WO2004078959A1

WO2004078959A1 - Pullulan breakdown enzyme, method for production thereof and use thereof

Info

Publication number: WO2004078959A1
Application number: PCT/JP2004/002567
Authority: WO
Inventors: Kazuhisa Mukai; Michio Kubota; Shigeharu Fukuda; Toshio Miyake
Original assignee: Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Priority date: 2003-03-04
Filing date: 2004-03-02
Publication date: 2004-09-16
Also published as: TW200502393A; JP2004261132A

Abstract

A novel pullulan breakdown enzyme; a method for producing said pullulan breakdown enzyme which comprises using a microorganism which belongs to Aureobasidium Genus and produces said enzyme; a method for degrading pullulan using said enzyme; and a method for producing a pullulan being reduced in its viscosity and/or its Mw/Mn ratio. The novel pullulan breakdown enzyme can be used for producing a pullulan which has a low viscosity and a small weight average molecular weight/number average molecular weight ratio and is easy to handle.

Description

Description Pullulan-degrading enzyme, its production method and application

The present invention relates to a pullulan-degrading enzyme, a method for producing the same, and a use thereof. More specifically, when acted on pullulan, an α′-1,4 darcoside bond on the reducing terminal side adjacent to an α′-1,6 glucoside bond is produced. A pullulan-degrading enzyme that reduces the viscosity by hydrolyzing it into an end-type, a method for producing the same, a method for degrading pullulan using the enzyme, and a microorganism having the ability to produce the enzyme and / or the enzyme The present invention relates to a method for producing pullulan in which the viscosity and / or the weight average molecular weight / number average molecular weight ratio is reduced. Background art

Pullulan is a viscous gelcan obtained by aerobically culturing Aureobasidium pullance (A ureobasidiumpu II u Ians) in a nutrient medium containing saccharides such as monosaccharides and oligosaccharides. It is a linear high molecular polysaccharide having a chemical structure in which maltotriose is used as a repeating unit and linked by a 1,6 glucoside bond, and usually has a wide molecular weight distribution width.

The industrial production method of pullulan is described in, for example, Japanese Patent Application Laid-Open No. 6-65302 and "Biotechnology and Bioengineering", Vol. Vol., Pp. 463 to 470, 1987, and the like. Usually, microorganisms having pullulan-producing ability are aerobic in a nutrient medium containing saccharides such as monosaccharides and oligosaccharides. After cultivation, the bacteria are removed and concentrated. Shrink and, if necessary, decolorize and desalinate and collect the pullulan-containing solution, or pulverize it and collect the pullulan-containing powder. Further, if necessary, high-purulance pullulan-containing substances can be obtained by performing molecular weight fractionation or separation by precipitation with an organic solvent.

Pullulan has characteristics such as water solubility, film forming properties, glossiness, transparency, gas barrier properties, oil resistance, salt resistance, tackiness, adhesion, moldability, edibility, and digestibility. In addition, it is used not only as a reagent, but also as a base material for various foods and drinks, cosmetics, pharmaceuticals, agriculture, forestry, fisheries and livestock products, industrial and industrial materials, adhesives, coatings, etc. It can be advantageously used in many applications as various molded products, for example, granules, tablets, sticks, films, sheets and the like. In particular, pullulan, which has a narrow molecular weight distribution range, is used as a plasma expander (see Japanese Patent Publication No. 61-214481) and a molecular weight standard reagent (see Japanese Patent Publication No. 2-485851). It is known to be useful in the fields of pharmaceuticals and chemicals.

Pullulan is generally a solution containing a relatively large molecular weight pullulan, although it is less viscous compared to other polysaccharides such as xanthan gum, cod gum, locust bean gum, sodium alginate, carboxymethylcellulose, etc. Because of its high viscosity, it has the drawback that when it is processed and used in various applications, it requires skill in handling and cannot be easily used in a wide range of applications. Furthermore, pullulan, which has a wide molecular weight distribution width, has a drawback that various properties such as film forming property and adhesiveness are likely to be poor. Therefore, a pullulan having the above-mentioned various properties, a low viscosity, and a narrow molecular weight distribution width is demanded in the art.

In order to obtain such pullulan, a method of hydrolyzing bonds between glucose molecules in pullulan by acid decomposition or enzymatic decomposition to fractionate pullulan having a desired molecular weight can be mentioned. Although acid decomposition is a simple method, There is a problem in that the taste is changed by the salt produced when neutralizing with acid after acid decomposition. In addition, when this salt is removed, the operation becomes complicated. Next, enzymatic degradation is a method that utilizes an enzyme that acts on pullulan to catalyze hydrolysis. Examples of enzymes include pullulanase (EC 3.2.1.41), amylase (EC) 3.2.1)) and dalcanase described in JP-B-53-31234.

