JPH0673466B2 - Method for producing maltooligosaccharide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing maltooligosaccharides, and more specifically to supplying a starch liquefaction liquid under a specific condition to a reactor filled with immobilized maltooligosaccharide-forming amylase. It relates to a method for producing a characteristic maltooligosaccharide.

[Problems to be Solved by Prior Art and Invention]

Decomposition of starch or amylose with acid or amylase gives various oligosaccharide mixtures. These are industrially produced in large quantities and consumed as starch sugars such as starch syrup and starch syrup, but it was difficult to efficiently produce high-purity maltooligosaccharides on an industrial scale except for maltose. .

In recent years, various maltooligosaccharide-forming amylases that act on starch or amylose to specifically produce maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose, and maltohexaose have been successively discovered,
Expectations are growing for efficient industrial production of maltooligosaccharides.

As the malto-oligosaccharide-forming amylase, the following are known in addition to β-amylase.

Maltotriose-forming amylase [Katsuo Wakao et al .: Starch Chemistry, 26,175. (1979), Streptomyces griseus origin; Yoshiyuki Takasaki: Showa
58th Annual Meeting of the Japanese Agricultural Chemistry, p169 (1983), from the genus Bacillus] Maltotetraose-producing amylase [JF Robyt and R.
J. Ackerman: Arch. Biochem. Biophys., 145,105 (19
71), Pseudomonas st
Originated from maltpentaose amylase [N. Saito: Arch.
Biochem. Biophys., 155,290 (1973), from the origin of Bacillus licheniformis; Shoichi Kobayashi et al .;
(1983); Naoki Yoshiyoshi et al .: Proc. Of Japan Agricultural Chemistry 1984, P584 (1984)] Maltohexaose-producing amylase [K. Kainuma et al .: FEB
S Lett., 26, 281 (1972), from the origin of Aerobacter aerogenes; JF Kenne.
dy and CA White: Strke, 31,93 (1979); Taniguchi Hajime: Starch Chemistry, 29,107 (1982); Y. Takasaki: Agri
c. Biol. Chem., 47, 2193 (1983)] However, these maltooligosaccharide-forming amylases have low productivity and are very expensive as compared with general amylases. Therefore, as various maltooligosaccharides produced by using these amylases, reagent grade products are only produced in a small amount and are expensive.

Further, these reagent-grade maltooligosaccharides are produced by a batch method and require a long time, for example, a reaction time of 24 to 72 hours, that the maltooligosaccharide-forming enzyme is disposable, and a target maltooligosaccharide. In addition to separation from other sugars, a separation step between an enzyme and a product is required, and the purification and separation operation is complicated and requires a lot of manpower.

Moreover, it has been clarified that the purification separation by the commonly used gel filtration method, carbon chromatography, etc. is not suitable for mass production because the load of the sample is small and the productivity is low [Shigeo Suzuki] , Edited by Michinori Nakamura; "Experimental Method of Starch Science", Asakura Shoten, 197-202].

Therefore, the present inventors have conducted extensive research to develop a method for producing maltooligosaccharides that has solved the above drawbacks.

[Means for solving problems]

The present inventors have previously proposed an immobilized enzyme having various maltooligosaccharide-forming amylase immobilized thereon, on the premise of a continuous method for producing maltooligosaccharide (Japanese Patent Application No. 60-81340).

Therefore, when a starch liquefied liquid was supplied to a reactor filled with these immobilized enzymes and examined, the starch liquefied liquid was supplied under a specific condition in accordance with the activity of the filled immobilized enzyme to perform hydrolysis. It was found that the malto-oligosaccharide can be efficiently produced by carrying out the method, and the present invention was completed based on such findings.

