase, and Figure 3 is a graph showing the pH stability of the enzyme. FIG. 4 is a graph showing the temperature stability of the enzyme, and FIGS. 5 and 6 are graphs showing the temperature stability of the enzyme at various enzyme concentrations. It shows the ratio of sugar produced. ('10) ('/,) Figure 2 ('/,) Figure 4 Figure 5

Claims (1)

[Claims] 1. A method for obtaining a xylan decomposition product mixture by hydrolyzing xylan with xylanase derived from a bacterium belonging to the genus Bacillus, in which xylobiose and xylotriose are obtained by adjusting the xylanase concentration in the hydrolysis medium. , and xylotetraose, a mixture containing multiple types of xylooligosaccharides in a desired ratio. 2. The Bacillus bacterium is Bacillus pumilus (Bacillus pumilus).
illus Pumilus) SAM0045 (Feikoken Bibori No. 8175).