JPH0824574B2 - Method for producing xyloglucan degrading enzyme, and xyloglucan degrading enzyme - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a xyloglucan-degrading enzyme that does not act on cellulose but specifically acts only on xyloglucan, and more specifically, relates to, for example, plant elongation. It can be used as an enzyme reagent used for elucidating the structure and function of xyloglucan, which is a biopolymer that plays an important role in differentiation.

[0002]

2. Description of the Related Art Xyloglucan is a heteropolysaccharide composed of glucose, xylose, galactose, fucose, arabinose and the like as constituent monosaccharides.
For the main chain that is linked by a large number of 1,4-glucoside bonds,
It is referred to as xyloglucan because xylose has a basic structure in which it is frequently branched and branched by α-1,6 bond. This xyloglucan plays an important role as a constituent component of the primary cell wall of plants and as a seed storage polysaccharide of certain tropical legumes, and its structure and function have been drawing attention. In particular, xyloglucan in the primary cell wall of plants is considered to exist in close association with cellulose, and the relative existence form with cellulose plays an important role in plant elongation and differentiation. Is supposed to exist. Various techniques have been used to analyze the role of xyloglucan, and a polysaccharide-specific degrading enzyme reagent has become an important and powerful analytical means.

As described above, since the structure of the main chain of xyloglucan is similar to that of cellulose, the endo-
It is known that it is degraded by 1,4-β-glucanase, so-called endo-type cellulase, and endo-type cellulases of various origins have been used for elucidation of the structure and function of xyloglucan.

[0004]

However, this endo-type cellulase decomposes not only xyloglucan but also cellulose. And since xyloglucan exists in close association with cellulose in the plant cell wall from its existing form, it cannot always be said that the xyloglucan specifically acts only on xyloglucan in the analysis using endo-type cellulase, It has complicated the interpretation of the analysis results. In order to eliminate these difficulties and to clearly understand the role of xyloglucan, it has been desired to develop a degrading enzyme that does not act on cellulose but specifically acts only on xyloglucan.

[0005]

The present inventors have found that xyloglucan and cellulose have large differences in their three-dimensional structures, and that enzymes generally have high substrate specificity. Focusing on, we newly screened endo-1,4-β-glucanase-producing microorganisms using xyloglucan as a substrate from nature, and further examined the enzyme system produced by the isolated microorganisms using xyloglucan and carboxymethylcellulose as degradation substrates. As a result, the possibility of the existence of a xyloglucan-degrading enzyme that specifically acts only on xyloglucan and not on carboxymethylcellulose was thoroughly investigated. As a result, it was found that one strain of the genus Penicillium (Penicillium genus M451 strain), which is a type of filamentous fungus, produces a novel xyloglucan-degrading enzyme (hereinafter referred to as the present enzyme), and the present invention was completed. I arrived.

The mycological properties of the Penicillium sp. Strain M451 used in the present invention are as follows.

Growth: Growth on a Czapek agar medium containing yeast extract reaches a diameter of 28 mm at 25 ° C. for 7 days. The colony becomes white at first and turns blue-green with sporulation, and becomes gray-green at the latter stage of culture. Large and conspicuous conidia stalk bundles with a height of 2 to 3 mm are formed from the center of the village to the periphery. This conidia peduncle bundle is single-threaded and dense. In addition, the rear surface of the community becomes dark in the latter half of the culture, and is particularly noticeable just below the conidia peduncle. It does not grow at 37 ° C even under the above conditions.

Morphology: The diameter of the hypha is 2.5 to 3.5 μm
It is colorless and the hyphae have septa. The mycelium surface is smooth.

Penicili: Asymmetric.

Mettle: 2 to 4 grow in groups.

Fear Ride: 3 to 5 rings.

Conidia: An ellipse of about 4 × 3 μm, a smooth surface, and a long chain are formed.