The pullulanase is an enzyme that specifically hydrolyzes the 1,6 glucosidic bond in pullulan to finally produce maltotriose, and is composed of maltotriose and maltotriose units when acted on pullulan. Since oligosaccharides are formed relatively quickly, the molecular weight distribution of the obtained pullulan-containing material is wide. In general, the molecular weight distribution width can be represented by a numerical value (MwZMn) obtained by dividing a weight average molecular weight (hereinafter abbreviated as Mw) by a number average molecular weight (hereinafter abbreviated as Mn). The larger the ratio of, the wider the molecular weight distribution width. Conversely, as the ratio of Mw / Mn approaches 1, it means that the molecular weight distribution width is narrower, and when the ratio of MwZMn is 1, it means that the molecular weight is single. For example, if the hydrolysis of pullulan by Purura kinase, M w of about 3 9 5, the ratio of 000 at 1 \ 1 _¾ 7 ZM n is about 1.3 3 pullulan M w of approximately 4 6, 00 0 Hydrolysis to pullulan produces a pullulan-containing material with a broad molecular weight distribution with an MwZMn ratio of about 11.15. Such a pullulan-containing material having a wide molecular weight distribution is a miscellaneous mixture of pullulan having a molecular weight of thousands to hundreds of thousands, so that various properties such as film forming property and adhesiveness are often poor. . Next, when monoamylase (EC 3.2.1. 1) is allowed to act on pullulan, it only acts on the 1,4 glucoside bond in the maltotraose structure that is slightly present in the pullulan molecule. The disadvantage is that there is a limit to decomposing to low molecular pullulan and a large amount of enzyme is required. There is.

Furthermore, the glucanase disclosed in Japanese Patent Publication No. Sho 533-31234 is derived from Aspergillus Jless' Ashieragi (A spergi II usniger; ATCG 62,5). Pullulan-containing substances obtained by allowing this enzyme to act on pullulan have a wide molecular weight distribution width and a molecular weight of several thousand to several hundred thousand, similar to pullulanase. Since it is a miscellaneous mixture, various properties such as film-forming properties and adhesion are often poor.

In view of such a situation, it has been established that a method for producing pullulan having low viscosity and a narrow molecular weight distribution width, that is, having a small MwZMn ratio and easy to handle, has been established.

An object of the present invention is to establish a pullulan-degrading enzyme capable of producing pullulan having a low viscosity, a small MwZMn ratio and easy handling, a method for producing the same, and a use in view of the conventional technology. Disclosure of the invention

In order to solve the above problems, the present inventors have searched for an unknown pullulan-degrading enzyme, and have searched a wide range of microorganisms that produce the enzyme. Unexpectedly, the present inventors have found that a pullulan-producing bacterium, Aureobasidium ( Microorganisms belonging to the genus A ureobasidium not only synthesize pullulan, but also produce a novel pullulan-degrading enzyme that reduces the viscosity of the produced pullulan, and the pullulan-degrading enzyme has the following physicochemical properties: And a method for producing the pullulan-degrading enzyme, a method for degrading pullulan by causing the pullulan-degrading enzyme to act on pullulan, and a method for producing the pullulan-degrading enzyme by acting the pullulan-degrading enzyme on pullulan. Establish a method for producing pullulan with reduced viscosity and / or MwZMn ratio The present invention has been completed. Physicochemical properties of the pullulan degrading enzyme

(1) Action

When it acts on pullulan, it reduces the viscosity by hydrolyzing the O-1,4 darcoside bond at the reducing end adjacent to the 1,6 darcoside bond into an end form.

(2) Substrate specificity

Not pullulan only, 6 ³ - O-one glucosyl maltosyl Bok Riosu, 6 ⁴ - O-one glucosyl maltosyl Te Toraosu, 6 ⁵ one 0- a - Gurukoshi Le maltopentaose, 6 ³ - .sigma. one Mazoreto trio sill malto Triose, and 6 ³ — (9—1 (6 ³ — Ο— 1 malto triosyl-malto triosyl) Act on 1 malto triose.

(3) Molecular weight

SDS—PAGE, about 67,000 to about 107,000 daltons.

(4) Isoelectric point

In electrophoresis containing ampholine, pI about 3.6 to about 4.6.

(5) Optimum temperature

pH 4.0, around 40 ° C for 60 minutes.

(6) Optimal pH

3 5. C, reaction at 60 minutes, pH about 4.0 to about 4.5.

(7) Temperature stability

Holds at pH 4.0 for 60 minutes and stabilizes at around 35 ° C.

(8) pH stability

Stable at pH approx. 3.8 to approx. 6.7 after holding at 35 ° C for 60 min.

(9) Activity inhibition Activity is inhibited by 1 mM Hg2 ⁺ , Pb2 ⁺ and Fe3 ⁺ Brief description of the drawings

FIG. 1 is a diagram showing the effect of temperature on the activity of pullulan degrading enzyme derived from Aureopacidium 'pullulans ΪFO 6353.

FIG. 2 is a graph showing the influence of ρΗ on the activity of pullulan degrading enzyme derived from aureopasidium ■ pullulan ίFO 6353.

FIG. 3 is a graph showing the temperature stability of pullulan-degrading enzyme derived from Aureobasidium 'pullulans iFO 6353.

FIG. 4 is a diagram showing the pH stability of pullulan-degrading enzyme derived from Aureopacidium 'pullulans IFO 6353. BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the microorganisms that produce the pullulan-degrading enzyme of the present invention include, for example, Aureobasidium (Aureobasidium) fe, Aureobasidium (Famentas), Variety (Famentas), and Aureobas ιd ιumfermentans variety ferm θ ntans. A 0, Aureobasidium fermentan svarietyfusca IFO 640, Aureobasidium puvar J ulance (A ureobasidium pu II u I ans) Ϊ FO 6 3 5 3 And microorganisms such as Aureobasidiumpullulans, IrO446, and mutants thereof.