That is, in the present invention, maltooligosaccharides (abbreviated as Gn, n represents an integer of 3 to 10) from maltotriose (abbreviated as G ₃ ) to maltodecaose (abbreviated as G ₁₀ ). When using malto-oligosaccharide-producing amylase (abbreviated as Gn-producing amylase), the specific surface area of pores having an affinity for the enzyme of 80% or more and a pore diameter of 100Å or more is 20 to 300 m ² Weight-based space velocity C / (A × B) hr per unit activity of immobilized enzyme determined by the following formula in a reactor filled with Gn-producing amylase immobilized on a porous carrier of / g ^-1 (IU / g) ^-1 (where A is the expression activity (IU / g), B is the immobilized Gn-producing amylase (g) packed in the reactor, and C is the starch liquefaction supplied to the reactor. Liquid volume (solid content) (g-solid content / hr) 1 x 10
^The method for producing maltooligosaccharides is characterized in that the malto-oligosaccharide is supplied under the condition of ^{-4 to} 3 × 10 ^-2 hr ^-1 (IU / g) ^-1 .

As the raw material starch used in the present invention, various ones can be used, but usually potato starch, sweet potato starch, corn starch,
Waxy cornstarch, cassava starch, etc. are used.
The glucose equivalent (DE) of the liquefied starch solution passed through the reactor is usually 1 to 35, preferably 5 to 20. Here, the DE of the starch liquefaction liquid is 1 or less in the case of waxy corn starch, and other starches
Those with a DE of 5 or less are severely aged, and in order to prevent aging, it is necessary to heat and hold at 70 to 80 ° C. or more, which is disadvantageous in the process. On the other hand, when DE is 35 or more, glucose,
It is not suitable because the production of low molecular weight sugars such as maltose increases and the yield of the target maltooligosaccharide decreases. The method of liquefying the various starches is not particularly limited, but is usually treated with an acid such as liquefied α-amylase or hydrochloric acid.

Next, the maltooligosaccharides are maltotriose, maltotetraose, maltopentaose, maltohexaose and the like, and the maltooligosaccharide producing amylase is an amylase which produces the above-mentioned various maltooligosaccharides.

Various methods are known as methods for immobilizing enzymes,
One of them is a carrier binding method including a physical adsorption method, an ionic binding method, a covalent binding method and the like. Among them, the physical adsorption method and the ionic binding method are easy to operate, the destruction of the active center of the enzyme protein or the change of the higher-order structure is small, and the carrier can be easily recycled. If possible, it can be said that this is a promising method for industrialization.

The immobilization means for maltooligosaccharide-forming amylase previously proposed by the present inventors (Japanese Patent Application No. 60-81340) is also an advantageous method. In this method, as a carrier for maltooligosaccharide-forming amylase, the affinity for the enzyme is 80% or more, and the specific surface area of pores having a pore size of 100Å or more is 2
A porous carrier that is 0-300 m ² / g is used. Here, the affinity between the enzyme and the carrier means a value obtained by the following method.

0.5 g of carrier and 25 IU of enzyme (2.0 ml of 50 mM phosphate buffer, pH 7.0)
In the 50 ml Erlenmeyer flask, the enzyme was immobilized on the carrier while shaking with 120 strokes, 4 cm width at room temperature for 1 hour, and then filtered to measure the activity in the obtained filtrate. Then, the affinity between the enzyme and the carrier is determined from the next knowledge.

The purity of the enzyme used for obtaining the affinity is not necessarily absolute, but it is preferable to use an enzyme having a specific activity of 30 IU / mg · protein or more. The affinity between the enzyme and the carrier was obtained at a level lower than the enzyme activity / carrier weight ratio in the industrial use, but it is important as a measure for knowing the affinity between the enzyme and the carrier. That is, the present inventors have found that an enzyme immobilized at an actually high enzyme activity / carrier weight ratio showing a high expression activity for a substrate also shows a high value for the above affinity. Regarding this affinity, it is necessary to be 80% or more as described above, but it is preferably 95% or more in the case of a carrier for maltotetraose producing amylase, and in the case of a carrier for maltopentaose producing amylase. It is preferably 80% or more. These values depend on the origin and specific activity of the enzyme used. Here, the expression activity is defined as follows.

