JPS60176585A - Biochemical method and composition - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for biochemically decomposing bentosan, an enzyme that can be used therefor, and a method for producing a composition containing the enzyme.

Prior Art Bentosan is a class of polymers of one or more naturally occurring pentose, found in various plants such as grains such as wheat and oats, tea, coffee, seaweed, fruits, vegetables, and sorghum. Contained in natural products. In many applications where natural products are used as raw materials, the presence of bentosan is not desired. For example, when the bentosan in the flour combines with water, the flour made using this flour becomes hard, and this drawback can be improved if the bentosan content in the flour is reduced. As a next example, during the production of instant coffee, soluble gums are extracted along with the coffee due to bentosan in the coffee. By reducing bentosan in coffee, the coffee extraction rate can be increased and a better product can be obtained.

Our UK Patent No. 1421127 is derived from barley and has β
Discloses a method for producing an enzyme related to -1,4/β-1,3-glucan. The enzyme obtained is mainly β-1,4/β
-1,3-glucanase, other β-glucanases, laminarinase, HirO-? It contains diethyl cellulase, cellulase and alpha-amylase, and is particularly useful in the treatment of barley. Penicillium emersonii (P. nicllimemers) as an enzyme source
onii) are used.

A particularly suitable microorganism has been deposited with the Commonwealth Medical Institute, UK as 1M1116815. The name of this microorganism is now Talaromyces emersonii (Talaromyae).
m emersonii).

The problem we were trying to solve: We were able to use this microorganism to produce other enzymes. It is a pentosanase and can catalyze the breakdown of bentosans, especially those derived from wheat.

This type of enzyme is well known, and various enzymes have been produced, e.g.
It is used in starch production, agriculture, brewing, extraction of plant tissues, and production of vegetable dyes. However, since high temperatures are used in many of these applications, thermostable pentosanases with good activity even at high temperatures are desired.

Means for Solving the Problems The enzyme according to the invention is suitable for use in Tayloromyces emersonii, in particular Tayloromyces emersonii Stolk, which has the ability to catalyze the decomposition of bentosan.
x h Obtained by culturing T. emma sonii Stokes IMI 116815 or a mutant strain thereof.

The following feature of the present invention provides an enzyme produced by culturing a microorganism belonging to Tayloromyces emersonii (hereinafter referred to as T. emersonii), particularly T. emersonii IMI 116815 or a mutant strain thereof, which has the ability to catalyze the decomposition of xylan. Ru. Xylan is a polymer of pentose xylose.

The new enzyme according to the invention is thermostable and has good activity at high temperatures, making it particularly useful for bentosan degradation at high temperatures. For example, in the stability test of the enzyme obtained by the method described in Example 1 below, in the presence of a substrate, 95%
The enzyme activity after heating at pH 5.0 for 6 minutes at ℃ was more than 50 times the original activity. The relationship between the activity of this enzyme and time was also good; when oat coat was used as a substrate, 8
Optimum activity was obtained at 2°C at 7°C, and at 45°C at 95°C. This finding is unusual for enzymes of fungal origin.

For example, the optimal activity of the β-glucanase mentioned above is 60-
It seems to be obtained at 70°C, but it decreases to 40q6 at 80°C.

The properties of the new enzyme according to the present invention, measured using a crude enzyme preparation obtained by the method described in Example 1 below, are as follows.

(a) p for optimal activity when measured at 50°C
H is 5.0, (bl The temperature for optimal activity is 87°C when measured at pH 5.0, 2°C, (c) Xylose added beforehand when measured at pH 5.0, 50°C, Glucose or maltose do not affect enzyme activity (determined using xylan from oat coat as pentose substrate).

The enzyme according to the invention has the ability to catalyze the decomposition of bentosan, such as T, Emersonii, IMI.
116815 or its mutant strain. However, by appropriately modifying the culture medium, the amount of protosanase produced can be reduced.
- It was found that it can be increased for glucanases. T
.

Emerson's good name is T, Emerson's good IMI
In the case of the enzyme obtained directly by culturing 1+6815, the level of pentosanase for β-1,4/β-11,3-glucanase was reduced by the method of the invention using a conventional medium as a result of the modification of the medium water. This is an improvement over when implementing .

Culture can be carried out by conventional methods. Therefore, T.

The cultivation can be carried out under aerobic conditions using the Emersonii strain, preferably submerged culture, with stirring in air or oxygen.

The medium used contains assimilable carbon sources, nitrogen sources and optionally growth promoters and inorganic salts.

As suitable carbon sources, bentosan-rich materials can be used, such as grains such as barley, wheat, Distillas solubles, other malt or grain distillation by-products, cellulose, such as vine floc or xylan.

