JP4197604B2 - Novel strain, novel α-glucosidase and method for producing the same - Google Patents

Description

【００２９】
【発明の効果】
本発明により、新規菌株であるアクレモニウム・エスピー（Acremonium sp.） YW1株が提供される。この菌株から産生されるα-グルコシダーゼは、α-１,３-グルコシド結合を効率的に生成することができる。このα-グルコシダーゼにより、α-１,３-グルコシド結合を有するニゲロオリゴ糖を得ることができる。
【図面の簡単な説明】
【図１】アクレモニウム・エスピー（Acremonium sp.） YW1株の産生するα-グルコシダーゼの反応至適温度を示す図である。
【図２】アクレモニウム・エスピー（Acremonium sp.） YW1株の産生するα-グルコシダーゼの反応至適ｐHを示す図である。
【図３】アクレモニウム・エスピー（Acremonium sp.） YW1株の産生するα-グルコシダーゼの温度安定性を示す図である。
【図４】アクレモニウム・エスピー（Acremonium sp.） YW1株の産生するα-グルコシダーゼのｐH安定性を示す図である。
【図５】アクレモニウム・エスピー（Acremonium sp.） YW1株の産生するα-グルコシダーゼを２０％マルトースに反応させた際のHPLC分析チャートである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel α-glucosidase, a method for producing the same, and a novel strain having a novel α-glucosidase production ability. More specifically, the present invention relates to an α-glucosidase that generates an α-1,3-glucoside bond by a transglycosylation reaction and does not generate an α-1,6-glucoside bond, a method for producing the same, and the novel α-glucosidase. The present invention relates to a novel strain having production ability.
[0002]
[Prior art]
α-Glucosidase is an enzyme possessed by many microorganisms, animals and plants, but its substrate specificity is wide. At present, α-glucosidase derived from Aspergillus is used for industrial production of isomaltoligosaccharides. However, in this transglycosylation reaction using α-glucosidase, an isomaltoligosaccharide having an α-1,6-glucoside bond is obtained as a main product, and other isomaltoligosaccharides having various α-glucoside bonds are simultaneously produced. Generated. Therefore, an oligosaccharide mixture having various binding modes is finally obtained.
[0003]
In recent years, in the synthesis of various oligosaccharides and glycosides, there has been a demand for an enzyme that can efficiently generate a target glucoside bond and obtain a target reaction product with high specificity. In particular, with regard to nigerooligosaccharide in which glucose is bound by an α-1,3-glucoside bond, there are specific functionalities such as an immunoregulatory function (see Non-Patent Document 1) and a natural pigment fading-inhibiting effect (see Patent Document 1). Has been found. Therefore, a highly specific enzyme that efficiently generates an α-1,3-glucoside bond has been demanded.
[0004]
An enzyme derived from Acremonium has been reported as a glycosyltransferase that catalyzes a glycosyltransferase reaction that efficiently generates an α-1,3-glucoside bond (see Patent Document 2). The enzyme described in Patent Document 2 acts on a substrate selected from starch and degradation products thereof, and has an activity of binding a glucose unit to α-1 → 3 at the 3-position of the non-reducing terminal glucose part, and thereafter It is a glycosyltransferase having an activity of reducing the molecular weight of an oligosaccharide obtained by cleaving adjacent α-1 → 4 bonds while maintaining α-1 → 3 bonds.
[0005]
[Non-Patent Document 1]
S. Murosaki et al., International Immunopharmacology 2, 2002, Netherlands, p. 151-159
[Patent Document 1]
Japanese Patent Laid-Open No. 2000-189101 [Patent Document 2]
Japanese Patent Application Laid-Open No. 5-59559
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to efficiently generate an α-1,3-glucoside bond, which can efficiently generate an oligosaccharide or glycoside such as a nigerooligosaccharide having an α-1,3-glucoside bond. To provide a novel α-glucosidase that can be produced. Furthermore, this invention aims at provision of the novel strain which can be used for the manufacturing method of such a novel alpha-glucosidase, and its manufacturing method.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors searched for α-glucosidase that newly generates an α-1,3-glucoside bond efficiently. As a result, a new strain belonging to the genus Acremonium produces a new α-glucosidase that efficiently generates α-1,3-glucoside bonds and does not generate α-1,6-glucoside bonds. The headline and the present invention were completed.