The medium used for culturing the above microorganisms may be any medium as long as these microorganisms can grow and have the ability to produce the pullulan-degrading enzyme of the present invention. Any of natural media may be used. As the carbon source, any of the above microorganisms can be used for growth, and is selected from, for example, starch, a partially degraded product thereof, glucose, maltose maltooligodisomaltooligosaccharide syrup, sucrose, fructose, isomerized syrup, and nectar. One or more kinds can be used. Nitrogen sources include, for example, inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen such as urea, glutamate, casein, peptone, yeast extract, corn and stipple. One or two or more selected from inclusions are used. In addition, as an inorganic component, for example, a phosphate, a magnesium salt, a potassium salt, a sodium salt, a j-unsalt, or the like is appropriately used. The concentrations of these carbon sources, nitrogen sources and inorganic components in the medium may be set as appropriate.

The culture conditions for the microorganisms are generally about 20 to about 35 ° C, preferably about 25 to about 30 ° C, and a pH of about 1 to about 9, preferably a pH of about 2 to about 8 It is usually performed aerobically, under the conditions chosen. The culturing time may be any time as long as the time at which the microorganism starts to proliferate, and is preferably from 20 hours to 150 hours. The concentration of dissolved oxygen in the culture solution is not particularly limited, but is preferably 0.5 to 20 ppm, and the flow rate is adjusted, agitated, oxygen is added to the flow, and the pressure inside the fermenter is adjusted. And other measures can be adopted. The culture system may be either batch culture or continuous culture.

After culturing the microorganism capable of producing pullulan-degrading enzyme of the present invention as described above, the pullulan-degrading enzyme of the present invention is recovered from the resulting culture. The enzyme of the present invention is mainly present in the orchid-removed solution obtained by removing the cells from the culture, and the bacteria-removed solution may be collected as a crude enzyme solution, or the whole culture may be used as the crude enzyme solution. Alternatively, the cells can be immobilized on a carrier as appropriate and used as a crude enzyme, and can be concentrated or purified according to a conventional method. From culture As a method for removing the cells, a known solid-liquid separation method, for example, a precoat filter, a membrane filtration method using a flat membrane or a hollow fiber membrane, a centrifugation method, or the like is appropriately employed. As a method for shaking, a salting out method of ammonium sulfate, an acetone and alcohol precipitation method, a membrane concentration method using a flat membrane or a hollow membrane, etc. are appropriately adopted. As the purification method, polyethylene glycol fractionation, ion-exchange column chromatography, water column chromatography, gel filtration chromatography, etc. are appropriately employed, and by appropriately combining one or more of these purification methods, An enzyme showing a single band by electrophoresis can be obtained.

The activity of the pullulan degrading enzyme of the present invention is measured as follows. Pull Run (trade name “Pull Run PI — 20”, sold by Hayashibara Shoji Co., Ltd.) by Stormeyer's method (S trumeyer, D.H., Analytical 'Baoke: East Trie (A na Iityea) IB iochemistry), Vol. 19, pp. 61-71, 1967), and the resulting reduced pullulan was obtained at a concentration of 2.0% (w / V ) and so as to 5 0 mM acetate buffer (dissolved in _P H 4. 0) as a substrate solution was added an enzyme solution 0. 4 ml to the substrate solution 0. 4 ml, at 3 5 ° C After holding for 60 minutes, the reaction was stopped by adding 0.05 ml of 1N sodium hydroxide solution, and the amount of reducing sugars generated in the reaction solution was measured using a modified Park-Johnson. Quantified by the method of Hizukuri et al., Carbohydrate Resear Gh, Vol. 94, pp. 205-213, 1989. The amount of enzyme required to produce a reduced sugar equivalent to 1 micole of glucose per minute under the above conditions was defined as 1 unit of enzyme activity.

As a substrate for the pullulan degrading enzyme of the present invention, pullulan having a larger Mw than that of pullulan obtained after the enzymatic reaction is usually used. Substrate The concentration is not particularly limited, and is preferably 1% (w / V) or more industrially. The reaction temperature may be appropriately set at a temperature at which the enzyme is not inactivated, for example, a temperature of 4 ° C to 50 ° C, preferably 25 ° C to 40 ° C. The reaction pH may be adjusted in the range of about pH 3.0 to about pH 7.0, and is preferably adjusted appropriately in the range of pH of about 3.0 to about 5.0. The reaction time may be appropriately selected depending on the progress of the enzyme reaction, and is usually about 0.1 to about 100 units of the enzyme per gram of the solid substrate, and the reaction time is about 0.1. About 100 hours.

The pullulan obtained in this way has a reduced viscosity to an appropriate level or a reduced ratio of MwZMn, preferably less than 3, and is easy to handle. Like conventional pullulan, it has water-solubility, film-forming properties, gloss, transparency, gas barrier properties, oil resistance, salt resistance, adhesiveness, adhesiveness, moldability, edibility, indigestibility, etc. ing.

The pullulan-containing solution obtained as described above can be decolorized with activated carbon, desalted with H-type and OH-type ion-exchange resins and concentrated to form a syrup-shaped pullulan product, or dried and powdered, according to a conventional method It is optional to make it into a shaped product. If necessary, further purification by molecular weight fractionation, separation by precipitation with an organic solvent, or the like can be advantageously carried out to obtain pullulan having a narrower molecular weight distribution width. The solution and powdered pullulan products and purified pullulan products obtained in this way can be advantageously used in many applications such as foods and drinks, cosmetics, pharmaceuticals, agriculture, forestry, fisheries and livestock products, as well as molecular weight standard reagents. it can.