Examples of suitable sources are barley, Distillas solubles, other malt or grain distillation by-products, soybean meal, nitrates or ammonium salts such as ammonium dihydrogen phosphate.

If it is desired to increase the level of bentosanase activity relative to β-glucanase activity, carbon or nitrogen sources such as Scotagran (mixture of solupulus and dried barley flour), ammonium nitrate, hot alesyrup, corn pellets ( mixture of dried corn flour and solubles), syrup (eurns 5yr
(up), corn stizo liquor, etc. can be used.

Examples of inorganic salts are potassium, magnesium or
Jium sulfate or hydrochloride.

A practical medium is barley at a concentration of 0.2-3.5% w/v;
Contains a concentration of 0.4-5.5 q6 w/v Distylates solubles or other malt or grain distillation by-products and 0.2-3.0% cellulose.

temperature, so that the maximum amount of the desired enzyme is accumulated in the medium.
Adjust culture conditions such as pH and time. For example, temperature 35-60°C (preferably 50-54'C), pH 3
, 5-4, 5 for 1-20 days (preferably 7-10 days).

After the culture is completed, the culture solution can be used as a crude enzyme preparation. Alternatively, a powder obtained by drying the culture solution can also be used. If desired, the pentosanase can be purified to some extent, for example by chromatography, to enhance the pentosanase activity, β-1,4/β-1,3-glucanase activity. Purification methods and method products of this type also fall within the scope of the invention.

Extracellular enzymes can be extracted from the culture, for example, by conventional methods. For example, as a first step, the formed mycelium is separated by a pre-coding method using, for example, a filter coated with a filter aid. An enzyme solution is obtained from the resulting f solution directly or preferably after concentration under reduced pressure. Although it can be used as an enzyme source, adding sodium benzoate or sodium pyrosulfite to the fermentation broth can enhance the shelf life, heat stability or preservative properties of the enzyme. Alternatively, the enzyme solution can be adsorbed with a suitable solid, such as wheat flour or barley flour.

In this case, a carrier such as carboxymethyl cellulose (Na) (CM(A) may be used in combination, if necessary.

The latent material thus obtained can also be dried to obtain an active enzyme preparation. If desired, the concentrate can be subjected to, for example, spray drying, freeze drying, or roller drying to obtain a dry enzyme formulation.

Since it is uneconomical to extract the enzyme under the strictest conditions, it is not necessarily necessary for the purpose of containing the enzyme, and it is practically sufficient to obtain a crude composition. The composition in this case generally also has other primary activities.

If a solid enzyme product is desired, it can be obtained from P solution or concentrate by conventional methods.

For this purpose, precipitation is effected by adding an excess of a water-miscible organic solvent, such as ethanol or a ketone, such as an acetone, to the P solution or concentrate. In addition to being directly precipitated, it can also be precipitated on a carrier. Examples of carriers of this type are starch, methyl cellulose or C
I'm an MC.

The seeds of T. emersonii used for culture were collected. For example, it can be collected from surface cultures of microorganisms by well-known methods. For example, microorganisms can be mixed with peptone (e.g. 0.5
% w/y), sodium chloride (e.g. 0.4%w
/v), glycerin (e.g. 0.75% W/V)
), molasses (e.g. 0.8% w/v), potassium dihydrogen phosphate (e.g. 0.006% w/v) and magnesium sulfate (e.g. 0.005% w/v),
A solid nutrient medium, such as an agar medium containing H2Ow/v), is sterilized, for example in an autoclave, at about 120° C. for 15 minutes and cooled. Microorganisms are inoculated into this medium and cultured at, for example, about 37° C. for about 10 days, and the resulting spores are inoculated into an aqueous medium. The aqueous medium is, for example, approximately 1
It is sterilized at 20℃ for 15 minutes and contains malt extract (e.g.
3.3chi v/v), yeast extract (e.g. 2.04
w/v ) and ammonium dihydrogen phosphate (e.g. 0
．． 6% w/v). Until good growth of mycelium is observed, for example, when the medium is 500-
Incubate for 2 hours at 45°C.

Transfer the culture to the next medium. The medium composition used for this purpose is similar to that of the main medium described above, and is sterilized, for example, by steam. A typical example of a rod medium is the medium shown in Table 1 below. For example, using about 401 medium, about 50
C. for about 72 hours with agitation (e.g., 420 r, p, m,) and aeration (e.g., 50 l/min). Next, the seed culture is inoculated into the above-mentioned main medium by a conventional method.