[0008]
Furthermore, the present inventors searched for microorganisms that produce the novel α-glucosidase from known strains, and acquired Acremonium genus Acremonium (A. luzulae), Acremonium fusidioides (A. fusidioides), A. sclerotigenum, and A. implicatum strains produce α-1,3-glucoside bonds efficiently and α-1,6-glucoside bonds The present inventors have found that α-glucosidase that does not produce glycan is produced and completed the present invention.
[0009]
That is, the present invention is as follows.
(1) Acts on a carbohydrate having an α-1,4-glucoside bond at the non-reducing end, generates an α-1,3-glucoside bond by transglycosylation, and forms an α-1,6-glucoside bond. An α-glucosidase characterized by not producing.
(2) Glucose is produced by hydrolysis reaction of α-1,4 glucoside bond, α-1,3 glucoside bond, α-1,2 glucoside bond, α-1,6 glucoside bond -Glucosidase.
(3) α-glucosidase having the following physicochemical properties.
(1) Action It acts on a carbohydrate having an α-1,4-glucoside bond at the non-reducing end, and generates α-1,3- and α-1,4-glucoside bonds by transglycosylation. -Does not produce 1,6-glucoside bonds. Glucose is produced by a hydrolysis reaction.
(2) A tetramer of 440000 is composed of two polypeptides having molecular weights of 51000 and 60000 as one unit.
(3) Isoelectric point pI 4.5
(4) Optimal temperature 50 to 55 ° C
(5) Optimum pH
pH 7.0
(6) Temperature stability Shows a residual activity of 90% or more at 45 ° C. or less.
(7) pH stability Shows a residual activity of 90% or more at pH 6-11.
(4) An α-glucosidase characterized in that the microorganism belonging to the genus Acremonium is cultured and the α-glucosidase according to any one of (1) to (3) is collected from the obtained culture. Production method.
(5) said microorganism is Acremonium sp. YW1 strain, A. luzulae, A. fusidioides, A. sclerotigenum, or The method for producing α-glucosidase according to (4), which is Acremonium implicatum (A. implicatum).
(6) Acremonium sp. YW1 strain.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The novel α-glucosidase of the present invention acts on a carbohydrate having an α-1,4-glucoside bond at the non-reducing end, and generates an α-1,3-glucoside bond by a transglycosylation reaction. , 6-glucoside bond is not produced.
The conditions for the transglycosylation reaction will be described in Example 4 described later.
Here, the saccharide having an α-1,4-glucoside bond at the non-reducing end is, for example, maltooligosaccharide or starch.
[0011]
Furthermore, the α-glucosidase of the present invention includes malto-oligosaccharides, oligosaccharides having different binding modes such as cordobiose and nigerose, artificial substrates such as paranitrophenyl-α-maltoside (pNPαG2), and weak but isomaltose and Glucose is produced by hydrolysis reaction of paranitrophenyl-α-glucoside (pNPαG).
Hydrolysis conditions were 37 for 1 mM maltose, nigerose, cordobiose, isomaltose, trehalose, sucrose, maltotriose, maltotetraose, pNPαG, pNPαG2 and 1 (w / v)% soluble starch. Under the conditions of ° C and pH 7, 0.005 U of this enzyme was added and reacted for 2 hours.
The α-glucosidase of the present invention is essentially a saccharide hydrolase, similar to the conventionally used α-glucosidase derived from Aspergillus. What is the glycosyltransferase described in Patent Document 2? It is separate.
[0012]
More specifically, the α-glucosidase of the present invention has the following enzyme chemical properties.
(1) Action It acts on a carbohydrate having an α-1,4-glucoside bond at the non-reducing end, and generates α-1,3- and α-1,4-glucoside bonds by transglycosylation. -Does not produce 1,6-glucoside bonds. Glucose is produced by a hydrolysis reaction.
(2) A tetramer of 440000 is composed of two polypeptides having molecular weights of 51000 and 60000 as one unit.
(3) Isoelectric point pI 4.5
(4) Optimal temperature 55 ° C
(5) Optimum pH
pH 7.0
(6) Temperature stability Shows a residual activity of 90% or more at 45 ° C. or less.
(7) pH stability Shows a residual activity of 90% or more at pH 6-11.
[0013]
The α-glucosidase of the present invention can be produced by culturing a microorganism belonging to the genus Acremonium and collecting the α-glucosidase of the present invention from the obtained culture. As a microorganism belonging to the genus Acremonium, for example, the novel strain Acremonium sp. YW1 strain of the present invention can be used.
[0014]
The mycological properties of the novel strain Acremonium sp. YW1 of the present invention are shown below.