Since the absolute value of the activity expressed by the immobilized enzyme varies greatly depending on the conditions of use, it was determined by setting the following measurement conditions. That is, 10 mg (wet) of immobilized enzyme was added to 0.5 ml of 10
mM phosphate buffer (pH 7.0) (in 50 ml Erlenmeyer flask)
In addition to the 10% w / v% soluble starch (M
(manufactured by erck Co., Ltd.) 5.0 ml was added, and an enzymatic reaction was carried out while shaking at 40 ° C. for 10 minutes with a shaker at 120 strokes / min and a width of 4 cm, and the produced reducing sugar was measured by the Somogyi-Nelson method,
The expressed activity was determined. 1 unit is 1μ per minute
It means the amount of enzyme that cleaves mol glucoside bond.

This carrier must further satisfy the following conditions. That is, the specific surface area of the pores having a pore diameter of 100 Å or more of the carrier is 20 ~ 300 m ² / g, preferably 40 ~ 200
It needs to be m ² / g. This specific surface area is 300 m ² / g
If it exceeds, the carrier becomes weak in strength, which is not preferable. In addition, in the carrier for amylase producing maltotetraose, it is desirable that the specific surface area of the pores having a pore diameter of 400 liters or more is 3 to 100 m ² / g, preferably 5 to 50 m ² / g. Here, the specific surface area is a value measured by the mercury penetration method.

It is not enough to satisfy any one of the above conditions, and only those satisfying all the conditions at the same time show high reactivity with the substrate.

If the porous carrier satisfies the above characteristics,
Regardless of the material and the manufacturing method, a porous synthetic resin such as a phenol-based adsorbent resin, a styrene-divinylbenzene-based adsorbent resin, a triazine-based adsorbent resin, an acrylic ester-based adsorbent resin, or a weakly basic anion exchange resin is usually used. .
More specifically, Duolite resin (Diamond Shamrock Co., Ltd.) product names “S-761”, “S-762”,
"ES-771", "ES-8610", etc .; Diaion resin (manufactured by Mitsubishi Kasei Co., Ltd.) under the product names "HP-10", "HP-"
20 "," HP-50 "," SP-206 ", etc .; Amber light resin (made by Rohm and Haas)" XAD "
-7 "," XAD-8 "and the like.

The particle size of the porous carrier is not particularly limited as long as the expression activity is high, but it is suitable to use one having a particle size of usually 0.05 to 1.5 mm, more preferably 0.2 to 1.0 mm. A small particle size is a preferable factor for enzyme immobilization and expression activity, but if it is too small, pressure loss will occur when the immobilized enzyme is filled in the reactor and the starch liquefaction liquid is supplied to it. Is large, which is inconvenient for driving. On the other hand, if the particle size is too large, it is not efficient in immobilizing the enzyme or in the expression activity.

Various methods can be applied to immobilize the enzyme on the carrier, but the most general method is to dissolve the enzyme in an appropriate buffer solution and mix the enzyme solution with the carrier and stir as described above. Is the way. Then, solid / liquid separation is performed by an operation such as filtration, and the obtained immobilized enzyme is washed with an appropriate buffer solution. As the buffer solution, a phosphate buffer solution (pH 6 to 7), a Tris hydrochloric acid buffer solution (pH 7 to 9), distilled water or the like is preferably used.

The amount of the enzyme immobilized varies depending on the enzyme to be used and its specific activity and the type of carrier, but usually 1 to 40 mg, preferably 2 to 15 mg of protein per 1 g of carrier is suitable.

When the amount of enzyme immobilized is less than 1 mg as protein per gram of carrier, the immobilization rate is high and immobilization can be performed efficiently, but the absolute value of the expression activity is small and the yield of the target component is low. On the other hand, if the amount of immobilization is more than 40 mg as protein per 1 g of carrier, the immobilization rate at the time of immobilizing the enzyme will decrease, so not only the efficiency of enzyme use will be poor, but the ratio used as expression activity will also decrease. , Not preferable.

In addition, the immobilized maltooligosaccharide-forming amylase obtained by the above method may be treated with an appropriate polyfunctional crosslinking agent, for example, glutaraldehyde, to improve the ability to maintain the enzyme's immobilized activity.

