JPH0387178A - Novel endodextrase, its production and production of isomaltooligosaccharide using the same enzyme - Google Patents

Abstract

NEW MATERIAL:Endodextrase. Action: hydrolyzes dextran in endo-formula and forms alpha-isomaltotriose; Substrate specificity: hydrolyzes bacterial polysaccharide dextran; Reactional pH: 4-11.5; Optimum pH: 6.4; Range of stable pH: pH4.5-10; Reactional temperature range: 20-55 deg.C; Optimum reactional temperature: 35 deg.C; Stability to temperature: 100% below 45 deg.C, exhibiting 25% residual activity at 50 deg.C and completely inactivates at 55 deg.C (treated at pH6.4 for 10min); Molecular weight: 88000; Isoelectric point: 4.5; Amino acid composition (mol%): Leu 6.82, Val 7.56, Ala 9.16, Gly 8.69, Glu 8.08, Asp 12.26, etc. USE:Efficiently produces isomaltooligosaccharide mainly composed of isomaltotriose and exhibits high titer. PREPARATION:The objective endodextrase is collected from the cultured substances of endodextrase-producing fungus belonging to genus Fusarium of mold fungus or mutant strain of said fungus [e.g. Fusarium sp. (FERM P-10976)].

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel endodextranase, a method for producing the same, and a method for producing isomalto-oligosaccharide using the present enzyme.

[Prior Art] In recent years, various oligosaccharides have attracted attention as functional food materials in the food industry. Isomaltotriose, a type of these oligosaccharides, is used as a difficult-to-tactile sweetener, a growth factor for bifidobacteria in the intestines, a research reagent, and as an effective moisturizer due to its extremely high retention properties. The development of an efficient manufacturing method is highly anticipated.

As a method for producing isomaltotriose, a method of partially hydrolyzing dextran with mineral acid has been known, but with this method, isomaltotriose cannot be obtained selectively and the yield is extremely low. low. Furthermore, the purification process was complicated and was not suitable as an industrial method.

As another method, production of isomaltotriose by an enzymatic method using dextranase, which hydrolyzes dextran and mainly produces isomaltotriose, is also being considered.

Brevibacterium fuscum (Brevibact) is an example of a dextranase that hydrolyzes dextran to mainly produce isomaltotriose.

erium fuscum var, dextran
Bacterial exo-enzyme [Bi
ochim, rib excretion □ Acta, 61-70 f1
974)] is known. However, dextranase originating from Brevibacterium was not suitable for industrial production due to the complicated purification process and low enzyme recovery rate and low specific activity of the purified enzyme.

[Problems to be Solved by the Invention] The present invention has been made in view of the problems of the prior art described above, and its purpose is to achieve high levels of endodextranase production by utilizing a new endodextranase-producing bacterium that is not derived from Brevibacterium. To provide a novel endodextranase with a high potency, 1) to provide a method for industrially producing endodextranase at a high yield using the endodextranase-producing bacterium; An object of the present invention is to provide a method for efficiently producing isomaltooligosaccharides mainly composed of isomaltotriose using dextranase.

[Means for Solving the Problems] The present inventors conducted a search for microorganisms that produce an enzyme that acts on dextran and produces isomaltotriose as the king, and found that the filamentous fungus Fusarium
The present invention was completed based on the recognition that a strain belonging to the genus I can efficiently produce an enzyme that mainly produces isomaltotriose by acting on dextran in large quantities outside the bacterial body.

The mycological properties of this strain are described below.

(1) Growth status on various culture media ■ Malt agar medium The growth rate of this fungus on malt agar medium is that the colony diameter reaches 0.5 to 1.5 cm in 13 days at 25°C.
It reaches 2.0-3.0 cm in 7 days. It is white and velvety in appearance, and conidia are well formed.

