JPH01171492A - Production of xylooligosaccharide derivative - Google Patents

Abstract

PURPOSE:To obtain a xylooligosaccharide derivative useful in various starch industries, in high efficiency, by contacting a specific enzyme with xylan or hydrolyzed xylan in the presence of an alcohol. CONSTITUTION:A microbial strain of genus Acremonium (e.g. Acremonium cellulolyticus) capable of producing xylooligosyl transferase A is cultured in a medium containing a vegetable biomass (e.g. bagasse) as a carbon source and a nitrogen source (e.g. peptone) at 20-40 deg.C for 2-15 days under aerobic condition. The cultured product is heat-treated and purified to obtain a xylooligosyl transferase A having a molecular weight of 51,000, an isoelectric point of about 5.0, a working pH of 3-6 and an optimum working temperature of 50 deg.C. The enzyme A is added to a reaction liquid containing xylotetraose and a 1-10C straight-chain alkyl alcohol, etc., and made to react at about 45 deg.C for about 2hr to obtain the objective xylooligosaccharide derivative.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for producing xylooligosaccharide derivatives using glycosyltransferases.

``Prior Art'' Xylan is a major hemicellulose component of plants of the Hematinaceae family, typified by broad-leaved trees and rice, and is an important biomass raw material that accounts for 20-30% of the constituents of these plants. This xylan is obtained in large quantities as a by-product of the pulp industry and from agricultural waste, especially corncobs, and has been used as a feed modifier or as a raw material for the production of xylitol. However, these demands are not so large, and there is a desire to develop higher value-added products using xylan as a raw material.

In recent years, xylooligosaccharide has attracted attention as a new product made from xylan, and attempts have been made to utilize its water activity regulating effect to apply it to foods. On the other hand, in the starch sugar industry, which is the most advanced field in the use of carbohydrates, methods are being developed to use glycosyltransferases to produce higher value-added transfer products from starch or starch melting products. be. Although the existence of enzymes that have a transglycosylation effect has long been known in the xylan family, the development seen in the starch sugar industry has not yet been seen. The glycosyltransfer activity is mostly due to the reverse reaction of hydrolase, and there is no glycosyltransferase with sufficient activity, and the only receptors other than sugars are primary alcohols that are soluble in water. This is partly due to the fact that it is only well known. If these problems are solved, it will be possible to enzymatically produce new substances using xylan as a raw material through transglycosylation, and products with even higher added value can be produced from xylan.

"Purpose" From the viewpoint of providing a more advanced use of xylan, which is contained in large amounts in plant biomass, the present inventors used xylan or xylooligosaccharide prepared from xylan as a donor, With the aim of producing useful glycosyltransferases through xylo-oligosaccharide transfer, which was previously unknown, we began the development of xylose-based glycosyltransferases. As a result, it was discovered that a strain of the genus Ammonium, a type of mesophilic fungus, produces a new glycosyltransferase that has xylooli, . We have discovered that alcohols can be widely used as receptors, and that sugar transfer can be carried out efficiently even when these alcohols have low solubility in water. In other words, by using various alcohols in addition to sugar as receptors for this enzyme,
The present inventors discovered that xylooligosaccharide derivatives with modified hydroxyl groups are produced as a result of the transglycosylation reaction, leading to the completion of the present invention.

"Structure" The present invention relates to a method for producing a hydroxyl group-modified xylooligosaccharide derivative from xylan or xylan melted product using xylooligosaccharyltransferase as an enzyme catalyst.

In the present invention, Acremonium cellulolyticus is effectively utilized as an exemplary strain thereof.

The mycological properties of the syltransferase A-producing bacteria are as follows.

Growth: Fast growth on malt extract agar medium at 30℃
It reaches a diameter of 70cm in 7 days. The colony is initially white and later becomes slightly yellowish. The vaporized hyphae are loosely swollen, wool-like, and sometimes form filamentous 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 Zapek agar, but the bulge of vaporized mycelium was smaller, and the growth and pi1 range was 3.
Optimum p is around 4 at 5-6.0, growth temperature range is 15℃
~43°C, with an optimal growth temperature of 30°C.

Form R: The diameter of the hyphae is 0°5-2.5μ.It is colorless and septa are observed in the hyphae. In addition, the hyphal surface is smooth.