In each medium of potato dextrose agar, oatmeal agar, and malt agar, the growth is slightly slow and reaches 17 to 18 mm in diameter at 25 ° C. for 1 week. The mycelium is fluffy and has a slightly low convex shape. The aerial mycelium color is white. In all the media, formation of white conidia is observed around 5 days after the start of the culture. Conidia are chain-like and change in color is not observed until 4 weeks of culture. Conidial stalk formation and soluble pigment production are not observed. The width of the aerial hyphae is almost constant and occasionally forms hyphae bundles. The conidial pattern stands upright and everything grows alone from the aerial hyphae. There are few conidia showing secondary branching. The phialide has a slightly bent structure and varies in size. The fare ride did not swell in the middle, and the width narrowed toward the tip. The surface of mycelium and phialide is smooth. Conidia are unicellular and smooth to slightly rough. The shape was spindle-shaped, and no curved one was observed.
[0015]
When the properties shown above are collated with the fungal classification method, the bacterium belongs to the genus Acremonium, and the present inventors named the bacterium Acremonium sp. YW1 strain.
This bacterium was deposited as FERM P-19011 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of September 13, 2002.
[0016]
As microorganisms belonging to Acremonium, in addition to Acremonium sp. YW1 strain (FERM P-19011), for example, A. luzulae (deposit number: IFO30535), which is a known microorganism, Acremonium fusidioides (deposit number: IFO31140), Acremonium sclerotigenum (deposit number: IFO8385), A. implicatum (deposit number: IFO30538), etc. Variants and mutants of these strains can also be used.
[0017]
The medium used for culturing the microorganism belonging to Acremonium to obtain the α-glucosidase of the present invention may be any nutrient medium that can grow microorganisms and can produce the α-glucosidase of the present invention. Any medium may be used. The carbon source and nitrogen source contained in these media are not particularly limited, and examples of the carbon source include starch, starch hydrolyzate, maltose, glucose, fructose, lactose, sucrose, reduced starch hydrolyzate, and the like. As the nitrogen source, for example, inorganic nitrogen compounds such as ammonium salts and nitrates, organic nitrogen compounds such as urea, corn steep liquor, casein, casamino acid, yeast extract, soybean flour, peptone, meat extract, etc. may be used. it can. Moreover, as an inorganic component, potassium salt, sodium salt, calcium salt, magnesium salt, phosphate, manganese salt, zinc salt etc. are used suitably, for example. Furthermore, amino acids, vitamins and the like can be added as necessary.
[0018]
The culture may be carried out under conditions that allow microorganisms to grow and produce the α-glucosidase of the present invention. For example, the culture is performed at a temperature of 4 to 40 ° C., preferably 25 to 35 ° C., and the culture time is preferably 12 hours to 10 days. Just be careful.
[0019]
After culturing the microorganism in this manner, the α-glucosidase of the present invention is recovered. Recovery of α-glucosidase from the culture can be performed according to a conventional method. For example, the cells can be removed by solid-liquid separation means such as centrifugation or filtration to obtain a crude enzyme solution. If necessary, a purified enzyme can be obtained by using usual enzyme purification means such as salting out, various column chromatography and ultrafiltration. The α-glucosidase obtained in the present invention can be used to prepare a sugar solution containing a nigerooligosaccharide as disclosed in JP-A-9-299095. -Glucosidase may be a crude enzyme or a purified enzyme. Purification of the α-glucosidase of the present invention can be performed using a known technique. As an example, DEAE-Toyopearl, Butyl Toyopearl, Toyopearl HW-55 is used after sterilizing the culture by centrifugation. By carrying out purification, an electrophoretically uniform enzyme can be obtained.
[0020]
Using α-glucosidase of the present invention as raw material for oligosaccharides and polysaccharides having α-1,4 glucoside bond, α-1,3 glucoside bond, α-1,2 glucoside bond, α-1,6 glucoside bond Can be obtained by carrying out a transglycosylation reaction at 45 ° C. and pH 7 for 12 to 72 hours. It goes without saying that a carbohydrate-related enzyme such as other hydrolase can be used in combination in the nigerooligosaccharide production reaction as necessary. The nigerooligosaccharide having an α-1,3-glucoside bond thus obtained has a mild sweetness and has various functions such as improving the taste of foods, suppressing the fading of natural pigments, and immunomodulating functions. This is a material that is expected to be used in a wider range of fields, mainly for food applications.
[0021]
【Example】
The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples.
