JPS61162181A - Production of thermostable xylanase - Google Patents

Abstract

PURPOSE:To collect thermostable xylanase from a culture mixture, by cultivating a microorganism belonging to the genus Acremonium, capable of producing thermostable xylanase. CONSTITUTION:A microorganism such as Acromonium cellulolyticus TN (FERMBP-685), etc. belonging to the genus Acremonium, capable of producing thermostable xylanase, is cultivated. It is cultivated aerobically in a medium containing a carbon source such as xylan, cellulose, bagasse, etc., a nitrogen source such as a nitrate, etc. and a metallic salt. A supernatant liquid or an extracted solution obtained after the cultivation is used as a crude enzyme solution. Crude enzyme powder can be obtained by acetone precipitation method, etc., impure protein is denatured and precipitated by heat treatment and removed, to give an enzyme solution having only xylanase activity.

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, a constituent monosaccharide, or xylooligosaccharide, a polymer of xylose. Available for 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 an enzymatic saccharification method of biomass using heptylulase is being considered. By doing this, not only glucose (; It can also be expected to have the effect of increasing the degradability of cellulose.

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, xylanases with optimal action temperatures in the high temperature range include Humicola lanuginosa (J, Fer■, Tschn
ol,, vol. 62 p63-69.1984) Cherabira Telestri X (Enzyi+e Microb
, τechno1. , Volume 6, p175-180゜Md4
) etc. are known. However, the optimal operating temperature of these xylanases in short-term reactions is around y-←, and the stable operating temperature in long-term reactions is estimated to be lower, and the reaction products are more likely to contain xylo-oligomers than xylose. Problems remain, such as high sugar content and incomplete saccharification.

〔the purpose〕

Therefore, the present inventors conducted a wide search 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 xylanase that is highly thermostable for the first time, and that this xylanase is a highly saccharifying xylanase with a strong xylose-producing ability at high temperatures. Having recognized this fact, the present invention has been completed.

〔composition〕

In other words, 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 contents will be explained in more detail.

In 1JQfil, examples of strains include Acremo, PigliT5 cellulolyticus (^creson).
ius csllulol-:voice tlJ g) is effectively used. Japan also has a cellulase complex characterized by having a strong cellulose saccharification ability by containing a significant amount of β-glucosidase as well as a heat-stable xylanase. It is a bacterium that produces

Next, the national characteristics of the heat-stable xylanase-producing bacteria used in the present invention are as follows.

Growth: Growth on malt extract agar is far reaching 70 degrees in diameter in 7 days at 30°C. The colony is initially white and later becomes slightly yellowish. The aerial hyphae are loosely raised and wool-like,
・Sometimes forms knitted 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 Czapek agar, but the male hyphae were less bulging, with a growth PI of 43.5
-6.0, maximum -PH is around 4, growth temperature range) +Ii
Mk -43°C, optimal growth temperature is 30°C.

The violet diameter of the moth and hyphae is 0, 5-2, 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 very unstable and easily disappeared by subculturing on Czapek agar and malt extract agar media. When observed at 0 isolation, the conidiophores protruded from the side of the vaporized hyphae and were colorless. The conidia are subspherical, smooth, colorless, and the chains are very loose and easily dispersed. Regarding the above mycological properties, W, Gam5 rcephalos
poriu@artige Schimmel-pil
ge J P84, G. Fisher (1971).
and C.H. Dickin-son, Mycol.
As a result of referring to Papers Volume 115 PIG (1968), the home country is Acremonium (＾arezen◎niu
It was considered appropriate to consider it as a filamentous fungus closely related to the genus ~S). It should be noted that there have been no known strong cellulase-producing bacteria in the Acremonium genus, and that the strain of the present invention has been deposited in the free space in the strong and characteristic cell-'i=,==. ing.

4. Heat resistance due to Acremonium bacteria of t*rkA light. +
In order to produce ν゛4''-exo→ze, normally xylan 5xa glucan, cellulose, Avicel, bran rice straw, bagasse and other vegetable biomass are used as carbon sources, and nitrates, nitrates and nitrogen are added as nitrogen sources. Using a liquid or solid medium containing an organic or inorganic nitrogen source such as ammonium salts or peptone, yeast extract, and small amounts of metal salts,
The cells are cultured aerobically at ℃ for about 2 to 15 days. 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 is, but it can be prepared by known methods such as ammonium sulfate salting out method or acetone precipitation method.
Crude enzyme powder can be obtained (,3jB4.+1-,
'::Vs'1l by taking advantage of the heat resistance of this enzyme.
By heat-treating for 2 hours at 65°C, it is possible to denature and precipitate impure proteins and remove them without impairing the activity of -if%C:llanase, making it easy to prepare an enzyme solution with only xylanase activity. It can be prepared as follows. The heat-stable xylanase preparation of the present invention thus obtained has the following properties.

