JP2964042B2 - Maltotriose-forming amylase, its production and use - Google Patents

Description

The present invention relates to a novel maltotriose-producing amylase that produces maltotriose as a main component from starch, a method for producing the same, and a maltotriose-containing sugar solution using the same. The method relates to a method for producing the same.

(Prior art)

Maltotriose is a sugar in which three glucose molecules obtained by partially decomposing starch with an enzyme or an acid are linked by α-1,4-glucosidic bonds. Maltotriose is 17% sweeter than sugar, but it has a mild sweetness, so it is used as a sweetener in foods. In addition, since it has high hygroscopicity and water retention, it can be used as an agent for retaining moisture of food, preventing drying, preventing precipitation of sugar crystals, and preventing starch from aging. In addition, it has recently been found that maltotriose has many good specificities in food processing, such as better thermal stability than glucose and maltose.

However, since there is no industrially established maltotriose production technology, by-products during the production of maltose (the remaining maltose obtained by saccharifying starch with β-amylase and pullulanase and then decomposing maltose with an ion exchange resin or the like) are currently available. (Sugar solution containing about 45% maltotriose) is commercially available, and this method has a limited supply of maltotriose.

Enzymes that produce maltotriose from starch include:
Streptomyces griseus (Streptomyc
es griseus) produced by the enzyme N-A468 [Japanese Patent Publication No. 57-691
5, Starch Science, Vol. 23, No. 3, pp. 175-181 (1979)] and amylase G of Bacillus subtilis.
3 (JP-B-59-37957, JP-B-60-15315) are known. However, these enzymes have not been produced industrially yet because the productivity of the enzymes by microorganisms is low and the enzymes do not have properties that can withstand industrial use.

[Means for solving the problem]

The present inventors have conducted a search for microorganisms for the purpose of industrially establishing a technique for producing maltotriose from starch or a derivative thereof. The identified bacteria were found to produce significant amounts of special amylase. This particular amylase has been found to hydrolyze starch to produce 50-70% maltotriose. An amylase that produces such a remarkable amount of maltotriose has not been known so far, and production of a maltotriose-producing amylase from a bacterium of the genus Microbacterium has not been known. The present invention has been made based on such findings, and the present inventors have produced and purified maltotriose-forming amylase, and further established the use thereof to complete the present invention.

Hereinafter, the physicochemical properties of the maltotriose-forming amylase of the present invention will be described.

(A) Action and Substrate Specificity Glucan consisting of α-1,4-glucoside bonds of amylose, amylopectin, glycogen, starch and their derivatives is decomposed in an endo-type to produce a sugar solution containing maltotriose as a main component. Generate. The production rate of maltotriose from liquefied starch reaches 50 to 70%, although it varies depending on the saccharification conditions, that is, the substrate concentration, the degree of starch liquefaction (DE), the presence or absence of a debranching enzyme, the pH, and the temperature. This enzyme does not substantially act on pullulan.

(B) Action pH and optimum pH It acts in a wide range of pH 5 to 9. The optimum pH was around 6.5 (4M in 0.05M phosphate buffer or acetate buffer).
Reaction at 0 ° C for 30 minutes). The result is shown in FIG.

(C) Working temperature and optimum temperature Works up to about 60 ° C. The optimum temperature was found at about 50 ° C. [reacted with 1% soluble starch, 0.05 M Tris buffer (pH 7.0) for 30 minutes]. The result is shown in FIG.

(D) Thermal stability Approximately 10% was inactivated when heated in a 0.05 M Tris buffer (pH 7.0) at 50 ° C. for 10 minutes, and almost inactivated by heating at 60 ° C. for 10 minutes. However, the stability is improved in the presence of calcium ions. FIG. 3 shows the results.

(E) pH stability After standing at 25 ° C. for 3 hours in a 0.1 M buffer (acetic acid or phosphate buffer), the residual activity was measured. As a result, pH 6 ~
9 was stable. The result is shown in FIG.

(F) Stabilization An increase in thermal stability was observed in the presence of calcium ions (see FIG. 3).

(G) Inhibitor 5 × 10 ⁻³ M of MnSO ₄ , CuSO ₄ , HgCl ₂ , FeCl ₃ , ZnSO ₄ , AlCl ₃ , respectively, about 87%, about 97%, about 98%, about 100%, about 100% ,
It was inhibited by about 100%. The inhibition was 8% and 19% by 1 × 10 ⁻³ M and 2 × 10 ⁻³ M monoiodoacetic acid, respectively.

(H) Molecular weight The molecular weight measured by the gel filtration method was about 40,000.