Regarding the above-mentioned mycological properties, Shunichi Udagawa [Antibacterial and Antifungal Volume 19 No. 12 pp 657-665]
(1991)], the result showed that this fungus was penicillium.
Claviforme (Penicillium clavi)
It is reasonable to think that it is a filamentous fungus closely related to
This bacterium is a Penicillium sp. Strain M451 (Penicill)
ium sp. It was named M451). This bacterium is FERM
P-13798 has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology.

In order to produce the present enzyme from the above-mentioned Penicillium sp. Strain M451, tamarind seed xyloglucan is usually used as a carbon source, and a nitrogen salt is added as a nitrogen source.
It is aerobically cultivated at 20 to 30 ° C. for about 4 to 10 days using a liquid or solid medium containing an inorganic or organic nitrogen source such as ammonium salt or peptone or yeast extract and a small amount of metal salt. Since this enzyme is an enzyme produced outside the bacterial cells, in the case of a liquid medium, after culturing, a supernatant obtained by filtration or centrifugation is used, and in the case of solid culture, after culturing, water or a suitable inorganic salt is added. The extracted liquid can be used as a crude enzyme liquid. Since the crude enzyme solution contains an enzyme activity similar to conventional endo-type cellulase, it needs to be removed. However, the co-existing endo-type cellulase activity can be easily removed by known chromatography. .

The present enzyme (purified product) obtained as described above has the following properties.

(1) Molecular weight and isoelectric point; the purified present enzyme has a molecular weight of about 28,000 and an isoelectric point pH of about 5.
0.

(2) Action: This enzyme acts on xyloglucan of various origins and xyloglucan-derived oligosaccharide having a degree of polymerization of 12 or more generated by partial decomposition of xyloglucan by endo-type cellulase, and decomposes it at a specific place. To do. On the other hand, with regard to cellulose having no xylose side chain, it does not act at all on absorbent cotton which is crystalline cellulose, Avicel which is microcrystalline cellulose, and non-crystalline cellulose prepared by treating purified pulp with phosphoric acid. It also has no effect on hydroxyethyl cellulose, which is a soluble derivative of cellulose. Furthermore, carboxymethylcellulose, which is a soluble cellulose derivative having an anionic dissociative group, acts only slightly, but its decomposition rate is 1% or less. It does not act on β-glucans other than the above at all. Similarly, it does not act on linear cellooligosaccharides having a degree of polymerization of 2 to 6 prepared by partial decomposition of cellulose. As described above, the present enzyme is an enzyme that specifically decomposes only xyloglucan and does not act on cellulose.

(3) Working pH and optimum working pH: When xyloglucan is used as a decomposition substrate, the working pH range of this enzyme is as shown in FIG. 1 (a) when it is allowed to act at 45 ° C. for 10 minutes. pH 3 to 6, optimum working pH is about 5.0
It was found in the vicinity.

(4) Stable pH range; the enzyme is citric acid-
As shown in FIG. 1 (b), the stable pH range was about 5.0 to 7.0 as viewed from its residual activity after leaving it in a phosphate buffer solution at 45 ° C. for 3 hours.

(5) Working temperature range and optimum working temperature: When xyloglucan is used as a decomposition substrate, the working temperature range of this enzyme is as shown in FIG.
As shown in (a), the action was observed up to about 55 ° C,
The optimum working temperature was 50 ° C.

(6) Thermostability: The present enzyme was heat-treated in a 50 mM acetate buffer (pH 5.0) at each temperature for 10 minutes, and the residual activity was up to 50 ° C. as shown in FIG. 2 (b). Almost no deactivation, about 10% at 55 ° C and about 85% at 60 ° C lost activity.

(7) Inhibitor: Of various metal ions,
This enzyme was strongly inhibited by 1 mM or more of mercury ion and copper ion.

(8) Purification method: This enzyme was prepared by concentrating and desalting the culture filtrate by ultrafiltration, and then using Q-sepharose and S.
-Most of the contaminating proteins were adsorbed and removed by column chromatography using Sepharose, and the active fractions not adsorbed on these columns were further subjected to anion exchanger PBE94.
By performing ion exchange and adsorption chromatography using the column of No. 3, and further by gel filtration with a Toyopearl HW50F column, it was possible to separate and purify to a uniform SDS electrophoresis.