■Potato sucrose agar medium Honkan's growth rate on potato sucrose agar medium is
At 25℃ for 3 days, the colony diameter was 1.0-2.0.
cm, and reaches 3.0 to 5.0 cm in 7 days.

It is white and velvety in appearance, and conidia are well formed.

■Oatmeal agar medium Honkan's growth rate on oatmeal agar medium is 25°
The colony diameter reaches 0.5-1 cm in 13 days and 1.5-2.0 cm in 7 days. It is white and velvety in appearance, with few conidia formed.

(2) Physiological properties ■Growth pH It grows in the pl+ range of pl+4.5 to 8.0, but the optimum pH for growth is 5.0 to 6.5.

■It can grow in a temperature range of 15 to 45°C, but the optimal temperature for growth is 20 to 30°C.

■Other notable features Endodextranase is induced and produced extracellularly when cultured in a dextran-containing medium.

(3) Morphological characteristics ■ Conidiophore Conidia are lateral.

■Fear Ride It is a monophial ride.

■Conidia Contains large conidia and small conidia. Large conidia are 25~
It measures 32 x 4 to 5 ILm, has bent arms, and has shoulders. Small conidia have six large conidia that are 5-7 x 2-3 μm and small conidia have 0 to 3 septa.

Based on the above findings, Illustrated Encyclopedia of Fungi Part 1 and Part 2, Yoshitoshi Uda
This strain was classified as Fusarium sp., written by Keisuke Tsubaki, published in 1977 by Kodansha, etc.

The above strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under the contract No. 10976 (FERMP-10976).
) has been deposited as.

The gist of the method for producing endodextranase in the present invention is that the filamentous fungus Fusarium (
Endodextranase-producing bacteria belonging to the genus Fusarium or their mutant strains are inoculated into a culture medium and cultured under predetermined culture conditions.The cells are removed from the culture, and high-density The reason is that dextranase with a high titer can be collected at a high yield.

The properties of the novel endodextranase obtained by the present invention are described below.

(1) Action This enzyme produces dextran and mainly produces α-isomaltotriose.

(2) Substrate specificity This enzyme mainly hydrolyzes bacterial polysaccharide dextran. On the other hand, it has no effect on soluble starch, amylopectin, pullulan, curdlan, laminarin, xylan, etc.

(3) Optimal pH and stable pH range This enzyme acts over a wide range of pH 4.0 to 11.5, and the optimal pH is around 6.4 (0.25% Pharmacia Dextran T2O00 at 30"C) , allowed to act for 7 minutes).

In 5 mM KCl-NaOH buffer, the enzyme was adjusted to pH
11.0 also showed a relative activity expression of about 40%.

Therefore, it was confirmed that this enzyme expresses high activity even in a highly alkaline region. In addition, this enzyme has a pH of 4,
Stable over a wide range of pH 5 to 10.0 (25
After standing in mM McT]vaine buffer at 4°C for 24 hours, the residual activity of each was measured).

(4) Action temperature range and optimal action temperature This enzyme acts in the range of approximately 20 to 55°C, and the optimal action temperature is around 35°C (0.25% dextran T2O
00, pH 6, 4, 5 minute reaction).

(5) Temperature stability After heat treatment of the enzyme at pH 6.4 for 10 minutes, the residual activity of each enzyme was measured. Its concentration shows 100% residual activity up to 45°C and about 25% residual activity at 50"C.
It was almost completely inactivated at 55°C.

(6) Enzyme titer measurement method The reaction composition for endodextranase activity measurement is 1
% Dextran D2000 solution 0.5 ml, 25 m
MMcilvaine buffer (pH 6,4) 1.0
ml and 0.5 ml of enzyme solution, and reacted at 30°C for a certain period of time.

Reaction mixture 1. The reducing sugar produced by the enzymatic reaction is quantified per Oml using the Somogyi-Nelson method.

Under these conditions, the amount of enzyme that generates a reducing power equivalent to ILL mol of glucose per minute is defined as 1 unit.