Of the above physicochemical properties, the main properties are:
The N-A468 enzyme reported in JP-B-57-6915 (hereinafter referred to as N
Shown as -A468. ) And amylase G3 (hereinafter referred to as A-G3) reported in JP-B-60-15315, and the results are shown in Table 1.

 The activity was measured by the following method.

To 0.5 ml of 2% soluble starch dissolved in 0.1 M phosphate buffer (pH 7.0), an appropriate amount of enzyme is added, and a total amount of 1.0 ml is reacted at 40 ° C. to produce maltotriose and other reducing sugars. Quantified by the Somogyi Nelson method. Under these conditions, the amount of an enzyme that produces a reducing sugar equivalent to 1 micromole of glucose per minute is defined as one unit.

N-A468 is an amylase that degrades starch in exo form in units of maltotriose similarly to β-amylase,
A-G3 and the enzyme of the present invention are a kind of α-amylase because the decomposed sugar is α-sugar, and the degraded product mainly containing maltotriose until starch finally loses most of the iodine reaction. Amylase that decomposes into Furthermore, NA-468 is not affected by stabilization in the presence of calcium ions, whereas A-G3 and the enzyme of the present invention have improved thermostability. Further, when comparing A-G3 and the enzyme of the present invention,
Although the pH, stable pH, optimum temperature and the like are similar, the stable temperature is slightly different, and the molecular weight of the enzyme of the present invention is completely different from that of the enzyme of the present invention, which is about 25,000 for A-G3. As described above, the enzyme of the present invention is a novel maltotriose-forming amylase completely different from the conventionally known enzymes.

Examples of the microorganism that produces the maltotriose-forming amylase of the present invention include Microbacterium.
SP AM-9581 (Microbacterium sp. AM-9581) or a mutant thereof can be advantageously used in the present invention.

Microbacte sp. AM-9581 (Microbacte
rium sp. AM-9581) was newly discovered and isolated from soil, and its bacteriological properties are as follows. still,
This bacterium has been deposited with the National Institute of Microbial Industry as National Institute of Industrial Science No. 11315.

(1) Morphology: thin and long (about 10μ × 40μ) → short rod-coccus (2) Gram staining: positive (3) Spores:-(4) Motility: + (5) Flagella: perihair (6) Oxidase: + (7) Catalase: + (8) OF test: O (weak) (9) Dye: yellow (insoluble) (10) Requirement of free oxygen: obligate aerobic (11) In hydrolyzate of all cells Meso-diaminopimelic acid:-(12) Diamino acid in cell wall: lysine (13) VP compatible:-(14) Utilizing citrate (simmons):-(15) Nitrate reduction: + (16) Starch degradation: + (17) ) Gelatin degradation: + (18) Litmus milk reaction: No change (19) Urease:-(20) Phosphatase:-(21) Hydrogen sulfide production:-(22) Arginine degradation:-(23) Heat resistance 63 ° C, 30 Min: -72 ° C, 15 min:-(24) Salt tolerance 4%: + 7%: -10%:-( 25) Optimal temperature (° C): 35-41 (26) Growth temperature (° C): 11-46 (27) Growth potential of pH9.2 liquid medium: + (28) Growth potential of pH5.0 liquid medium: − (29) Indole production:-(30) Esculin degradation:-(31) Phenylalanine deamination:-(32) Methyl red reaction:-(33) Tyrosine degradation:-(34) Lecithinase reaction:-(35) sabouraud- dextrose development:-(36) Alkaline production in citrate medium:-(37) Gas from nitrate under alkaline anaerobic:-(38) Final pH of VP medium: 5.2 (39) Cell diameter: 0.4-0.8μ ( 40) Spores:-(41) Gas from glucose:-(42) Development MacConkey:-SS:-TSI (acid, slope / high layer): + (weak) /-(43) Dioxyacetone production:-(44 ) Production of acid from sugar Inulin:-Shoe cloth: + Trehalose: + Glycerol: + Raffy Source:-glucose: + fructose: + galactose: + mannose: + maltose: + cellobiose: + inositol:-sorbose:-rhamnose: + (weak) lactose: + (slow) mannitol:- , Bergey's Mannual of Sy
Referring to stematic Bacteriology, Vol. 2 (1986) and comparing the properties, lysine and glycine were found on the bacterial wall and mesodiaminopimelic acid was not found. Therefore, this bacterium was of the genus Microbacterium. Bacteria. This bacterium is closest to Microbacterium imperiale in terms of its properties, but does not coincide with the bacterium in the following points.