Examples of foods and drinks include confectionery, lactic acid beverages, jellies, liquid foods, viscous beverages and seasonings, processed foods such as laver and delicacies, bifidobacterium growth promoters, dietary fiber foods, low-calorie foods, and film-like foods. Or cosmetics such as sheet foods, for example, toothpaste, milky lotion, cream, It can be advantageously used as a raw material or an intermediate product for packs, hairdressing products, shampoos, rinses, treatments, lipsticks, bath preparations, refreshing films in the mouth, etc., and as pharmaceuticals, for example, ointments, tablets, films, capsules, plasma substitutes, etc. Agriculture, forestry and livestock products include, for example, coated seeds, granulated pesticides, molded fertilizers, molded feed, etc., and industrial materials include, for example, paper processing materials such as sizing agents, sizing agents, wastewater treatment agents, welding rods, It can be used advantageously as a raw material or intermediate product for molds and the like. Hereinafter, the present invention will be described in detail by experiments. Experiment 1 Preparation of pullulan degrading enzyme from Aureobasidium 'pullulans Experiment 1 — 1 Culture of Aureobasidium' pullulans

Sucrose (trade name “Granu sugar”, manufactured by Taito Co., Ltd.) 10.0% (wZv), yeast extract (trade name “Yeast Extract S”, manufactured by Asahivir Co., Ltd.) 0.2% ( wZ v;), ammonium sulfate 0.06% (wZV), dibasic potassium phosphate 0.2% (wZV), sodium chloride 0.1% (wV), magnesium sulfate ■ 7-hydrate 0. 0 4% (w ZV), iron sulfate ■ 7-hydrate 0.000% liquid medium consisting of 1% (w V) and water 200 ml in 500 ml Erlenmeyer flasks, each autoclave Sterilize at 121 ° C for 15 minutes, cool, inoculate Aureobasidium pullance iFO 6353, and incubate at 27 ° C and 230 rpm for 48 hours. After shaking culture, the resulting culture was mixed with the whole to obtain a seed culture.

Sucrose (trade name “Gran sugar”, manufactured by Taito Co., Ltd.) 1 2.15% (w / V), yeast extract (trade name “Yeast Extract S”, Asahibi 0.2% (w / V), sodium glutamate monohydrate 0.12% (\ ^ ノ \), nanomonium phosphate 0.06% (wZv;), Ammonium sulfate 0.06% (w / v), dipotassium phosphate 0.15% (w / V), sodium chloride 0.05% (w / V), magnesium sulfate-7 hydrate 0. A liquid medium comprising 0.4% (NO), 0.001% (wZv) of iron sulfate '7-hydrate and water was prepared. Put about 20 L of this medium in a fermenter with a capacity of 30 and sterilize it at 115 ° C for 15 minutes, cool to 27 ° C, and reduce the seed culture to 5% (v / v). And cultured with aeration and stirring at 27 ° C for 96 hours. The resulting culture was centrifuged (8,000 rpm, 15 minutes) to separate the cells, and about 19 L of culture supernatant was obtained. Experiment 11-2 Measurement of the viscosity reducing ability of pullulan

Using the culture supernatant obtained in Experiment 1-1, the ability of pullulan to reduce the viscosity was measured. First, pullulan (trade name “Pullulan PI — 20”, sold by Hayashibara Shoji Co., Ltd.) is dissolved in 50 mM acetate buffer (pH 4.0) and contains 10% (wZv) of pullulan. A solution was prepared. To 1.5 ml of this pullulan-containing solution was added 0.5 ml of the culture supernatant obtained in Experiment 1-1, and the mixture was kept at 35 ° C for 180 minutes. 5) was added thereto, and the mixture was kept at 100 ° C. for 10 minutes to inactivate the enzyme, and then cooled to 30 ° C. The viscosity of the obtained reaction solution was measured using a rotary E-type viscometer (manufactured by Tokyo Keiki Co., Ltd.). At the same time, a solution treated in the same manner as above except that the solution was not kept at 35 ° C. for 180 minutes was used as a control solution, and its viscosity was measured in the same manner as above.

As a result, while the viscosity of the control solution was 2 14 cP, the viscosity of the reaction solution added with the culture supernatant obtained in Experiment 1-1 and kept at 35 ° C for 180 minutes was 167 cP, and the viscosity of the solution containing pullulan was It has been found that substances with the ability to reduce exist. Experiment 11-3 Measurement of pullulan degrading enzyme activity

Experiment 11 Ammonium sulfate was added to about 19 L of the culture supernatant obtained in Step 11 so as to obtain a saturation of 80% (w / V), salted out, and centrifuged (8,000 rpm, After 15 minutes), the supernatant was collected and filtered with filter paper to obtain about 22 L of the filtrate. With respect to this filtrate (about 22 L), pullulan degrading enzyme activity was measured according to the enzyme activity measuring method described in the definition of enzyme activity described above. Experiment 2 Purification of pullulan degrading enzyme Experiment 2-1 Hydrophobic chromatography

Approximately 22 L of the filtrate obtained in Experiment 13 was previously equilibrated with a 20 mM acetate buffer (pH 6.0) containing 2 M ammonium sulfate (Butyl-Toyopear I650). M ”gel (manufactured by Tosoichi Co., Ltd.) and applied to a column for hydrophobic chromatography (gel volume: 880 ml) (flow rate: 3 ml / min). The protein was eluted at a concentration gradient decreasing from 0 to 0 M. The pullulan degrading enzyme activity of the eluted fraction obtained was measured in the same manner as in Experiments 13 to 13. Pullulan degrading enzyme activity was detected in the eluted fraction at a concentration of about 1.2 M ammonium sulfate. Was collected as a pullulan-degrading enzyme active fraction (1). The pullulan-degrading enzyme activity fraction (1) was measured for its ability to reduce the viscosity of pullulan in the same manner as in Experiments 1-2, and was found to have pullulan viscosity-reducing ability.