Conidia: The ability to form conidia is very unstable and easily disappeared by subculturing on Czapek agar and malt extract agar media. When observed at 0 isolation, conidiophores protruded from the sides of vaporized hyphae and were colorless. The conidia are subglobose, smooth, colorless, and the chains are Osporlus artlg.
e Schi-melpHge) p84. G
, edited by FIsher (1971) J and C, Il, Di.
ckinson r MycologicalPap
As a result of referring to ers volume 115 plO (1968) +, the home country is Acremonium.
We considered it appropriate to consider it to be a filamentous fungus closely related to the genus. Furthermore, in addition to xylo-oligosyltransferase, the native country produces a significant amount of cellulase, and since no bacteria of the Acremonium genus were previously known to have a high cellulase-producing ability, the native country is known as Acremonium. Acremonium cellul.

1ytlcus) are named FEBN P-6.
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as No. 867.

In order to produce the xylooligosyltransferase A produced by the genus Acremonium of the present invention, vegetable biomass such as xyloglucan, cellulose, avicel, xylan, bran, rice straw, and bagasse is usually used as a carbon source, and a nitrogen source is added to this. For example, using a liquid or solid medium containing a free or organic nitrogen source such as nitrates, ammonium salts or peptone, yeast extract, and small amounts of metal salts, at 20-40°C.
The cells are then cultured aerobically for about 2 to 15 days. Since xylooligosyltransferase A is an enzyme produced outside the bacterial cells, in the case of a liquid medium, the supernatant after filtration or centrifugation is used, and in the case of a solid culture, the supernatant is added to water or an appropriate inorganic solution after culturing. A solution extracted with salts can be used as a crude enzyme solution. The crude enzyme solution may be used as it is, but an enzyme powder can be obtained by a known method such as ammonium sulfate salting out method or acetone precipitation method. Furthermore, since this enzyme is thermostable, this property can be used to
By performing heat treatment at 965°C for 2 hours and 30 minutes, impurities can be denatured and precipitated and removed without impairing the activity of xylooligosyltransferase A, resulting in an enzyme solution with increased purity of xylooligosyltransferase^ activity. can be easily prepared. The thus obtained xylooligosyltransferase A specimen has the following properties.

(1) Molecular weight and isoelectric point (2) Action Xylo-oligosyltransferase A acts on xylan and xylo-oligosaccharides of xylobiose or higher prepared from xylan, and uses this as a xylosyl or xylo-oligosyl donor to transfer alcohol to the acceptor. It is transferred to a hydroxyl group to produce a xylosyl or xylooligosyl transfer product. To specifically describe this transfer effect using the case of xylopentaose as an example, the present donor is utilized by the enzyme as a donor that causes xylotriosyl transfer and xylobiosyl transfer. The probability of each transition occurring is approximately 85% for xylotriosyl transfer and approximately 15% for xylobiosyl transfer. Table 1 shows the probability of xylo-oligosyl transfer occurring in the initial reaction for other linear xylo-oligosaccharides, including xylopentaose.

Table 1 In the table, X- to x6- each indicate xylotriosyl transfer from xylosyl residue.

As shown in the table, when a xylooligosaccharide of xylohexaose or more is used as a donor, transfer products of xylotriosyl or higher are generated, but as the reaction time becomes longer, these are re-decomposed and finally xylobiosyl Gives the transfer product and the xylotriosyl transfer product.

This enzyme has clear transfer activity when the substrate oligosaccharide concentration is as low as 1%, and can transfer oligosaccharide residues of xylobiosyl or higher to the alcoholic hydroxyl group of the acceptor.

A glycosyltransferase having such an action was completely unknown until now, and the disclosure of the present invention is the first one.

Therefore, this enzyme was named xylooligosyltransferase^.

(3) Donor Although xylooligosaccharides of xylobiose or higher and xylan can be used as donors, transfer activity is sufficient when xylooligosaccharides of xylotetraose or higher and xylan are used as donors.

(4) Acceptor Xylo-oligosaccharides of xylobiose or higher can be used as acceptors, and they also act as donors, so if these are used as acceptors, this will effectively rearrange the xyloside bonds of oligosaccharides, so-called Disproportionation occurs. Sugars such as xylose and glucose are also not suitable as acceptors.

Among various alcohols other than sugars obtained by organic chemical synthesis methods or natural products, water-soluble and slightly water-soluble alcohols act as acceptors, and xylo-oligosyl transfer occurs to the hydroxyl group, resulting in xylo-oligosaccharide derivatives. generate.

(5) Action pi (and optimal action pH) The action pH range of this enzyme is pH 3 to 6, as shown in Figure 1a, when the enzyme is allowed to act for 10 minutes at 45°C, and the optimum action pH is approximately 5. Admitted.

(6) Stable pH range citric acid-phosphate &! The stable pH range when the solution was left at 25° C. for 24 hours in street liquid was about pH 2.5 to 8.5, as shown in Section 111b.