(7) Endodextranase purification method 0 Fusarium sp. in a liquid medium at 25°C, 5
Culture using a jar fermenter for 1 day, and use the culture solution 3
℃ and high-speed refrigerated centrifugation (8,000×H) to sterilize. At 4°C, add cold ethanol to the two-seed liquid and incubate at 30-6
The fraction precipitated at 0% concentration was subjected to high-speed refrigerated centrifugation (8,000x
g) and collected. The ethanol precipitate was then immediately dissolved in 25 mM McIlvajne buffer (
A column packed with DEAE-1-Yobal 650S dissolved in pH 6.4 and sufficiently equilibrated with the same buffer (pH 6.4) was used.
4.4 x 55 cm). 100mM Mc
The active fraction eluted with Ilvaine buffer (pH 6) was collected. After concentrating and dialyzing both of these, 25mM
A column (+, 8
x 1'20 cm) and gel filtration was performed.

The resulting single active fraction was collected and concentrated to obtain highly purified endodextranase.

(8) Molecular weight 5DS-polyacrylamide gel electrophoresis (SOSPA)
The molecular weight of endodextranase estimated by the GE method is approximately 88,000.

(9) Isoelectric point (pT) 1, Isoelectric focusing lightning phoresis rack (110ml) manufactured by Kr3
The isoelectric point of purified endodextranase, measured using a column for sterilization, is 4.50.

(10) Inhibition and Activation The activity of the enzyme is inhibited by 5 mM of heavy metal ions such as mercury, silver, iron, copper, etc., and by the same concentration of N-bromosuccinimide.

On the other hand, the activity of the enzyme is activated by 5 mM calcium ions.

(11) Amino acid composition (mole ratio %) Arginine 4.24, Lysine 4.31. histidine 2
,■5, Phenylalanine 4.04, Tyrosine 373,
Leucine 6.82, inleucine 5.87, methionine 047, valine 7.56, alanine 9.16, glycine 8.69, proline 6.34, glutamic acid 8.08,
Serine 6.5+, threonine 7.31. Aspartic acid 12.26, tryptophan 1.94, cystine 0.5
2 Next, a culture method for producing the endodextranase will be described.

In order to produce dextranase, the filamentous fungus Fusarium (such as the above-mentioned filamentous fungus FusΩriom sp.
An endodextranase-producing bacterium belonging to the genus Fusarium or a mutant strain thereof is inoculated into a culture medium and cultured. Regarding the cultivation method of water spores, either liquid culture or solid-state culture is possible, but aerobic liquid culture is preferable for industrial production.

As the nutrient source for the culture medium of the bacteria, those commonly used for culturing microorganisms can be used. Furthermore, when bacterial polysaccharide dextran is used as the sole carbon source, the endodextranase can be induced to be produced.

As nitrogen sources, various amino acids, organic nitrogen-containing substances,
Meat extract, peptone, malt extract, urea, etc. can be used, and as inorganic nitrogen compounds, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonium sulfate, etc. can be used alone or in combination. others,
It is recommended to add inorganic salts, bicumins, etc. as appropriate.

A suitable culture temperature is 20 to 30°C, more preferably 24 to 27°C. pH at the start of culture is 4.5-8
．． pH 0 is suitable, but pH 5.0 to 6.0 is more preferable.
5 is good. The number of days for culturing is preferably 5 to 7 days.

A crude enzyme solution can be obtained by removing bacterial cells by centrifugation from a culture that has sufficiently produced dextranase under the above culture conditions.

The crude enzyme solution obtained in this way can be used as a product as it is, but the crude enzyme solution can be further processed by known separation and purification methods, such as ultrafiltration membrane concentration method, organic solvent precipitation with ethanol, etc. A purified enzyme solution of the endodextranase is collected by a method such as salting out method using ammonium sulfate, column chromatography fractionation method, affinity chromatography fractionation method, isoelectric focusing method, etc. alone or in an appropriate combination. Is possible. An example is shown below.

