JPH0121957B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for producing heat-stable xylanase using bacteria of the genus Acremonium.

[Prior art]

Xylanase acts on xylan, which is part of the hemicellulose that makes up plant cell walls, and breaks it down into xylose, which is a constituent monosaccharide, or xylooligosaccharide, which is a polymer of xylose. It can be used for various purposes.
That is, by acting on xylan in plant biomass, it can be used to improve the edibility of feed, and the extractability of coffee extract from coffee beans. In addition, in recent years, the development of methods for converting biomass into energy or chemical raw materials as an alternative resource to petroleum has been progressing, and enzymatic saccharification of biomass using cellulase is being considered. Not only glucose but also xylose can be recycled. Furthermore, by decomposing the hemicellulose matrix surrounding cellulose with xylanase, cellulase becomes more accessible to cellulose, and the effect of increasing the degradability of cellulose can be expected.

On the other hand, when using such xylanase,
The following two properties are required for xylanase. That is, it must have an optimum operating temperature as high as possible in order to prevent contamination by bacteria during the decomposition reaction, and it must have an excellent ability to saccharify xylan.

Conventionally, Humicola lanuginosa (J. Ferm.
Technol., Vol. 62, p63-69, 1984)
Terrestris (Enzyme Microb. Technol., 6
Vol., p175-180, 1984). However, the optimal operating temperature for these xylanases in short-time reactions is around 65°C, and the stable operating temperature in long-term reactions is estimated to be lower. There are many problems such as incomplete saccharification.

〔the purpose〕

Therefore, the present inventors searched widely in nature for microorganisms that decompose plant biomass, and found that a strain of Acremonium, a type of mesophilic fungus, produced xylanase in culture, which has an optimal action temperature of 80°C. We discovered that mesophilic filamentous fungi produce highly thermostable xylanase for the first time, and that this xylanase is a new xylanase with extremely high saccharification ability and strong xylose-producing ability at high temperatures. The present invention has been completed by recognizing the above.

〔composition〕

Specifically, the present invention relates to a method for producing heat-stable xylanase, which comprises culturing Acremonium bacteria that produce heat-stable xylanase, and collecting heat-stable xylanase from the culture.

The content of the present invention will be explained in more detail below.
In the present invention, Acremonium celluloliticus (Acremonium celluloliticus) is used as an exemplary strain.
cellulolyticus) is effectively utilized. This bacterium is a bacterium that produces a cellulase complex characterized by having a strong ability to saccharify cellulose by containing heat-stable xylanase and a significant amount of β-glucosidase.

Next, the mycological properties of the thermostable xylanase-producing bacteria used in the present invention are as follows.

Growth: Growth is fast on malt extract agar at 30℃7
It reaches a diameter of 70mm in days. The colony is initially white and later becomes slightly yellowish. The aerial hyphae are loosely swollen, wool-like, and sometimes form rope-like hyphal bundles.
In the later stages of cultivation, the underside of the colony becomes pinkish-brown to reddish-brown. Almost the same growth was observed on Tuapetsk agar, but the growth of aerial mycelia was smaller. Growth PH
The range is 3.5 to 6.0, the optimum pH is around 4, and the growing temperature range is
The optimum growth temperature is 30°C between 15°C and 43°C.

Morphology: The diameter of the hyphae is 0.5 to 2.5 μm, colorless, and septa are observed in the hyphae. In addition, the hyphal surface is smooth.

Conidia and the ability to form conidia were extremely unstable and were easily lost by subculturing on Czapetsk agar and malt extract agar media. When observed during isolation, the conidiophores protrude from the sides of the aerial hyphae and are colorless. Conidia are subglobose, smooth, colorless, and have very loose chains and are easily dispersed. Regarding the above mycological properties, W. Gams' description of Cephalosporium [Cephalosporium Alteige Schimmelpilge]
artige Schimmelpilge, G. Fisher (Eds.), page 84, 1971] and Dickinson (CH
Dickinson's research [Mycological Paper, Vol. 115, p. 10, 1968
As a result of referring to [2013], we concluded that it is appropriate to consider this fungus to be a filamentous fungus closely related to the genus Acremonium. In addition, since no strong cellulase-producing bacteria have been known in the Acremonium genus, and because the strain of the present invention produces a strong and characteristic cellulase at the same time as xylanase, the bacteria Acremonium It was named Acremonium cellulolyticus TN. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FFRMBP-685.

In order to produce the heat-stable xylanase by Acremonium bacteria of the present invention, xylan,
The carbon source is plant biomass such as xyloglucan, cellulose, Avicel, bran rice straw, and bagasse, and the nitrogen source is organic or inorganic nitrogen sources such as nitrates, ammonium salts, peptone, yeast extract, and a small amount of metal salts. The cells are cultured aerobically at 20 to 40°C for about 2 to 15 days using a liquid or solid medium containing the following. Thermostable xylanase is an enzyme produced outside the bacterial cell, so in the case of a liquid medium, the supernatant after filtration or centrifugation is used after cultivation, and in the case of solid culture, it is added with water or an appropriate inorganic salt after cultivation. The extracted solution can be used as a crude enzyme solution. The crude enzyme solution may be used as it is, but a crude enzyme powder can be obtained by a known method such as ammonium sulfate salting out method or acetone precipitation method. Furthermore, taking advantage of the heat resistance of this enzyme, by heat treatment at PH4.9 and 65℃ for 2 hours, impure proteins can be denatured and precipitated and removed without impairing xylanase activity. An enzyme solution with only activity can be easily prepared. The heat-stable xylanase preparation of the present invention thus obtained has the following properties.