The mutant strain of T. emersonii used for the purpose of the present invention is irradiated with X-rays, gamma rays, ultraviolet rays in the presence of a photosensitive substance such as 8-methoxyzopsoralen, silicon oxide, hydroxy amines, pyrimyline-based analogues such as 5-bromouracil, acridine, alkylating agents such as ethyl methanesulfonate, or chemicals such as mustard gas, heat treatment, or various genetic methods (recombination, transduction, (transformation, lysogenization, lysogenization, and random selection of mutant strains, etc.).

A simple method of measuring the reducing sugars produced by the action of the enzyme on the bentosan substrate can be used to measure the activity of other desired enzymes on the culture. As an example, the enzyme sample to be measured is appropriately diluted and heated for 10 minutes at 50 DEG C. in an acetate buffer at pH 5.0 containing excess bentosan xylan. In order to measure the produced sugar content in terms of malt name, 1 alkaline 3,5-dinitrosalicylic acid reagent was added, heated in a boiling water bath for 5 minutes, rapidly cooled in ice water, and the absorption at 540% m was measured. , convert the measured value in Malteau space. The standard calibration curve used for the conversion is obtained by reacting a known water maltose standard dilution with the dinitrosalicylic acid reagent under the conditions described above. # of 1 unit is 50℃,
Under test conditions of pH 5.0, sugar equivalent to maltose 19 is produced.

The present invention further provides a method of reducing the bentosan content of a bentosan-containing material. The method consists of contacting said material with an enzyme according to the invention.

Typical examples of bentosans that can be degraded by the enzyme of the present invention include those contained in grains such as wheat and oats, those contained in plants such as tea and coffee and their extracts, fruits, It is found in vegetables, seaweed, and sorghum. In some cases, the bentosan is xylan, such as that contained in oat hulls. It can also break down bentosan contained in wheat.

Accordingly, the present invention provides a method for reducing xylan in xylan-containing materials. The method consists of contacting said material with an enzyme according to the invention.

The enzyme composition provided by the present invention can be used in various methods known in the enzymatic art.

Thus, for example, the enzyme can be contacted with an aqueous medium containing the substrate. It may be in the form of a suspension, mixture or solution. The reaction temperature varies depending on the extractability of the materials used, but is generally 35-93°C, preferably in the high temperature range, and in some cases above 90°C. The pH of the reaction mixture is, for example, 4.0-6.5, preferably 4.0-6.5.
5-5.5 (pH around 5.0 2. If desired, α
- Other enzymes such as amylase can also be used in combination.

The butterbur strain of T. Emersonii used in the present invention is
Cλ(1-
C; Re-deposited as No. G-290604. Since the two strains are the same, either one may be used.

EXAMPLES In the following non-limiting examples illustrating the invention, temperatures are given in °C and /q-centages are given in w/v.

Example I Y-10 Mrs. Emanii IM1116815 was incubated at 37°C for 10 days on an agar medium sterilized in an autoclave (0.51 pezotons, 0.4'fi sodium chloride, 0°75% glycerin, o, s molasses, 0.006%
KH□PO4 and 0.005% Mg804-n
120) Te cultured. The spores produced were transferred to the medium in a flask. The medium is sterilized in an autoclave; 3.
3 T malt extract, 2.0 T yeast extract and 0.3 T malt extract.
Contained 6% ammonium dihydrogen phosphate.

The flasks were kept at 45°C for 3 days and incubated on a rotary shaker (210r, p, m; step 4.9).

The resulting culture (400 ml) was placed in a steam-sterilized culture medium 407 in a 5° stainless steel fermenter.
Transferred to medium A) in Table 1 and stirred at 50°C for 75 hours (4
20 r, p, m,) aeration (cultured at 501/min).

The resulting culture (4,5 l) was placed in a steamed medium (40 g) in a 501 stainless steel 7-armenter with a capacity of
Table 1: Transfer medium A) K and stir at 500C for 75 hours (
420 r, p, m,) aeration (507/min) culture.

The obtained culture (4, 51) was transferred to a steam-sterilized medium (40 A') in a stainless steel fermenter with a capacity of 5 ol.
; Transfer the medium ARK in Table 1 and stir at 50°C for 36 hours (4
Culture was carried out at 20 r, p, m, ) aeration (501!/min).

The obtained culture (16)) was transferred to a medium (32A'; IE1 table medium B) in a stainless steel fermentor with a capacity of 50A' and stirred vigorously ( 420r, p, m,) was cultured. An average of 1°5 of sterile water was added to the culture medium each day.

The concentration of extracellular purpura according to the present invention in the culture is 36 u/I
It was i.