(9) Method for measuring activity: This enzyme specifically decomposes only xyloglucan. Therefore, a 0.5% aqueous solution of xyloglucan prepared from tamarind seeds 500 μm
An appropriate amount of enzyme was added to 1 (pH 5.0) to make the total amount 1000 μl, and the mixture was reacted at 45 ° C. for 10 minutes, and the reducing sugar produced was measured by the Nelson-Somogy method to determine the activity. Under this condition, the amount of enzyme that produces a reducing power corresponding to 1 μmol of glucose per minute was defined as 1 unit.

[0025]

The present invention will be described in detail below with reference to illustrated embodiments.

Example 1 First, tamarind seed xyloglucan 1%, peptone 0.8% and magnesium sulfate 0.
05%, monopotassium phosphate 0.2%, yeast extract 0.0
200 ml of 20 ml of medium (pH 6.0) containing 5%
Put in a Erlenmeyer flask and sterilize by a conventional method, and then Penicillium sp. Strain M451 (FERM P-13798)
Was inoculated and aerobically cultured at 30 ° C. for 6 days. Then, the bacteria were removed by filtration, and the activity of the resulting supernatant was measured by the above-mentioned activity measuring method.
Units.

Example 2; 4000 ml of a culture solution having the same composition as in Example 1 was prepared, and Penicillium sp.
The strain was inoculated and cultured in the same manner to obtain a culture filtrate. The total enzyme activity in the culture was 200 units. The culture filtrate was concentrated and desalted by ultrafiltration, followed by Q-sepharose and S-
Most of the contaminating proteins were adsorbed and removed by column chromatography using Sepharose, and the active fractions not adsorbed on these columns were further subjected to ion exchange and adsorption chromatography using a column of anion exchanger PBE94. Purified by gel filtration on a HW50F column. The purified enzyme was SDS-electrophoretically uniform, and the yield of activity was 12% based on the culture filtrate. The activity per mg of the purified enzyme protein was 24.1 units.

Example 3; 0.1 unit of the purified enzyme obtained in Example 2 was added to 0.5% tamarind seed xyloglucan, 0.5% carboxymethyl cellulose, 0.5%
PH 5.0, 45 for hydroxyethyl cellulose
The decomposition rate was calculated by measuring the direct reducing sugars produced by the decomposition action by the Nelson-Somogy method and the total sugars by the phenol-sulfuric acid method by allowing them to act at 0 ° C. and collecting a part thereof with time. As a result, as shown in FIG. 3, the purified enzyme rapidly decomposes only tamarind seed xyloglucan (XG), and other soluble cellulose derivatives such as carboxymethyl cellulose (CMC) or hydroxyethyl cellulose (HE).
Little or no decomposition of C).

Example 4; 0.1 unit of the purified enzyme obtained in Example 2 was applied to xyloglucan of various origins, celluloses and various polysaccharides shown in Table 1 in the same manner as in Example 3, After reacting for 18 hours, the decomposition rate was measured in the same manner. As a result, it was shown that this enzyme specifically decomposes only xyloglucan and has little or no effect on cellulose and other polysaccharides.

[0030]

[Table 1]

Example 5: 0.1 unit of the purified enzyme obtained in Example 2 was added to Example 3 in 0.8% solutions of various xyloglucans and cellulose-related oligosaccharides having the structures shown in Table 2. After the same action and reaction for 18 hours, the decomposition products were analyzed by high performance liquid chromatography using a lychrosolv NH ₂ column. As a result of measuring the decomposition rate with the added substrate completely disappeared as the decomposition rate of 100%,
As shown in Table 2, a polymerization degree of 7 to 7, which is a complete degradation product of linear cellooligosaccharide or xyloglucan by endo-type cellulase
It did not act on oligosaccharides of No. 9 at all, but acted only on higher heterooligosaccharides of polymerization degrees of 14 and 18 obtained by limited decomposition of tamarind seed xyloglucan by endo-type cellulase.