Ammonium sulfate was added to the above pullulan-degrading enzyme-active fraction (1) to a final concentration of 2 M, and the “Buty I-To® The protein was subjected to hydrophobic chromatography (gel volume 30 ml) using a “yopear I650M” gel, and then the protein was eluted with a concentration gradient in which the ammonium sulfate concentration decreased linearly from 2 M to 0 M. . The obtained eluted fraction was measured for pullulan degrading enzyme activity in the same manner as in Experiments 13 to 13. Pullulan degrading enzyme activity was detected in the eluted fraction at a concentration of about 1.2 M ammonium sulfate, and this fraction was detected. Was collected as a pullulan degrading enzyme active fraction (2). Experiment 2-2 Ion exchange chromatography

This pullulan degrading enzyme active fraction (2) was dialyzed against 20 mM acetate buffer (pH 6.0), and the dialysate was centrifuged to remove insolubles. The sample was subjected to anion exchange chromatography (gel volume: 30 ml) using “DEAE—Toyopearl 65 S” gel, and then the concentration of sodium chloride increased linearly from 0 M to 0.3 M. The protein was eluted with a gradient. When the obtained eluted fraction was measured for the activity of pullulan degrading enzyme in the same manner as in Experiments 13 and 13, the activity of pullulan degrading enzyme was detected in the eluted fraction at a sodium chloride concentration of about 0.15 M. The fraction was collected as a pullulan degrading enzyme active fraction (3).

This pullulan-degrading enzyme active fraction (3) was dialyzed against 20 mM acetate buffer (pH 6.0), and the solution was purified using a “Mono QHR 5 No. 5” column manufactured by Amersham Bioscience. The protein was subjected to ion exchange chromatography (gel volume: 1 ml), and then the protein was eluted with a concentration gradient in which the concentration of sodium chloride increased linearly from 0 M to 0.3 M. When the pullulan degrading enzyme activity of the obtained eluted fraction was measured in the same manner as in Experiment 13-1, the pullulan degrading enzyme activity was detected in the eluted fraction at a sodium chloride concentration of about 0.15 M. This fraction was collected as a pullulan degrading enzyme active fraction (4). Pullulan degrading enzyme activity in each of the above purification steps, Table 1 shows the specific activity and yield.

table 1

The purity of the purified pullulan-degrading enzyme sample obtained as the pullulan-degrading enzyme fraction (4) was assayed by polyacrylamide gel electrophoresis, and confirmed to be a single band. It was found to be an enzyme preparation. Experiment 3 Properties of pullulan degrading enzyme

Using the purified pullulan degrading enzyme preparation (pullulan degrading enzyme active fraction (4)) obtained in the same manner as in Experiment 2, the following physicochemical properties were examined. Experiment 3-1 Action

Pullulan having an M w of about 422,000 was dissolved in 50 mM acetate buffer (pH 4.0) to a concentration of 16% (w / v) to obtain a substrate solution. A purified pullulan-degrading enzyme preparation was added to 1 OmI of the substrate solution so as to act on 0.1 unit per 1 g of the solid substrate, and the mixture was allowed to react at 30 ° C for 48 hours. This reaction solution was sampled in small amounts over time, the viscosity of the sampled reaction solution was measured in accordance with the method described in Experiment 1-2, and the Mw and Mn of pullulan were determined by the following high-performance liquid chromatography ( Hereinafter, abbreviated as HPLC. Based on the results of the analysis, the following calculation was made. HPLC conditions

(1) Column: Two columns of “TS K gel GM PWXL column” (manufactured by Tosoh Toshiki Company) are used.

(2) Eluent: water

(3) Column temperature: 30 ° C

(4) Flow velocity: 0. Sm l Zm in

(5) Detection: Differential refractometer "RID-10A" (manufactured by Shimadzu Corporation) Standard molecular weight product

Pullulan P—5 (Mw = 5, 900), Pullulan P—10 (Mw = 111, 800), Pullulan P—20 (Mw = 22, 800), Pullulan P-50 (M w = 47, 3 0 0), Pullulan P-100 (M w = 1 1, 000), Pullulan P-200 (M w = 2 1 2, 00 0), Pullulan P-400 (Mw = 404, 000), Pullulan P-800 (Mw = 788, 00), Pullulan P-1600 (Mw = 1, 600, 0) 0), and Pullulan P-250 (Mw = 2, 520, 000) (all manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) were used.

Calculation method of M w and M n

(1) Creating a molecular weight curve

Each pullulan molecular weight standard was subjected to the above-mentioned HPLC, and a molecular weight curve was created with the elution time of each molecular weight standard on the horizontal axis and the common logarithm of the Mw of each molecular weight standard on the vertical axis. I asked.

(2) Calculation of M w and M n The fractionated reaction solution was subjected to the above-described HPLC, and the time from the start time to the end time of all detected peaks was divided at intervals of 0.2 minutes, and the area (S i) of each divided section was determined. Next, the intermediate value of the elution time of each divided section was inserted into the regression equation of (1), and the corresponding molecular weight (M i) was calculated. The obtained values of S i and M i were entered into Equations 1 and 2, and M w and M n of the fractionated reaction solution were calculated.

(Equation 1) Mw = ∑ (S i x i) / ∑ S i

(Equation 2) M n = ∑ S i / ∑ (S i / M i)

Table 2 shows the obtained results.