(1) Produces yellow pigment (insoluble).

(2) An acid is produced from glycerol and rhamnose.

(3) reducing nitrates;

(4) Arginine is not dispersed.

It is the latest Bergey's Man mentioned above that the strain is at the optimal growth temperature of 35-41 ° C and the maximum growth temperature is 45-46 ° C.
Starting from the Manual of Systematic Bacteriology, Vol. 2 (1986), it has not been described in any Microbacterium species on the IJSB list since then.

Based on the above, the strain was determined to be a new strain, and Microbacterium sp. AM-9581 (Microbacterium sp.
81). In order to produce maltotriose-forming amylase by culturing the bacterium, as a nitrogen source, an organic nitrogen source usually used for culturing microorganisms such as meat extract, peptone, corn steep liquor, casein, and ammonium chloride are used. And an inorganic nitrogen source such as ammonium sulfate. In addition, as a carbon source, starch, liquefied starch,
Dextrin, maltose, glucose, sucrose and the like are used. Various metal salts such as phosphates, magnesium salts, steel salts and the like are used as supplementary medium components.

The cultivation is performed at pH 5-9, preferably pH 6-8, at a temperature of 25-50.
C., preferably at 30.degree. C. for about 2 to 6 days.

Since maltotriose-forming amylase is an enzyme produced outside the cells, after culturing, the bacteria are removed by filtration or centrifugation, and the supernatant is collected and concentrated. If necessary, salting out with ammonium sulfate, sodium sulfate, etc.
Alternatively, an organic solvent such as acetone, isopropanol, ethanol or the like is added, and the enzyme is recovered as a precipitate and dried and stored.

This enzyme can be purified electrophoretically to a single enzyme by a conventional enzyme purification method. For example, ammonium sulfate salting out (硫酸 40% saturation), DEAE-Sepharose, DE
It can be purified by ion-exchange chromatography such as AE-Sephadex and DEAE-Toyopearl and gel filtration using biogel, Sephadex, TSK gel and the like.

The production of maltotriose from starch using this enzyme is carried out as follows.

As the starch, corn starch, potato starch and the like are usually used, and the starch is liquefied by a conventional method using a starch liquefying enzyme (α-amylase) produced by a bacterium belonging to the genus Bacillus (about DE 2 to 15). 20 to 40% of the liquefied starch
Saccharification is carried out using the maltotriose-forming amylase at a concentration of 6 to 8 and a temperature of 50 to 60 ° C. In the saccharification by this enzyme, the presence of a debranching enzyme such as pullulanase or isoamylase promotes saccharification and increases the yield of maltotriose.

 Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 Corn steep liquor 10%, soluble starch 2%, K ₂ HP
O ₄ 0.2%, MgSO ₄・ 7H ₂ O 0.1%, CaCl ₂ 0.01%, NaNO ₃
40 ml of medium (pH 7.0) consisting of 0.1% and Tween 40 0.1%
Is placed in a 200 ml Erlenmeyer flask, sterilized by a conventional method, and then Microbacterium sp.
No. 11315), and cultured with shaking at 30 ° C. for 3 days. After the culture, the bacteria were removed by a centrifuge, and the resulting supernatant was measured for the produced maltotriose-forming amylase. As a result, the amount was 5 units per 1 ml of the culture medium.

Example 2 In Example 1, as a medium, fish meat extract 3%, K _{2
HPO 4 0.3%, MgSO 4 ·} 7H 2 O 0.1%, using a medium containing CaCl ₂ 0.01%, micro Corynebacterium sp AM-9581
(Microbial Deposit No. 11315) and cultured with shaking at 30 ° C. for 3 days. After the culture, the centrifuged supernatant was measured for the produced maltotriose-forming amylase. As a result, the concentration was 8 units per 1 ml of the medium. 5 l of the supernatant was concentrated using an ultrafiltration membrane to 250 ml. To this was added 75 g of ammonium sulfate to dissolve and left at 5 ° C. overnight. A precipitate was obtained by centrifugation. A small amount of water was added to the obtained precipitate and centrifuged to obtain about 50 ml of a supernatant. This is TSK-GEL Toyopearl HW
The sample was loaded on a -40E column (2.4 cm in diameter × 110 cm in length), and desalted by gel filtration. The activated fraction was adsorbed on a DEAE-Toyopearl column (23 cm in diameter x 21 cm in length), washed with 0.02 M Tris / hydrochloric acid buffer (pH 7.0), and then 0 → 0.5 with potassium chloride solution containing the buffer. It eluted by linear gradient to M. The active fraction of this DEAE-Toyopearl chromatography was concentrated to 5 ml, and TSK-GEL Toyopearl HW65F
Gel filtration chromatography was performed on a column (2.4 cm in diameter × 110 cm in length), and a single enzyme could be obtained by disk electrophoresis and HPLC. G2000SWX
The molecular weight was estimated to be about 40,000 by HPLC using an L column (manufactured by TOSO). Table 2 shows the liquid volume, activity, recovery rate, etc. in the purification process.