Table 1 Ingredients Medium B (hook) Barley flour 1.0 3.38 Listilas solubles 2.25 5.06 Solkm Floe 2.25 2
．． 53(NH4)2H2PO40,340,77 to 2S
o4 0.12 0.27 Mg804・7H200,010,11Nice10
, 11 0 , 11 Mn80. '4H,OO,00050,0oIF e
S 04・7) (a OO-0040, OO8ZnSO
a ・7f(go 0 ,004 0.008CuSO
45) (,00,00050,001H,PO40,5
1002 Add water to 100% Example 2 Example of change in pentosanase to glucanase ratio during fermentation (a) Spores obtained by the method of Example 1 were transferred to a 250 Gof flask containing 40 mJ of medium A sterilized in an autoclave, Stirring at 45°C for 72 hours (20 Or, p, m,
) was cultured. The resulting culture (4 M) was transferred to autoclaved medium B2O in a 250 mJ flask and stirred (20 Or-p, m,) at 45°C for 12 days.
Cultured. After completion of the culture, the activity values of extracellular enzyme pentosanase (P) and barley β-glucanase (G) in the culture solution were measured, and the ratio of P:G was determined.

(b) Solca flocs were removed from the medium, oat-derived xylan (2.2%) was added, and the process proceeded to the above (&). The P2G ratio was 71 chi higher than that of (,).

Example 3 To measure the pentosanase activity of the enzyme obtained by the method described in Example 1, an ILs pentosan solution was maintained in a buffer at 50° C. and pH 5.0. A test system was prepared by adding enzyme to the solution, and a control system was prepared by adding an equal amount of water. The bentosan substrate used was xylan derived from oat hulls.

Samples of the test system were taken after 10 min, 30 min, and 22.5 h, rapidly cooled to prevent additional reactions, and spotted onto a 20 (JTYB square) thin layer chromatography plate coated with 0.25 Kieselder. .Selected standard sugar solutions and controls were also spotted.

The mobile phase was n-butanol/pyridine/ethanol/water (
20/15/25/10). Results were determined by spraying 5-thiosulfuric acid containing ammonium 5-thimolybdate onto the dry plates and then heating at 105° C. for 15 minutes.

All test products are Rf generated by standard monosaccharide solution Φ Jiro 7
The same spot as the value was shown.

This spot cannot be considered to be due to xylo-/min contained in the substrate or its chemical hydrolysates, but must be attributed to enzymatic decomposition of bentosan.

Example 4 The following comparative tests were carried out to demonstrate that the enzyme according to the invention can be applied to the enzymatic degradation of wheat starch.

Enzyme according to Example 1 (2me), low grade wheat flour (155,
S'), Sodium chloride (2,!/), Calcium chloride (0,17/), Bacterial alpha amylase (Product name: 'I'armamyl, Novo Indu)
mtriA/'S; 1 serving) and water (250ml)
was placed in a glass flask and thoroughly shaken. The pH of the solution was adjusted to 6.5 with caustic soda. To reduce evaporation, keep the flask in place for 5 hours.
It was kept at 0°C. Hydrolysis of starch was confirmed by YoP test. The viscosity of the decomposition product was 43a.

The final viscosity of the decomposition product of the control product according to the present invention without using enzymes is 1271! It was pI.

The following examples are for determining the properties of the enzyme according to the invention. In each example, an enzyme obtained by the method described in the example was used. However, small amounts of sodium benzoate and sodium pyrosulfite were added as bacteriological stabilizers. In each example, an aqueous solution of xylan derived from oat husk was used as a trap and bentosan substrate to measure enzyme activity, and after incubation for 10 minutes, the increase in reducing activity value was measured using a kyjirose calibration curve. .

The pH and temperature are listed in each example.

Example 5 Hydrolysis was observed.

Example 6 Enzyme samples were kept at 60, 70, 80, 90 and 95° C., no substrate was used, and the amount of # element was reduced in a sequential manner, and a series of records showing residual activity versus time was obtained. Residual activity at pH 5
, 0, measured at a temperature of 50°C.

At 60°C, no decrease in activity value was observed after 80 minutes.

At 70° C., 70 chi of the original activity remained after 1 hour.

At 80°C, 70% of the original activity value remained after 30 minutes.

At 90°C, 50% of the original activity value remained after 8 minutes.

At 95°C, 50+ of the original activity remained after 6 minutes.

Example 7 The relationship between temperature and enzyme activity was measured at pH 5.0.

The activity versus temperature curve showed that the peak activity value was at 87±20G. The activity at each temperature was measured and shown in the following table, with the maximum value set as 100.