(1) Multicomponent Xylanase Thermostable xylanase is separated into at least three components by disk electrophoresis, and each component is distinguished by its molecular weight and isoelectric point. Xylanase A has a molecular weight of approximately 51,000
So the equal point is 5.05, and similarly B is about 46.000.4
．． 57, C is approximately 36,000.3.55, and xylanase is a composite of these components.

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

(3) Action pH and Optimal Action pH The action pH range of this enzyme complex is pH 3-6, as shown in FIG. 1(a), and the optimum action pH was found to be approximately 5.

(4) Stable pH range The stable pH range when left for 24 hours at 25° under a citric acid-phosphate surgical solution is approximately 2.5-8.5 as shown in Figure 1 (C). Met.

(5) Action temperature range and optimum temperature range All components of the present enzyme complex can act at high temperatures up to about 90°C, but as shown in Figure 1(b), 0.25! When reacted for 10 minutes under xylan and 0.05M acetic acid solution (PH 4, 9), the optimum working temperature was approximately 80'C. - #f-f7I elemental complex was dissolved in 0.1M acetic acid solution (
As a result of heat treatment at 11→ for 10 minutes under p) 14.9), as shown in Figure 1(d), there was almost no deactivation up to 70°C, and approximately 60χ and 85 °C
Approximately goz lost activity after heating for IO minutes.

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

(8) Purification method This enzyme complex is prepared by heating the culture filtrate at 65°C for 2 hours, denaturing the impure proteins contained therein, and removing the resulting precipitate by centrifugation. Using Sing's column chromatography, each component could be separated and purified to homogeneity using disk electrophoresis.

(9) Activity measurement method Substrate suspension (pi44.9) in which 0.5% xylan prepared from rice straw (containing about 9z arabinose) was suspended in 0.1M acetate buffer (0-5ml) A quantity of enzyme was added, the total volume was made up to 1.0% with distilled water, and the reaction was carried out at 60°C. And the reducing sugar produced is somogyi ne; Measured by # method.

Under the conditions, 1μg of xylose is added to the phase, f
The amount of enzyme that generates 4t'F reduction power was defined as 1 unit.

As described above, the thermostable xylanase of the present invention has an optimal action temperature around 80°C, and its enzymatic properties have not been known so far, as the main product from xylan is xylose. It is much superior to other enzymes. 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 acid 0.12%,! ! 7-Road FR1 Potassium 1.20%, leaded, sulfate + 7:: Contains 0.001% copper sulfate. ,,p
H4, 0) After culturing 20 ml in a 200 ml electrolayer, the cells were sterilized using a centrifuge, and the xylanase activity of the obtained supernatant was measured.
It was 050 units.

Example 2 In Example 1, instead of cellulose, xyloglucan (Glyloid 3s, manufactured by Dainippon Pharmaceutical Co., Ltd.) was cultured with aeration at 230'C for 8 days. The xylanase activity of the supernatant obtained after centrifugation after culture was measured and found to be 2400 units per ml of culture solution.

The pH of this culture supernatant was adjusted to 4.9, and the precipitate formed after heat treatment at 65°C for 2 hours was removed by centrifugation.
An enzyme preparation with only xylanase activity was obtained. The recovery rate of xylanase was 92.4%.

Example 3 The xylanase enzyme preparation obtained in Example 2 was adjusted to a final concentration of t 7
10% xyla, it to be qoo units/ml
It was added to Solution J and saccharified at 65°C for 48 hours.

Regarding a portion of the obtained sugar solution, the amount of reducing sugar was measured and found to be 60 mg/m1 as xylose.

This value is the value obtained by decomposing xylan of the same concentration with sulfuric acid (6
8mg/ml), the decomposition rate was 88.2%. In addition, the decomposition products at this time were identified by paper chromatography and were found to consist mainly of xylose.
It also contained a small amount of xylobiose.

-5 = A (ts of the thermostable xylanase produced (a) "
Best: suitable -, indicates poor lpH stability and (d) thermal stability.

Government Office Application Procedure Dispute Manual (Method) 1. Indication of Case Patent Application No. 349 of 1985
0 No. 2, Name of the invention Process for producing thermostable xylanase 3, Person making the amendment 6, Type of "Detailed description of the invention" in the specification to be amended Paper O Specification, page 5, line 13 to the same page Line 17: “More than that...
As a result of ..., delete "and add the following phrase.

Claims (1)

[Claims] 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.