Example 3 Using the enzyme obtained in Example 2, amylose (DP = about
17) The reaction was carried out using amylopectin, soluble starch and the like as substrates, and the sugar composition of the degraded product was examined by HPLC. The reaction conditions were as follows: substrate concentration 2.5%, enzyme amount 2 units / g substrate, reaction temperature 55 ° C, reaction p
H7.0 (5 mM-Tris / HCl buffer containing 5 mM CaCl ₂ ) was used.

As a result, amylose was almost completely decomposed in 24 hours, and there was no oligosaccharide or limit dextrin higher than G4. The sugar composition was 5.5% for G1, 26.2% for G2, 68.3% for G3, and the reaction time was 48%. There was no change in the sugar composition at -96 hours.

In amylopectin (maize and sigma) and soluble starch (manufactured by Wako Pure Chemical), limit dextrin remains,
It could not be completely disassembled. After 48 hours of amylopectin, the sugar composition of G1 was 1.7%, G2 was 9.9%, G3 was 36.2%, and the sugar composition of soluble starch after 48 hours was G1 22%, G2 11.6%, G3
40.8%. In addition, when pullulanase (manufactured by Amano Pharmaceutical) is added at 5 u / g during these saccharification reactions,
At 24 hours, the substrate was almost completely degraded as in the case of amylose. The sugar composition at this time is G1 in amylopectin
5.6%, G2 26.4%, G3 68.0%, soluble starch G1
5.3%, G2 was 255% and G3 was 69.2%.

From these results, maltotoliose-forming amylase cannot decompose α-1,6-glucosidic bonds that decompose α-1,4-glucosidic bonds of amylose, amylopectin, starch and the like. Therefore, when saccharification is performed by adding pullulanase or a debranching enzyme capable of decomposing α-1,6-glucosidic bond, starch is almost completely decomposed and a saccharified solution having a G3 content of 60 to 70% and containing no limit dextrin is obtained. can get.

Example 4 Potato starch was liquefied using Bacillus subtilis α-amylase. A saccharification test was performed using liquefied starch of DE 4.2.

The maltotriose-forming amylase prepared in Example 1 was added to the above liquefied starch in an amount of 1, 2 or 5 units per solid, and a 20% concentration (w / w) in the presence of 1 × 10 ⁻² M CaCl _2. w
%), PH 7, and a temperature of 50 ° C. The results obtained by quantifying the sugar composition of the saccharified solution 48 hours and 72 hours after the reaction by high performance liquid chromatography are shown in Table 3.

As is clear from the table, when 5 units were used per 1 g of liquefied starch solids, about 72 hours after saccharification, maltotriose was about 50.6%, maltose was about 14.8%, and glucose was about 3.6%.
%, G4 and 31.0% of other sugars were obtained.

Example 5 Using 5 units of enzyme per 1 g of liquefied starch of DE4.25, saccharification was carried out at a reaction temperature of 50, 55 and 60 ° C. in the same manner as in Example 3. Table 4 shows the obtained results.

As is clear from the table, the reaction at 55 ° C. was faster than that at 50 ° C. to produce maltotriose faster, and a sugar solution containing about 52% maltotriose could be obtained.

Example 6 In Example 4, liquefied starch of DE 4.25 was added as substrate and 5 units per enzyme liquefied starch solids were added at concentrations of 5, 10, 15, 20, and 30% (w / w%), respectively. pH 7, temperature 5
Saccharification was performed at 0 ° C for 72 hours. Table 5 shows the obtained sugar compositions.

As is clear from the table, as the reaction was performed at a higher substrate concentration, a saccharified solution having a lower glucose content and a lower maltose content was obtained. A saccharified solution having a high maltotriose content was obtained even at a high substrate concentration such as 30%.

Example 7 In this example, results of saccharification in the presence of pullulanase in saccharification of liquefied starch with the present enzyme will be described.

In Example 4, DE 2.38,4.25,5.81,8.
Using liquefied starch of 05 and 12.6, under each substrate concentration 20%,
Enzyme is added at 5 units per 1 g of each substrate.
Saccharified for hours. Table 6 shows the obtained results.