Table 2

As is clear from the results in Table 2, it was found that the addition of the pullulan-degrading enzyme of the present invention reduced the viscosity of the solution containing pullulan and the Mw and Mn of pullulan over time. Further, since the viscosity of the pullulan-containing solution was significantly reduced in the initial stage of the reaction, it was revealed that the pullulan-decomposing enzyme of the present invention acts on pullulan in an end type. Experiment 3—2 Substrate specificity

Using various carbohydrates as substrates, the action of purified pullulan degrading enzyme preparations was tested. table Each of the carbohydrates described in 3 is dissolved in water to prepare an aqueous solution of the substrate at a concentration of 1 o / o (wZv), and the purified solution of purified pullulan-degrading enzyme is added to this substrate solution in 1 g of solid substrate. Each unit was added, and the yeast was allowed to react at 27 ° C. and pH 4.0 for 16 hours. In order to investigate the presence or absence of enzymatic action on each carbohydrate, a mixture of n-butanol, pyridine and water (volume ratio 6: 4: 1) was used as a developing solvent, and “Kieselgel 60” (manufactured by Merck) as a thin plate Silica gel thin layer chromatography (hereinafter abbreviated as TLG) using an aluminum plate (10 x 20 cm) was performed, and the reaction product was detected by the sulfuric acid-methanol method. Table 3 shows the results.

Table 3

Substrate Enzyme 1 Use Substrate Substrate Action Malto "Nigerose 1 Maltotriose Isomaltose-Maltotetraose 1 Isomaltotriose 1 Maltopenose Oxide 1 Dextranose-Maltohexaose 1 Amylose-Maltoheptaose- Soluble starch

Isopass-Pullulan ++ Cooma

No, north b ~ Ό ~ kunorekonrumororatoru 31.10, otrehalose 6,100-α-glucosinoremaltotetraose + + + treha mouth 6. One 0—a—Darcosyl maltopentaose + Kojibiose 6 ³ — 0—One maltotriosyl maltotriose + +

6 ^a -0- a-(6 ^a — 0— a

-Maltotriosyl) -maltotriose + + + Note)

"One" indicates "no change",

`` + J indicates `` substrate spots are slightly reduced and other products are observed ''

"++ J indicates that" substrate spots are significantly reduced and other products are found "

"10 ++ J" indicates that "substrate spots have almost disappeared and other products are observed".

As is clear from the results shown in Table 3, pullulan decomposing enzymes of the present invention, 6 ^three to .sigma. 0 -? Glucosyl maltosyl triose, 6 ⁴ - .sigma. one Darukoshiruma Rutote Toraosu, 6 ⁵ - .sigma. Gurukoshirumaru Bok pentaose, 6 ³ — σ— one malto triosil malto triose, 6 ³ — σ— one (6 ^3- σ-a? -maltotriosyl-maltotriosyl) It was found to act on maltotriose and pullulan. For these saccharides, the solution after the enzyme reaction is subjected to gas chromatography (hereinafter abbreviated as GC method j), and the retention time of the reaction products is compared with the retention time of known saccharides. The GC analysis was performed using a GC device, “GC-16A” manufactured by Shimadzu Seisakusho Co., Ltd., and the column was manufactured by “El” Science Co., Ltd. Using a stainless steel column (3 mm 0 x 2 m) filled with 2% silicon OV—17 / chromosolve, a nitrogen gas as a carrier gas is flowed at a flow rate of 40 mI / min, and the temperature is reduced to 160 ° C. The temperature was raised from 7.5 ° C to 32 ° C at a rate of 7.5 ° CZ, and the detection was performed using a flame ion detector.

Table 4

Table 4 Results by Li, pullulan decomposing enzymes of the present invention is not pullulan only, 6 ³ - .sigma. one Darukoshirumaruto Bok Riosu, 6 ⁴ -. .Sigma. one glucosyl Marutote tiger Orth, 6 ⁵ - O-one Darukoshirumaru Bok Pentaose, 6 ³ — O— and one malto triosyl malto trios, 6 ³ — Ο— Of — (63 ³ — O— one malto triosyl malto triosyl) found. Further, the pullulan degrading enzyme of the present invention has a In the structure of the substrate shown in Fig. 4, it was found that it has an action of hydrolyzing the 1,1 darcoside bond on the reducing terminal side adjacent to the -1,6 glucoside bond. Experiment 3—3 Molecular Weight

The purified pullulan-degrading enzyme sample was subjected to SDS-polyacrylamide gel electrophoresis, and the molecular weight of the enzyme was measured by comparing it with the molecular weight marker (manufactured by Nippon Bio 'Lad' Laboratories Co., Ltd.) which was simultaneously electrophoresed. A single band was detected in the range of about 67,000 to about 107,000 daltons. Experiment 3—4 Isoelectric point

The purified pullulan-degrading enzyme preparation was subjected to isoelectric focusing polyacrylamide gel containing 2.2% (w / V) amphorine (manufactured by Amersham Biosciences), and electrophoresed simultaneously. When the isoelectric point of the enzyme was determined in comparison with 'Bioscience'), the pI was about 3.6 to about 4.6. Experiment 3—5 Optimum temperature

Investigation was performed according to the activity measurement method. As shown in FIG. 1, the result is a reaction at pH 4.0 for 60 minutes, and the optimum is about 40 ° C. Experiment 3-6 Optimal pH