(Example 1) Acremonium sp. YW1 strain cultured corn starch (Nihon Shokuhin Kako Co., Ltd.) 0.5% (w / v), Shefton D (Sheffield) 0.75% (w / v), dipotassium hydrogen phosphate 0.1% (w / v), sodium carbonate 1.0% (w / v) and water adjusted to pH 8.0 and 100 ml in a 500 ml Erlenmeyer flask. And autoclaved at 121 ° C. for 20 minutes. The cooled liquid medium was inoculated with Acremonium sp. Strain YW1 and cultured at 28 ° C. and 130 rpm for 24 hours as a seed culture solution.
[0022]
4 L of a culture solution having the same composition as above was prepared, and 1 L was placed in a 5 L Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. The cooled liquid medium was inoculated with 5% (v / v) seed culture and cultured at 28 ° C. and 130 rpm for 96 hours. The enzyme activity in the sterilized culture broth was 0.003 units / ml.
[0023]
(Example 2) Purification of enzyme The culture broth obtained in Example 1 was centrifuged at 10000 rpm under cooling at 4 ° C. to obtain about 4 L of a supernatant excluding bacterial cells. The supernatant was concentrated to 200 ml using a PM-10 membrane (Millipore) and packed with DEAE-Toyopearl 650M buffered with 20 mM phosphate buffer (pH 7.0) (2.6 φ × 45 cm). And washed with the same buffer until the unadsorbed protein was completely eluted. Thereafter, the enzyme was eluted with a linear gradient having a salt concentration of 0 → 1M. The obtained active fraction was dialyzed against 20 mM phosphate buffer (pH 7.0), followed by the same buffer containing 2 M ammonium sulfate, and concentrated to 10 ml using a PM-10 membrane. This enzyme solution was placed on a column (1.6φ × 60 cm) packed with butyl-Toyopearl 650M buffered with 20 mM phosphate buffer (pH 7.0) containing 2M ammonium sulfate, and unadsorbed protein was added in the same buffer. Washed until elution was complete. Thereafter, the enzyme was eluted with a linear gradient of 2 → 0M ammonium sulfate concentration. The active fraction was collected, dialyzed against 20 mM phosphate buffer (pH 7.0), and then concentrated to 2 ml using a PM-10 membrane. This enzyme solution was placed on a column (1.6φ × 95 cm) packed with Toyopearl HW-55F buffered with 20 mM phosphate buffer (pH 7.0), and the enzyme was eluted with the same buffer. The active fraction was collected and concentrated to 2 ml using a PM-10 membrane to obtain a purified enzyme preparation. The obtained purified enzyme preparation had a total activity of 5.27 units and a specific activity of 13.1 units / mg protein.
The obtained purified enzyme preparation showed a single protein staining band in polyacrylamide gel electrophoresis, indicating that it was a highly purified preparation.
[0024]
(Example 3) Analysis of enzyme properties When the purified enzyme preparation obtained in Example 2 was subjected to SDS-polyacrylamide gel electrophoresis, it showed two protein-stained bands with molecular weights of 51000 and 60000. there were. Moreover, the molecular weight calculated | required with the gel filtration method showed 440000. This enzyme was suggested to be a tetramer with two polypeptides of 51000 and 60000 as one unit. Further, when the purified enzyme preparation was subjected to isoelectric focusing, a single protein staining band was shown, and the isoelectric point was 4.5.
[0025]
When the influence of the temperature and pH of this enzyme was examined, the optimum reaction temperature was 50 to 55 ° C. as shown in FIG. 1, and the optimum reaction pH was 7.0 as shown in FIG. Regarding the temperature stability of the enzyme, the enzyme solution was treated for 15 minutes at each temperature, and then the remaining enzyme activity was measured. The results are shown in FIG. Moreover, about pH stability, it processed at 5 degreeC for 24 hours using the Briton-Robinson buffer solution of each pH, and the residual activity after returning pH to 7.0 with a phosphate buffer solution was measured. The results are shown in FIG. The temperature stability and pH stability of this enzyme were around 45 ° C. and pH 6-11, respectively.
The enzyme activity was measured under the following conditions.
Measurement of enzyme activity (1) Place 1.0 ml of diluted enzyme solution accurately in a test tube and leave it at 37 ° C ± 0.2 ° C for 5 minutes to keep it warm.
(2) Incubate the 2% maltose solution at 37 ± 0.2 ° C for 5 minutes.
(3) Accurately measure 1.0 ml of a 2% maltose solution that has been kept warm, add it to 1.0 ml of the enzyme preparation diluted solution that has been kept warm, and stir.