As is clear from the table, a maltotriose sugar solution having a high content of about 50% was obtained using any of the liquefied starches in the range of DE 2.38 to 12.6.

Example 7 In this example, the result of saccharification in the presence of pullulanase in liquefaction precipitation saccharification with the present enzyme will be described.

The liquefied starch of DE 4.25 used in Example 4 was each 200 mg, maltotriose-forming amylase 1 unit (5 units per 1 g of substrate), and commercially available Bacillus sp. To 1 ml with water and pH
7. Saccharification at 50 ° C for 48 hours. No. 7
It is shown in the table.

The presence of pullulanase accelerates the saccharification reaction. As is clear from the table, when 5 units of pullulanase are added per 1 g of starch, a saccharified solution having a maltotriose content of 63% is obtained, which is about 12% higher than that of no addition. Was done.

The pullulanase activity was measured at pH 5.1 under 1% pullulan.
The reaction was carried out at 0 and a temperature of 40 ° C., and one unit was defined as an enzyme that produced a reducing power equivalent to 1 micromol of glucose per minute.

[Brief description of the drawings]

FIG. 1, FIG. 2, FIG. 3 and FIG. 4 show the optimum pH, optimum temperature, thermal stability and pH stability of the maltotriose-forming amylase of the present invention, respectively. In FIG. 3,-●-indicates stability in the presence of CaCl ₂ .

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A maltotriose-forming amylase having the following physicochemical properties: (A) Action It acts on starch to mainly produce maltotriose. (B) Substrate specificity It acts on amylose, amylopectin, glycogen and starch, and does not substantially act on pullulan. (C) Optimum pH 40 ℃, around pH 6.5 for 30 minutes reaction (d) Optimum temperature pH 7.0, around 50 ℃ for 30 minutes reaction (e) Thermal stability 40 ℃ at pH 7.0, 10 minutes To the neighborhood. However, in the presence of calcium ions, 90% of the activity remains at 50 ° C. (F) pH stability Around pH 6 to 9 in 3 hours at 25 ° C. (g) Stability and inhibition Thermal stability is improved by calcium ions. Substantially inhibited by manganese, copper, mercury, iron, zinc and aluminum ions. (H) Molecular weight Approximately 40,000 by gel filtration. 2. A method for producing maltotriose-forming amylase, comprising culturing a microorganism belonging to the genus Microbacterium and producing maltotriose-forming amylase, producing maltotriose-forming amylase, and collecting the maltotriose-forming amylase. . 3. A maltotriose-containing sugar solution obtained by reacting a starch or a derivative thereof with a maltotriose-forming amylase having the following physicochemical properties and collecting the obtained maltotriose-containing sugar solution. Manufacturing method. (A) Action It acts on starch to mainly produce maltotriose. (B) Substrate specificity It acts on amylose, amylopectin, glycogen and starch, and does not substantially act on pullulan. (C) Optimum pH 40 ℃, around pH 6.5 for 30 minutes reaction (d) Optimum temperature pH 7.0, around 50 ℃ for 30 minutes reaction (e) Thermal stability 40 ℃ at pH 7.0, 10 minutes To the neighborhood. However, in the presence of calcium ions, 90% of the activity remains at 50 ° C. (F) pH stability Around pH 6 to 9 in 3 hours at 25 ° C. (g) Stability and inhibition Thermal stability is improved by calcium ions. Substantially inhibited by manganese, copper, mercury, iron, zinc and aluminum ions. (H) Molecular weight Approximately 40,000 by gel filtration. 4. A maltotriose characterized in that a maltotriose-forming amylase and a debranching enzyme having at least the following physicochemical properties are allowed to act on starch or a derivative thereof, and a maltotriose-containing sugar solution obtained is collected. A method for producing an aus-containing sugar solution. (A) Action It acts on starch to mainly produce maltotriose. (B) Substrate specificity It acts on amylose, amylopectin, glycogen and starch, and does not substantially act on pullulan. (C) Optimum pH 40 ℃, around pH 6.5 for 30 minutes reaction (d) Optimum temperature pH 7.0, around 50 ℃ for 30 minutes reaction (e) Thermal stability 40 ℃ at pH 7.0, 10 minutes To the neighborhood. However, in the presence of calcium ions, 90% of the activity remains at 50 ° C. (F) pH stability 25 ° C., pH 3 to around 9 for 3 hours (g) Stability / inhibition Calcium ions improve thermal stability. Substantially inhibited by manganese, copper, mercury, iron, zinc and aluminum ions. (H) Molecular weight Approximately 40,000 by gel filtration.