Investigation was carried out according to the activity measurement method. As a result, as shown in FIG. 2, the reaction was carried out at 35 ° C. for 60 minutes, and the pH was optimally about 4.0 to 4.5. Experiment 3—7 Temperature stability

The enzyme solution was kept at each temperature for 60 minutes, cooled with water, and the remaining enzyme activity was determined according to the activity measurement method. As shown in Fig. 3, the result is stable at about pH 4.0 at around pH 35 ° C. Experiment 3—8 pH stability

After maintaining the enzyme solution in 100 mM acetate buffer of each PH at 35 ° C for 60 minutes, adjust the pH to 4.0, and measure the remaining enzyme activity according to the activity measurement method. I checked. The results are stable at a pH of about 3.8 to about 6.7, as shown in FIG. Experiment 3—9 Inhibition of activity

Investigation was carried out according to the activity measurement method. The activity is inhibited by 1 mM Hg2 ⁺ , Pb2 ⁺ and Fe3 ⁺ . Experiment 4 Pullulan decomposition test

[\ 1 \ «was added to an aqueous solution containing about 35,000 pullulan (final concentration 1% (wZv)), and the purified pullulan-degrading enzyme preparation obtained in Experiment 2 was added at 0. The reaction solution, which was operated for 1 unit and kept at 30 ° C and pH 4.0 for 24 hours, was sampled with time. After heating the sampled reaction solution at 100 ° C for 10 minutes to deactivate the enzyme, the molecular weight of pullulan in the reaction solution was determined using the TSKgeI GM PWXL column described in Experiment 3-1. ] (Tosoh Co., Ltd.) was used to measure the sugar content, and the sugar composition was determined by using two “MC gel C04 SS columns” (manufactured by Mitsubishi Chemical Corporation). HPLC was performed using water as eluent, column temperature was 80 ° C, flow rate was 0.4 mI Zmin, and detection was differential refraction. Analysis was performed using a total RID-10A (manufactured by Shimadzu Corporation). Simultaneously, pullulanase (trade name "Promozym 400", manufactured by Nopozymes Japan Ltd.) was applied to pullulan in the same manner as above except that the reaction pH was changed to 5.0. The molecular weight of pullulan in the reaction solution was measured and the composition was analyzed. Table 5 shows changes in the molecular weight and sugar composition of pullulan by the pullulan degrading enzyme of the present invention, and Table 6 shows changes in the molecular weight and sugar composition of pullulan by pullulanase.

Table 5

D in the table means the degree of glucose polymerization. Table 6

DP in the table means the degree of glucose polymerization.

From the results in Tables 5 and 6, when pullulanase was allowed to act on pullulan, the Mw / Mn ratio of the solution containing pullulan increased from about 4 to 11, whereas the pullulan of the present invention increased. When a degrading enzyme is applied to pullulan, the ratio of Mw ZM n. Is maintained at less than 3, and pullulan having a lower molecular weight than pullulan as a substrate can be produced without increasing the molecular weight distribution of pullulan. Clear I'm sorry. Furthermore, when 1 \ 1 \ ^ produces about 46,000 pullulan, the amounts of oligosaccharides produced by both enzymes with a degree of glucose polymerization (DP) 21 or less are compared. In the case of pullulanase, the value was 1.8%, whereas in the case of pullulanase, it was 11.3%, which was about 7 times the value of the pullulan-degrading enzyme of the present invention. From the above results, the pullulan-degrading enzyme of the present invention has the property of easily decomposing pullulan in a unit having a high degree of glucose polymerization, and therefore, without increasing the molecular weight distribution width of pullulan, this property was obtained. It was presumed to produce low molecular pullulan rather than pullulan as a substrate. Incidentally, when the pullulan degrading enzyme of the present invention was allowed to act on the polymer pullulan-containing solution having a large ratio of MwZMn of about 10 as a substrate solution, the Mw and Mn of pullulan were similarly determined as described above. Gradually decreased, and the viscosity of the solution containing pullulan and the ratio of MwZMn also gradually decreased. Hereinafter, examples of the present invention will be described. Example 1

Pullulan (1 \ 1 \ / \ / is about 458,000, 1 \ 1 门 is about 66,800, and the ratio of Mw / Mn is about 6.9) / V) was dissolved in 100 mM acetate buffer (PH 4.0) to prepare a substrate solution having a viscosity of 120 cP. To this substrate solution, add the purified pullulan-degrading enzyme preparation obtained by the method of Experiment 2 so that it acts at 0.1 unit per gram of solid pullulan, and hold at 30 ° C for 8 hours to decompose the pullulan. A pullulan solution was obtained.

This product is a pullulan solution with low viscosity of 8.6 GP, low Mw / Mn ratio of 3.9, narrow molecular weight distribution width, and easy to handle. Gloss, transparency, gas barrier properties, oil resistance, salt resistance, stickiness, adhesion, moldability, edibility, indigestibility, etc., as well as molecular weight standard reagents, food and beverages, cosmetics , Pharmaceuticals, agriculture, forestry and livestock products, It can be advantageously used in many applications such as industrial materials. Example 2

A pullulan solution was obtained in the same manner as in Example 1 except that the holding time of 8 hours was set to 24 hours.