As a result of the temperature of 4050607080 (87±2) 9095 or higher, as shown in Example 5, it is recognized that the enzyme according to the present invention has inherent thermostability. It is clear that the enzyme can be used at temperatures above 90°C.

In contrast, crude β-
The optimum temperature for glucanase is 60-70'C, so
At ℃, only 40 chips with a large activity value remain.

Example 8 In the case of a typical enzymatic degradation by an industrial method, in order to investigate whether the enzyme activity is affected by the presence of reaction products and other sugars, three kinds of Maltose) was added separately to the enzyme and then measured at 50'C1 pH5°O. An amount of each sugar was added corresponding to the amount of xylose produced from the substrate under the assay conditions.

As a result, no increase or decrease in enzyme activity was observed. It was therefore found that increasing the monosaccharide does not affect the enzyme activity, contrary to what occurs in many industrial applications.

Effects of the Invention It has been found that the enzyme according to the present invention has high productivity and can produce the desired enzyme at a high enough concentration for practical use.

The enzyme according to the invention can be used in a variety of applications where other pentosanases can be used. For example, when used in the production of wheat flour, the amount of natural pentozan in the flour is reduced, thereby improving the shelf life of the flour. When used in starch production or hydrolysis, the viscosity of starch slurry is reduced because the pentosan content of grains is reduced. This viscosity adversely affects starch production efficiency and cost, and is therefore also disadvantageous for hydrolysis. The enzyme can also be used in agriculture to improve the properties of animal feed, such as mixed feed fermented in silos. When used in brewing, for example, wheat,
It is possible to improve the production and extraction of fermentable sugars and the storage and processing of certain fermentation liquids when using wort containing sorghum.

The enzyme can also be used for the extraction of plant tissues, such as tea, coffee, fruit extraction, alginate extraction from seaweed, and the production of natural vegetable dyes. In view of the temperatures used for these purposes, the thermostability of the enzyme is particularly advantageous.

Claims (14)

[Claims] (1) Enzyme 0 obtained by fermentation of a microorganism belonging to Tayloromyces emersonii or its mutant strain that has the ability to catalyze the decomposition of bentosan. (2) When measured using oat coat-derived xylan as a sun substrate, the pH for optimal activity at 50°C is 5.0, and the temperature for optimal activity is 8.
7±2°C and has an activity remaining after heating at pH 5.0 for 6 minutes at about 95°C and about 50% of the original activity. (3) β-1,4/β-1, and an enzyme obtained by fermentation of a microorganism belonging to Tayloromyces emersonii or a mutant strain thereof having the ability to catalyze bentosan decomposition.
β-1,4/β-glucanase in the form of a mixture with β-1,4/β-glucanase, by modifying the medium in which both enzymes are produced.
2. The enzyme composition of claim 1, wherein the level of pentosanase activity is increased relative to the level of 1,3-glucanase activity. (4) The microorganism is Tayloromyces emmanii IM
I 116815 t or CMI-C:G-2906
04. The enzyme according to claim 1, which is 04. (5) a step of producing a culture containing an enzyme having pentosanase activity by culturing a microorganism belonging to Tayloromyces emersonii in a medium;
A method for producing a #element composition having pentosanase activity. (6) The method according to claim 5, wherein the level of pentosanase activity relative to the level of β-1,4/β-1,3-glucanase activity is increased by modifying the medium. (7) The method of claim 5, wherein the level of pentosanase activity relative to the level of β-1,4/β-1,3-glucanase activity is increased by a purification step subsequent to the fermentation step. (8) The microorganism is Tayloromyces emersonii IMI
116815 or GMI-CG-290604 or a mutant strain thereof. (9) A method for reducing the bentosan content of a bentosan-containing material, which comprises the step of contacting the bentosan-containing material with the enzyme according to claim 1 or the enzyme composition according to claim 3. (10) The method according to claim 9, wherein the bentosan-containing material is a grain selected from wheat or oat, a plant selected from teapot or coffee, or an extract thereof, or a fruit or vegetable. (11) The method according to claim 9, wherein the bentosan-containing material is starch. (12) The method according to claim 9, wherein the bentosan-containing material is wheat or a wheat-derived material. (13) Enzyme and bentosan-containing material at a temperature of 35-9
10. The method according to claim 9, wherein the contacting is carried out at 50C, pH 4.0-6.5. (14) Using a composition containing pentosanase derived from a microorganism belonging to Tayloromyces emersonii,
A method for reducing bentosan content in grains selected from wheat and oats, plants selected from tea and coffee or extracts thereof, fruits or vegetables.