(7) Working temperature range and optimum working temperature Using 0.1% xylotetraose as donor and acceptor, pH 4
, 9 for 10 minutes, as shown in Fig. 2a, the temperature of action decreased to a high temperature of about 90°C. occurred, and in the presence of 50% propyl alcohol, it worked up to 55°C, and the optimum action temperature was 50°C.

(8) Thermostable xylooligosyltransferase^ was added to 0.1M acetate buffer (p
The residual activity after heat treatment for 1 hour at each temperature under H4,9) was the third [! As shown in Ia, there is almost no deactivation up to 70°C, about 40% at 75°C, and 85°C,
Approximately 95 gourds lost their activity after one hour of heating. Also, 50%
When heat-treated at pH 4.9 in the presence of n-propyl alcohol, as shown in Figure 3, there is almost no inactivation up to 45°C, about 25χ at 55°C, and about 25χ at 70°C.
Approximately 95 pieces lost their activity after one hour of heating.

(9) Inhibitor Among various metal ions, this enzyme was strongly inhibited by 1 sM or more of mercury ion and copper ion.

(10) 1 Preparation Method This enzyme is prepared by heating the culture solution at 65°C for 2 hours and 130 minutes, denaturing the impure proteins contained therein, removing the resulting precipitate by centrifugation, and then DEAL! -) Yopal, ion exchange and adsorption chromatography using an anion exchanger PBE 94 column, and Biogel A
By gel filtration using a 0.5i+ column, it was possible to separate and purify to homogeneity by disk gel electrophoresis.

(11) Activity measurement method Since this enzyme transfers using xylooligosaccharides as acceptors and donors, 100 μr of a 0.11 aqueous solution of xylotetraose prepared from xylan (pH 4
, 9), add an appropriate amount of enzyme to make the total amount 150μ!
The reaction was carried out at 45° C. for 10 minutes, and the generated transfer products of xylopentaose or higher were separated and quantified by high performance liquid chromatography using a Licrosolp MH2 column to determine the activity. Under these conditions, the amount of enzyme that produces xylooligosaccharides of 1 μg or more of xylopentaose per minute is defined as 1 unit.

"Effect J" As described above, the xylooligosyltransferase A disclosed in the present invention is a novel enzyme that performs xylooligosyltransfer using xylooligosaccharide prepared from xylan or xylan as a donor, and is It has the useful property of acting stably and producing xylo-oligosaccharide derivatives.The xylo-oligosaccharide derivatives produced in this way have a hydrophilic sugar moiety and a hydrophobic modified moiety, so they have a surfactant effect. It is estimated that there are many compounds with the same properties, and this opens the door to the production of hitherto unimaginable useful xylooligosaccharide derivatives from xylan by enzymatic reactions.

Next, examples of the present invention will be shown.

Example 1 Cellulose 4x, peptone 1 cup, potassium nitrate 0.6%
, potassium chloride o, 15x, magnesium chloride 0.1
2χ, potassium phosphate 1.2π and zinc sulfate, copper sulfate, Acremonium cellulolyticus after sulfate (FIR
MP-6867> was inoculated and cultured with aeration at 30°C for 6 days. After culturing, bacteria were removed by centrifugation, and the xylooligosyltransferase A activity of the resulting supernatant was measured, and the result was 220 units per 11 culture fluids.

Example 2 In Example rN1, the carbon source was replaced with cellulose, and 4% xyloglucan (Glyloid 33, manufactured by Dainippon Pharmaceutical Co., Ltd.) was used.
Acremonium cellulolyticus (FERN P-6867) was inoculated into the medium to which Acremonium cellulolyticus (FERN P-6867) was added, and cultured with aeration at 30°C for 8 days. The xylooligosyltransferase activity in the centrifuged supernatant was 1200 units per ml of culture solution.

The PL of this culture supernatant was adjusted to 4.9, and after heat treatment at 65°C for 2 hours and 30 minutes, the resulting precipitate was removed by centrifugation to obtain an enzyme preparation with xylooligosyltransferase A activity. . Xylooligosyltransferase "A" cycle.

Example 3 The heat-treated enzyme preparation obtained in Example 2 was concentrated, desalted, tIkD
I! AI! -) Ion exchange chromatography was performed using Yovar M65G and anion exchanger PBE 94 ram to obtain an active fraction. This active fraction was further subjected to gel filtration using a Biogel A0.5 column to purify xylooligosyltransferase A. The purified enzyme was disk electrophoretically homogeneous and the yield of activity was 22% relative to the culture supernatant. In addition, 1 m of lyophilized enzyme specimen
The xylooligosyltransferase^ activity per g was 61 units.