This product has a low viscosity of 4.7 cP and a ratio of Mw / Mn of 2.9, has a narrow molecular weight distribution width, and is an easy-to-handle pullulan solution. , Gloss, transparency, gas barrier properties, oil resistance, salt resistance, stickiness, adhesion, moldability, edibility, indigestibility, etc. It can be advantageously used in many applications such as cosmetics, pharmaceuticals, agriculture, forestry, fishery products, and industrial materials. Example 3

Aureobasidium 'purulance was cultured in the same manner as in Experiment 11 except that the culture time was 72 hours. Subsequently, the pH of the culture was adjusted to 4.5 using sodium hydroxide, and the temperature was set to 35 ° C. The pullulan-degrading enzyme obtained by the above method was added in an amount of 0.05 unit per 1 mI of the culture solution, and maintained for 4 hours with gentle stirring to decompose pullulan. The resulting culture was centrifuged (8,000 rpm, 15 minutes) to separate the cells, and a supernatant containing pullulan was obtained. It was pulverized to obtain pullulan powder.

This product is a pull-run that has an M π of about 100,000 and a ratio of VI w M n of 2.85 to 3.85, has a molecular weight distribution width, and is easy to handle. Water-soluble, film-forming, glossy, transparent, gas-barrier, oil-resistant, salt-resistant, tacky, adhesive, moldable, edible, resistant to digestion, etc. Not only can it be used as a standard reagent, but it can also be advantageously used in many applications, such as food and drink, cosmetics, pharmaceuticals, agriculture, forestry, fisheries, and industrial materials. Example 4

A pullulan powder was obtained in the same manner as in Example 3, except that the 4-hour holding time was changed to 10 hours.

This product is an easy-to-handle pullulan with a Mn of about 40,000 and a ratio of MwZMn of 2.90, a narrow molecular weight distribution width, and water solubility, film forming property, It has glossiness, transparency, gas barrier properties, oil resistance, salt resistance, adhesiveness, adhesiveness, moldability, edible properties, indigestibility, etc. It can be used to advantage in many applications such as cosmetics, pharmaceuticals, agriculture, forestry, fisheries, and mining materials. Industrial potential

As described above, the present invention relates to a pullulan-degrading enzyme, a method for producing the same, and a use thereof. More specifically, when acted on pullulan, the present invention relates to a 1,1 darcoside on the reducing terminal side adjacent to a 1,6 glucosidic bond. Use of pullulan-degrading enzyme that hydrolyzes the bond into an end form and reduces its viscosity, a method for producing the same, a method for degrading pullulan using the enzyme, and a microorganism having the ability to produce the enzyme or the enzyme And a method for producing pullulan having a low viscosity and a narrow molecular weight distribution width. According to the present invention, pullulan having a low viscosity and a narrow molecular weight distribution width can be easily produced without a change in taste quality. Such pullulan has a low viscosity, a small ratio of Mw / Mn, is easy to handle, and is water-soluble, film-forming, glossy, transparent, gas-poor, oil-resistant, salt-resistant, Adhesive, adhesive, moldable, edible, indigestible, etc., not only reagents, but also foods, beverages, cosmetics, pharmaceuticals, It can be advantageously used for many purposes such as agriculture, forestry and livestock products, and industrial materials.

The present invention is an invention exhibiting such remarkable functions and effects, and is a very significant invention that contributes to the art.

Claims

The scope of the claims

1. Pullulan degrading enzyme having the following physicochemical properties:

(1) Action.

When acted on pullulan, the viscosity is reduced by hydrolyzing the 1,1 darcoside bond at the reducing end adjacent to the 1,6 darcoside bond into an end type.

(2) Substrate specificity

Not pullulan only, 6 ³ - σ- over glucosyl malt triose, 6 ⁴ - 0 - one glucosyl Marthe Te Toraosu, 6 ⁵ - σ- one Gurukoshi Rumaru Bok pentaose, 6 ³ - Ο- Fei one maltotriosyl Marthe trio over 6 ³ — σ— 1 (6 ³ — σ— 1 malto triosyl-malto triosyl) Acts on 1 malto triose.

(3) Molecular weight

SDS—PAGE, about 67,000 to about 107,000 daltons.

(4) Isoelectric point

In ampholine-containing electrophoresis, pI about 3.6 to about 4.6.

(4) Optimum temperature

pH 4.0, around 40 ° C for 60 minutes.

(5) Optimal pH

In a reaction at 35 ° C for 60 minutes, the pH is about 4.0 to about 4.5.

(6) Temperature stability

Holds at pH 4.0 for 60 minutes and stabilizes at around 35 ° C.

(7) pH stability

Stable at pH approx. 3.8 to approx. 6.7 after holding at 35 ° C for 60 min.

(9) Activity inhibition The activity is inhibited by 1 mM Hg2 ⁺ , Pb2 ⁺ and Fe3 ⁺ .

2. The pullulan-degrading enzyme according to claim 1, wherein the pullulan-degrading enzyme is an enzyme derived from a microorganism belonging to the genus Aureobasidium.

3. A microorganism belonging to the genus Aureobasidium capable of producing the pullulan-degrading enzyme according to claim 1 or 2, is cultured, and the pullulan-degrading enzyme is collected from the culture. A method for producing a pullulan-degrading enzyme.

4. A method for degrading pullulan using the pullulan degrading enzyme according to claim 1 or 2.

5. The pullulan degrading enzyme according to claim 1 or 2, and Z or a microorganism having the pullulan degrading enzyme activity are brought into contact with a pullulan-containing solution, and the viscosity and the ratio of Z or weight average molecular weight Z number average molecular weight A method for producing pullulan, which comprises producing pullulan with reduced amount and collecting the pullulan.

6. The method for producing pullulan according to claim 5, wherein the ratio of the weight average molecular weight and the Z number average molecular weight is less than 3.