First, bacterial cells and the like in the culture solution are removed by centrifugation to obtain a cell-free supernatant. It is also possible to use this supernatant as crude enzyme. Add cold ethanol to this two-separate solution to precipitate at a concentration of 30-60% to 25mM.
Dissolved in Mcrlvaine buffer (pH 6,4),
r] E A E Toyopearl 650S column.

IF]lImMMcI]vainc buffer (pH 6,1
) is subjected to gel filtration using a Biogel P-100 column to obtain purified endodextranase.

The method for producing isoflu-1-oligosaccharide of the present invention is characterized in that the endodextranase thus obtained is allowed to act on dextran to produce isomalto-oligosaccharide mainly composed of isomaltotriose. In this case, endodextranase can act on dextran as a culture cultured as described above, as a crude enzyme solution prepared from the culture, or further as a purified enzyme.

The enzyme reaction for producing isomaltotriose using the endodextranase thus produced is preferably carried out at 30-50°C, p+ (5,0-8,0, reaction time 4-18 The reaction time and dextran concentration are preferably 52 to 8%.The composition of the produced sugars in the reaction solution is approximately 30% isomaltotetraose or higher, approximately 60% isomaltotriose, approximately 7% isomaltose, Glucose was about 3%.

Additionally, if the purified endodextranase is applied to dextran T2O00 which has been partially hydrolyzed with dilute hydrochloric acid etc., isomalto-oligosaccharides mainly consisting of isomaltotriose can be efficiently produced and obtained within a short period of time. found.

[Example] Next, J:Su, Kinai IVl will be specifically explained in the following example.

Example 1 Preparation of crude enzyme solution 1 10% dextran (octasaccharide 70), 0.2% NH4
N0. , O1%KH□PO4,0,05%ugso4
・7H□0.0.04% (:aC1z2H20,0,3
A culture medium (pH 5, 513°C) consisting of % polypeptone and 0.3% yeast extract was placed in a jar fermenter at 5C.
sp, 1.1 vvll, 25 at 110 rrlm
The cells were cultured at ℃ for 5 days. After culturing, the culture solution was subjected to high-speed cooling continuous centrifugation to remove bacterial cells and obtain a cell-free supernatant. This second serum liquid contained dextranase at approximately 5.2 Qt/ml. This activity is clearly higher than the dextranase (42 units/ml) produced by Brevibacterium mentioned above.

Example 2 Preparation of crude enzyme solution 2 The sterilizing solution obtained in Example 1 was treated with cold ethanol at a concentration of 30-60%, and the resulting precipitate was treated with 25 mM McI 1v.
aine buffer (pH 6.41) to obtain 50 ml of a crude enzyme solution. This enzyme solution contained 58.1 units/ml of endodextranase activity.

Example 3 Enzyme Purification Of the +11Ql stock solution obtained in Example 2, 48.4
ml of 25 mM McTIvainc i3-TJA
I'll [i, 41 A column packed with DEAE-1-Yopal 650S that has been sufficiently equilibrated with
After adsorbing the enzyme on 4.4x 55 cml, InnmM McTIvainc weak flI solution (
Elute at pHli, Il.

The active fractions were collected. After concentrating and separating this active fraction, 25 mM Mcllvair+a buffer (pI)
Gel filtration was performed using a column (1.8 x 120 cml) filled with Biogel P-100, which had been sufficiently equilibrated as described in et al. 4).

The resulting single active fraction was collected and concentrated in a collodion bag to obtain purified endodextranase. This purified enzyme was analyzed by polyacrylamide gel electrophoresis (PAGE).
) and SO3-polyacrylamide gel electrophoresis (S
When DS-PAGE) was performed, only a single protein staining band was observed in each case. Through these purification operations, it was revealed that the present endodextranase was purified to an electrophoretically homogeneous standard.