(1) Multicomponent nature of xylanase Thermostable xylanase is separated into at least three components by disk electrophoresis, each of which is distinguished by its molecular weight and isoelectric point. xylanase A
has a molecular weight of approximately 51,000 and an isoelectric point of 5.05, and similarly B
is about 46000, 4.57, C is about 36000, 3.55,
Xylanase is made up of a composite of these components.

(2) Action The xylanase complex acts on soluble and insoluble xylan contained in plant biomass to produce xylose and xylooligosaccharides. In addition to the above, oligosaccharides consisting of arabinose and xylose are also produced from arabinoxylan containing arabinose. This enzyme complex also acts effectively on xylobiose and decomposes it into xylose. The final product therefore consists primarily of xylose.

(3) Action PH and optimal action PH The action PH range of this enzyme complex is PH3-6, as shown in Figure 1a, and the optimal action PH is approximately 5.
was recognized.

(4) Stable PH range The stable PH range when left for 24 hours at 25°C under a citric acid-phosphate buffer was about 2.5-8.5, as shown in Figure 1c.

(5) Action temperature range and optimal temperature range All components of the present enzyme complex can work at high temperatures up to about 90°C, but as shown in Figure 1b,
The optimal working temperature is approximately 80°C when reacted for 10 minutes under 0.25% xylan and 0.05M acetate buffer (PH4.9).
℃ was observed.

(6) Thermostability When this enzyme complex was heat-treated at each temperature for 10 minutes under 0.1M acetate buffer (PH4.9), it showed almost no loss up to 70°C, as shown in Figure 1d. About 60% of the activity was lost after heating at 80°C for 10 hours, and about 90% when heated at 85°C for 10 minutes.

(7) Inhibitor Among various heavy metal ions, it was strongly inhibited by 1mM or more of mercury ion and copper ion.

(8) Purification method This enzyme complex is produced by heating the culture filtrate at 65°C for 2 hours, denaturing the impure proteins contained therein, removing the resulting precipitate by centrifugation, and then applying it to DEAE Sepharose and chromatofocusing columns. By chromatography, each component was able to be separated and purified to homogeneity by disk electrophoresis.

(9) Activity measurement method Add an appropriate amount of enzyme to 0.5 ml of a substrate suspension (PH4.9) in which 0.5% xylan (containing about 9% arabinose) prepared from rice straw is suspended in 0.1M acetate buffer. and make the total volume to 1.0 ml with distilled water, and make 60
The reaction was carried out at ℃. The reducing sugar produced was measured by the Somogyi-Nelson method.

Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μg of xylose per minute was defined as 1 unit.

〔effect〕

As described above, the thermostable xylanase of the present invention has an optimal action temperature around 80°C, and its enzymatic properties are different from those of previously known enzymes, as the main product from xylan is xylose. It's much better than. These characteristics of the enzyme of the present invention have brought about significant technological progress in the industrial use of xylanase.

Next, examples of the present invention will be shown.

Example 1 Cellulose 4%, peptone 1%, potassium nitrate
0.6%, potassium chloride 0.16%, sodium chloride 0.16
%, magnesium sulfate 0.12%, monopotassium phosphate
200ml of 20ml of medium (PH4.0) containing 1.20% and 0.001% each of zinc sulfate, manganese sulfate, and copper sulfate
Acremonium celluloliticus TN (FERM BP) was sterilized using a conventional method.
-685) and cultured with aeration at 30°C for 6 days.
After culturing, the cells were sterilized using a centrifuge, and the xylanase activity of the resulting supernatant was measured to be 1050 units per ml of culture solution.

Example 2 In Example 1, xyloglucan (Dainippon Pharmaceutical Glyroid 3S) 2 was used instead of cellulose.
Acremonium celluloliticus (FERM P-6867) was inoculated into a medium supplemented with 5% and cultured at 30° C. for 8 days with aeration. As a result of measuring the xylanase activity of the supernatant liquid centrifuged after culture, culture liquid 1
It was 2400 units per ml.

Adjust the pH of this culture supernatant to 4.9, and heat at 65℃2.
The precipitate formed after the heat treatment for several hours was removed by centrifugation to obtain an enzyme preparation having only xylanase activity. The recovery rate of xylanase was 92.4%.

Example 3 The xylanase enzyme preparation obtained in Example 2 was added to a 10% xylanan suspension at a final concentration of 300 units/ml, and saccharified at 65°C for 48 hours. The amount of reducing sugar in a portion of the obtained sugar solution was measured and found to be 60 mg/ml as xylose. This value was converted to the value obtained by decomposing the same concentration of xylan with sulfuric acid (68mg/
ml), the decomposition rate was 88.2%. Furthermore, the decomposition products at this time were identified by paper chromatography and were found to consist mainly of xylose, with a small amount of xylobiose.

[Brief explanation of drawings]

Figure 1 shows Acremonium TN (FERM BP-
(a) optimal action PH, (b) optimal action temperature, (c) PH stability, and (d) thermostability of the thermostable xylanase produced by 685).

Claims (1)

[Claims] 1. A method for producing heat-stable xylanase, which comprises culturing Acremonium bacteria that produces heat-stable xylanase, and collecting heat-stable xylanase from the culture.