The conventionally known endo-type cellulase is
In contrast to the degradation of linear cellooligosaccharides, this enzyme is distinct from known endo-type cellulases in that it does not act on them. In addition, xyloglucan oligo 1
It is known that 4, 18 saccharides are decomposed into oligo 7-sugar and 9-sugar by known endo-type cellulases, respectively, and the degradation products of these oligosaccharides by this enzyme are also known as the degradation products of known endo-type cellulases. It was similar. Therefore, the cleavage site of this enzyme for xyloglucan was on the reducing end side of the glucose residue having no xylose side chain.
However, the fact that it does not act on linear cellohexaose but acts on xyloglucan oligo-14 sugar strongly suggested that the xylose side chain is important for the substrate recognition of this enzyme. This result showed that this enzyme has an entirely different substrate specificity from the conventionally known endo-type cellulase.

[0033]

[Table 2]

Example 6; 0.1 unit of the purified enzyme obtained in the above Example 2 was added to the higher heterooligosaccharide having a degree of polymerization of 14 used in Example 5 to limit the action of the isoprimeverose-forming enzyme. Was prepared. After polymerization, various oligosaccharides having the structures shown in Table 3 of 8 to 14 were allowed to act in the same manner as in Example 5, and the decomposition rate was measured in the same manner. As a result, this enzyme requires at least two isopimeverose (xylosyl α-1,6-glucose) units on the non-reducing end side of the substrate cleavage site, and it is shown that it does not act on substrates shorter than that. Was done. On the other hand, known endo-cellulase is known to be able to degrade all oligosaccharides having the structures shown in Table 3, and the results also show that the enzyme has a substrate specificity completely different from that of conventional endo-cellulase. It was shown to have.

[0035]

[Table 3]

The present invention has been described above based on the embodiments, but the present invention is not limited to the above-mentioned embodiments, and can be carried out in any manner as long as the configuration described in the claims is not changed. You can

[0037]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a novel xyloglucan-degrading enzyme that does not act on cellulose at all or hardly acts without using any special technique or device.

The obtained enzyme can be used as a powerful enzyme reagent for elucidating the relationship between cellulose and xyloglucan in the primary cell wall of plants and for elucidating the structure of xyloglucan.

Further, since it does not act on cellulose at all and can decompose and remove xyloglucan, which is considered to exist around cellulose, it is expected that pulp with a higher degree of purification can be produced.

[Brief description of drawings]

FIG. 1 is a graph showing the optimum action pH (a) and pH stable range (b) of the xyloglucan-degrading enzyme of the present invention at 45 ° C.

FIG. 2 is a graph showing the optimum action temperature (a) and thermal stability (b) of the xyloglucan-degrading enzyme of the present invention in an acetate buffer (pH 5.0).

FIG. 3 shows xyloglucan (XG) and carboxymethyl cellulose (CM) produced by the xyloglucan-degrading enzyme of the present invention.
It is a graph which shows the time-dependent change of the decomposition of C) and hydroxyethyl cellulose (HEC).

Claims (2)

[Claims] 1. A Penicillium strain M451 strain (FERM
P-13798) is cultivated, and the coexisting endo-cellulase-like enzyme activity is removed from the resulting crude enzyme solution by chromatography or the like, thereby producing a xyloglucan-degrading enzyme. 2. A xylogle having the following physicochemical properties:
Can-degrading enzyme. (1) Substrate specificity: Only xyloglucan is specifically hydrolyzed to give cellulose.
Does not decompose. (2) Optimum pH and stable pH range: The optimum pH is pH 5 when xyloglucan is used as a substrate.
It is 0. The stable pH range is pH 5.0 to 7.0. (3) Optimum temperature range of action: It is in the range of 30 ° C to 55 ° C. (4) Molecular weight The molecular weight is about 28,000.