Table 1 does not show changes in the total protein amount, total enzyme activity amount, activity recovery rate, specific activity, etc. due to the above enzyme purification process. As shown in this table, 16. Omg was obtained with an activity recovery of 86.7%. The titer (specific activity) of this purified enzyme preparation is 152 units/mg
It was protein. Moreover, this purified enzyme had the above-mentioned properties.

(Left below) 8 Example 4 10α dextran T2O00 (manufactured by Pharmacia)
in 12.5 mM Mcllvaine buffer (pH 6).
, 4125 (] mm, Fusarium s
p, the original endodextranase per mg of substrate
．． 085 units were added and heated at 35°C for 30 minutes, 1.3, 6.
Reactions were carried out for 18.27 hours, respectively. Figure 1 shows the results of analysis of changes in the composition of sugar produced in the reaction solution over time by high performance liquid chromatography. The composition of the produced sugar at a reaction time of 30 minutes was approximately 47.8% isomaltotetraose or higher, approximately 51% isomaltotriose, approximately 1% isomaltose, and approximately 0.2% glucose. Further, the composition of the produced sugar after a reaction time of 6 hours showed a distribution of about 33% isomaltotetraose or higher, about 61% isomaltotriose, about 4% isomaltose, and about 2% glucose. Thereafter, the yield of isomaltotriose tended to decrease slightly as the reaction time progressed. Note that the conditions for high performance liquid chromatography are as follows.

Column: Am1nex HPX-42A (7,8mm
φx 300 mm10 Mobile phase: Deionized water flow rate -0.6 ml/min Column pressure: 30 kg/cm" Column temperature near 0°C Detector: Differential refractometer, manufactured by Kansei Seisakusho, product name rRID
-6AJ The structure of the branched oligosaccharide was confirmed by paper chromatography and high performance liquid chromatography using the acid partial hydrolyzate of dextran as a standard substance, and it was found that all of the branched oligosaccharides were isomalto-oligosaccharides consisting of α-1,6-glucosidic bonds. there were.

Example 5 Dextran T2O00 (manufactured by Pharmacia) 5.0
Dissolve g in 80 ml of 0.2N hydrochloric acid and incubate at 95°C for 4 hours.
After hydrolysis for a period of time, 2N sodium hydroxide was added to adjust the pH to 6.1. Deionized water was then added to bring the total volume to 125 ml. 1. Add 0.085 units of purified endodextranase per 1 mg of the above-mentioned dextran partial hydrolyzate and heat at 35°C for 30 minutes.
Reactions were carried out for 6.18 hours. Changes in the sugar composition produced in the reaction solution over time were analyzed using high performance liquid chromatography. The composition of the produced sugar at a reaction time of 30 minutes was about 19% isomaltotetraose or higher, about 51% isomaltotriose, about 15% isomaltose, and about 15% glucose. Furthermore, the composition of the produced sugar after a reaction time of 3 hours showed a distribution of about 8% isomaltotetraose or higher, about 60% isomaltotriose, about 15% isomaltose, and about 17% glucose.

Note that the conditions for performing high performance liquid chromatography are the same as in Example 4.

Example 6 10% sucrose, 0.05% MgSO4.7H70.0,
A liquid medium (pH 7.0, 121) consisting of 2% 2HP, 4.1% polypeptone, and cariogenic streptococci (Streptococcus
tococcus mutansl and incubated at 37°C.
Culture for 5 days. Insoluble substances produced in the culture solution were filtered off and thoroughly washed with deionized water. The suspension of the residue (approximately 80 ml) was concentrated under reduced pressure using a rotary evaporator and lyophilized to 60"C1. Through this series of operations, a lyophilized sample of insoluble glucan was obtained from the 1°C culture solution. 1.2 g of target was obtained.