Example 4 Purified enzyme obtained in Example 3. The '4 position was reacted with lπ xylotriose, xylotetraose, xylopentaose, and xylohexaose at 60° C. for 15 minutes and 2 hours, and the reaction products were analyzed using a Biogel P-2 column. As shown in Figure 4a, xylohexaose is obtained from xylopentaose, xylooctaose is obtained from xylohexaose, and xylononaose and xylodecaose are obtained from xylohexaose as the main transfer products, and this enzyme is a xylooligomer. It was shown to catalyze syl transfer. Furthermore, at a reaction time of 2 hours, as shown in Figure 4, the rearrangement reaction progresses, and a large number of rearrangement products of xyloundecaose or less, which have a higher degree of polymerization than the added substrate, are produced, and the intermolecular sugar It was shown that a transfer reaction was occurring.

Practical HFI Example 5 0.5 unit of the purified enzyme obtained in Example 3 was incubated at 45°C in a reaction solution containing 1% xylotetraose and 25% each of linear alkyl alcohol having 1 to 10 carbon atoms.
After reacting for 2 hours, the transfer product was analyzed by high performance liquid chromatography using a Licrosolve NH2 column. As a result, 0.5 unit of the purified enzyme obtained in Example 3 of Example 4 was added to 1% xylotetraose. and a reaction solution containing 25% of each linear alkyl diol having 2 to 8 carbon atoms and hydroxyl groups at both ends at 45°C.
After reacting for 2 hours, the rearranged product was analyzed as in Example 5. As a result, alkylated xylobiose having a hydroxyl group at the end of the alkyl group was obtained from each diol at the yield relative to xylotetraose shown in Table 3.

Table-3 0tetraose and 5ee-butyl alcohol at 25%
After reacting at 45° C. for 2 hours in a reaction solution containing x, the transfer product was analyzed in the same manner as in Example 5. As a result, a transition product in which xylobiose was transferred to the hydroxyl group of 5ee-butyl alcohol was obtained with a yield of 26% based on xylotetraose.

Example 8 After reacting 0.5 unit of the purified enzyme obtained in Example 3 at 45°C for 2 hours in a reaction solution containing 25x of B xylotetraose and benzyl alcohol, the transfer product was converted to Example 5. As a result, a transfer product in which xylobiose was transferred to the hydroxyl group of benzyl alcohol was obtained with a yield of 32% based on xylotetraose.

Example 9 In place of xylotetraose in Example 5, xylotetraose was used
0 After reacting 0.5 units of the purified enzyme obtained in Example 3 at 45°C for 18 hours in a reaction solution containing 1% xylan (derived from barley) and 25% h-propyl alcohol, the transfer product was analyzed in the same manner as in Example 5. As a result, a transfer product in which xylobiose was transferred to the hydroxyl group of propyl alcohol was obtained at a yield of 8 times higher than xylan.

[Brief explanation of the drawing]

Figure 1 shows Acremonium cellulolyticus (FER)
Optimum action of xylooligosyltransferase A produced by M
b) are shown respectively. Figure 2 shows Acremonium cellulolyticus (FEB)
The xylooligosyl transition figure 3 produced by Acremonium cellulolyticus (FEB
xylooligosyltransferase A produced by NP-6867) in acetate buffer (&), and 50! Thermal stability in n-propyl alcohol (b) is shown. Figure 4 shows xylotriose (x3) to xylohexaose (x6) as donor and acceptor for 15 minutes (
&) and 2 hours (b) shows the elution pattern of the reaction product in gel permeability analysis using Biogel P-2 when reacted with xylooligosyltransferase. 1 to 12 shown at the top of the figure indicate the degree of polymerization of xylo-oligosaccharides, and A to E indicate the main transfer products in the 15 minute reaction, indicating that they are xylotetraose to xylodecaose. There is. Figure 1: pH Figure 2: Reaction temperature (c°C) Figure 3: Processing temperature ("C) Drawing engraving/eluate volume (ml) Office application procedure amendment (method) % formula % 1. Indication of incident 1988 Patent application No. 328470
No. 2, Name of the invention Process for producing xylo-oligosaccharide derivatives 3, Relationship with the case of the person making the amendment Patent applicant address: Mr. 1-3-1 Kasumigaseki, Chiyoda-ku, Tokyo
Name (114) Director of the Agency of Industrial Science and Technology Kozo Iizuka 44,
Designated agent 〒305

Claims (1)

[Claims] Cultivating Acremonium bacteria having the ability to produce xylooligosyltransferase A, and contacting the resulting culture or xylooligosyltransferase A collected from the culture with xylan or xylan hydrolyzate in the presence of alcohol. A method for producing a characterized xylooligosaccharide derivative.