(4) After leaving this exactly at 37 ± 0.2 ° C. for 10 minutes, add 1.0 ml of 0.5N hydrochloric acid and stir.
(5) Add 0.5 ml of 0.5N sodium hydroxide solution and stir.
(6) Add 1.0 ml of coloring reagent with Pipetman and stir.
(7) Immediately leave it in a constant temperature water bath at 37 ± 0.2 ° C for 25 minutes to perform the color reaction.
(8) After stirring, the absorbance at 505 nm is measured. At this time, ultrapure water is used as a control.
[0026]
(Example 4) Reaction of the enzyme Using the purified enzyme preparation obtained in Example 2, the reaction of the enzyme with maltose was examined. Maltose (Nihon Shokuhin Kako Co., Ltd.) is dissolved in 20 mM phosphate buffer (pH 7.0) to a concentration of 20% (w / w), and 0.5 unit is added per gram of maltose solids. The reaction was carried out at 50 ° C. for 48 hours. The reaction solution was left in boiling water for 10 minutes to inactivate the enzyme, then desalted with an ion exchange resin, and the reaction product was analyzed by HPLC. The HPLC analysis was performed using Asahipak NH2P-50 (manufactured by Showa Denko KK) as a column and 75% (v / v) acetonitrile and an RI detector as an eluent. As a result, as shown in FIG. 5, a peak different from maltose was observed in the disaccharide fraction, and it was confirmed that this peak was nigerose by comparison with a commercially available standard carbohydrate, and its production rate was 5.2%. there were.
[0027]
(Example 5) Production of α-glucosidase and production of oligosaccharides from known strains Among known microorganisms, strains belonging to the genus Acremonium and having confirmed the ability to produce α-glucosidase of the present invention are shown. It is shown in 1. When these strains were cultured in the same manner as in Example 1, and the α-glucosidase production ability of the culture supernatant was used, and the reaction was performed using maltose as a substrate in the same manner as in Example 4 using the culture supernatant as a crude enzyme. Table 1 also shows the production rate of oligosaccharides other than maltose. In addition, the production rate of oligosaccharide was calculated | required by making the whole area into 100% in the analysis using HPLC.
In the case of using any of the strain enzymes, nigerose was produced as in the case of α-glucosidase produced by Acremonium sp. Strain YW1, and isomaltose was not produced.
[0028]
[Table 1]

[0029]
【The invention's effect】
According to the present invention, a novel strain Acremonium sp. YW1 is provided. The α-glucosidase produced from this strain can efficiently generate α-1,3-glucoside bonds. By this α-glucosidase, a nigerooligosaccharide having an α-1,3-glucoside bond can be obtained.
[Brief description of the drawings]
FIG. 1 is a graph showing the optimum reaction temperature of α-glucosidase produced by Acremonium sp. Strain YW1.
FIG. 2 is a graph showing the optimum pH of α-glucosidase reaction produced by Acremonium sp. Strain YW1.
FIG. 3 is a graph showing the temperature stability of α-glucosidase produced by Acremonium sp. Strain YW1.
FIG. 4 is a graph showing the pH stability of α-glucosidase produced by Acremonium sp. Strain YW1.
FIG. 5 is an HPLC analysis chart when α-glucosidase produced by Acremonium sp. YW1 strain is reacted with 20% maltose.

Claims (3)

Α-glucosidase having the following physicochemical properties:
(1) Action It acts on a saccharide having an α-1,4-glucoside bond at the non-reducing end, and generates α-1,3- and α-1,4-glucoside bonds by transglycosylation. Does not produce -1,6-glucoside bonds. Glucose is produced by a hydrolysis reaction.
(2) Molecular weight
By SDS-polyacrylamide gel electrophoresis, a tetramer of 440000 is composed of two polypeptides of 51000 and 60000 as one unit.
(3) Isoelectric point pI 4.5
(4) Optimal temperature 50 to 55 ° C
(5) Optimum pH
pH 7.0
(6) Temperature stability Shows a residual activity of 90% or more at 45 ° C. or less.
(7) pH stability Shows a residual activity of 90% or more at pH 6-11. Acremonium sp. YW1 strain (FERM P-19011), Acremonium ruzulae, Acremonium fusidioides, Acremonium sclerotigenum (A. scleroigen) A method for producing α-glucosidase, wherein the α-glucosidase according to claim 1 is collected from a culture obtained by culturing monium implicatum (A. implicatum) . Acremonium sp. YW1 strain (FERM P-19011) .

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