25 mM McT containing 0.2% of the above insoluble glucan
lvaine buffer (pH 6,412, 4 ml, Fu
100 μm of purified endodextranase (8 liquids (2.5 units)) originating from Sarium sp was added and reacted at 37°C. Reducing sugars produced over time under this reaction condition were quantified using the Somogyi-Nelson method. did.

Additionally, the total sugar in the insoluble glucan was determined by the phenol-sulfuric acid method, and the decomposition rate of the insoluble glucan was calculated.

Combining the obtained results, Fusarium sp.
The endodextranase of origin is S L r e f
It was confirmed that about 25% of the insoluble glucan produced by O. c.

[Effect of the invention] As explained above, according to one aspect of the present invention, filamentous fungi F
A novel high-titer endodextranase produced by P. usarium sp.

According to another aspect of the present invention, an endodextranase-producing bacterium belonging to the genus Fusarium (Fusarium genus) or a mutant strain thereof is cultured to produce endodextranase from the culture in high yield. can be obtained industrially.

Furthermore, according to another aspect of the present invention, isomalto-oligosaccharides mainly composed of isomaltotriose can be efficiently produced by allowing the endodextranase to act on dextran.

This isomalto-oligosaccharide is used as a hard-to-tactile sweetener, preservative,
It can also be effectively used in the food industry, cosmetics industry, etc. as a factor that activates bifidobacteria in the intestine.

Furthermore, the endodextranase of the present invention has 5tre
It also has the ability to decompose insoluble glucans produced by tococcus mutans, so it can be used in toothpastes, denture cleaners,
It can also be used in oral hygiene-related products such as bad breath removers.

[Brief explanation of drawings]

FIG. 1 is a graph showing changes over time in the sugar composition produced when purified endodextranase was allowed to act on a 2% dextran T2O00 solution as a substrate.

Claims (1)

[Claims] 1. A novel endodextranase characterized by having the following physicochemical properties. (1) Action This enzyme hydrolyzes dextran in an endo-mode to mainly produce α-isomaltotriose. (2) Substrate specificity This enzyme mainly hydrolyzes bacterial polysaccharide dextran. (3) Optimal pH and stable pH range This enzyme acts over a wide range of pH 4.0 to 11.5, and the optimal pH is around 6.4. Also, pH 4.5 to 10.0
It is stable within the range of . (4) Action temperature range and optimum action temperature This enzyme acts in a temperature range of 20 to 55°C, and the optimum action temperature is around 35°C. (5) Temperature stability When this enzyme is heated at pH 6.4 for 10 minutes at various temperatures, it shows 100% residual activity up to 45°C, about 25% at 50°C, and is almost completely inactivated at 55°C. do. (6) Molecular weight The molecular weight of this enzyme is approximately 88,000. (7) Isoelectric point (p\I\) The isoelectric point of this enzyme is 4.50. (8) Amino acid composition (mole ratio %) Arginine 4.24, Lysine 4.31, Histidine 2.
15, phenylalanine 4.04, tyrosine 3.73,
Leucine 6.82, Isoleucine 5.87, Methionine 0.47, Valine 7.56, Alanine 9.16, Glycine 8.69, Proline 6.34, Glutamic acid 8.08
, serine 6.51, threonine 7.31, aspartic acid 12.26, tryptophan 1.94, cystine 0.
522, an endodextranase-producing bacterium belonging to the filamentous fungus Fusarium genus or a mutant strain thereof is inoculated into a culture medium, cultured under predetermined culture conditions, and endodextranase is extracted from the culture. A method for producing endodextranase, which comprises collecting the endodextranase. 3. A method for producing isomalto-oligosaccharides, which comprises allowing the endodextranase obtained by the method according to claim 2 to act on dextran to produce isomalto-oligosaccharides mainly composed of isomaltotriose. 4. The method for producing isomalto-oligosaccharide according to claim 3, wherein the endodextranase is made to act as a culture containing this enzyme, a crude enzyme solution prepared from this culture, or a purified enzyme.