The present inventors have conducted various studies on conditions for efficiently producing maltooligosaccharides using various immobilized enzymes, and as a result, have found that the following factors have a great influence. That is, the type and concentration of the substrate to be used and its supply amount, the type (physical properties) of the immobilized carrier, the amount of immobilized enzyme, the amount packed into the packed column, the temperature of the reaction system, the conditions such as pH and the like.

As a result of systematically examining these, it was found that the influence of these factors and the like can efficiently produce maltooligosaccharides within the range of the expressions and conditions shown below.
That is, according to the method of the present invention, the immobilized maltooligosaccharide-forming amylase is charged into a reactor, and the starch liquefaction liquid described above has a weight-based space velocity per immobilized enzyme unit activity of 1 × 10 ⁻⁴ to.
3 × 10 ⁻² hr ⁻¹ (IU / g) ⁻¹ , preferably 3 × 10 ⁻⁴ to 1 × 10
^By supplying under the condition of ^-2 hr ^-1 (IU / g) ^-1 , the malto-oligosaccharide is efficiently produced. Here, the weight-based space velocity per unit activity of the immobilized enzyme is a value obtained as follows. First, the same immobilized maltooligosaccharide-forming amylase 10 mg (wet) that is filled in the reactor.
Was added to 0.5 ml of 10 mM phosphate buffer (pH 7.0) (in a 50 ml Erlenmeyer flask) and allowed to fully adapt to the same substrate (the same kind and concentration of starch, etc.) as the one supplied to the reactor 5.0 m
l, and 120 at the same temperature as the reactor by a reciprocal shaker.
Enzymatic reaction is performed while shaking with strokes / min., 4 cm width, and reducing sugar produced is measured by Somogyi-Nelson method, or malto-oligosaccharide produced directly by an analytical instrument such as high performance liquid chromatography is measured and expressed. The activity is determined (this expression activity is used as AIU / g-carrier). When the immobilized maltooligosaccharide-forming amylase packed in the reactor is Bg (wet) and the amount of starch liquefied liquid supplied to the reactor is Cg-solid content / hr, the weight-based space velocity per unit activity is Is calculated as C / (A × B) hr ⁻¹ (IU / g) ⁻¹ . In addition, when DE is large, the target maltooligosaccharides and sugars smaller than them are contained in the raw material, so it is more practical to use the solid content excluding them as the value of C. If the weight-based space velocity per unit activity is larger than 3 × 10 ^-2 hr ^-1 (IU / g) ^-1 , that is, if the reaction time in the reactor is short, the hydrolysis reaction does not sufficiently occur. Therefore, the yield of maltooligosaccharide is deteriorated, which is not preferable. Further, when the weight-based space velocity per unit activity is smaller than 1 × 10 ^-4 hr ^-1 (IU / g) ^-1 , that is, when the reaction time in the reactor becomes long, it is apparent in the following publications. As described above, the produced maltooligosaccharides are further over-decomposed, so that low-molecular-weight sugars such as glucose and maltose are produced, and not only the purity of the product is significantly lowered, but also the efficiency in the case of subsequent purification and separation is improved. It is not preferable because it worsens.

Regarding the above-described malto-oligosaccharide over-degradation, the maltooligosaccharide-forming amylase specifically produces each oligosaccharide (maltotriose, maltotetraose, maltopentaose, maltohexaose, etc.) at the initial stage of the reaction, It has been clarified that the product itself decomposes in the later stage of the reaction [T.Nakakuki et al; C
arbohydro. Res., 128,297 (1984)].

According to the present invention, the use of the immobilized maltooligosaccharide-forming amylase can be expected to expand the reaction conditions as compared with the original non-immobilized maltooligosaccharide-forming amylase. For example, in the case of an immobilized maltotetraose-forming amylase proposed by the present inventor (disclosed in Japanese Patent Application No. 60-18340),
The temperature stability spreads to about 10 ° C on the high temperature side, and the pH stability spreads to about 1 on the low pH side. Also, the optimum temperature is fixed
It has been found that the temperature rises by 10 to 15 ° C, and the optimum pH curve also spreads to the acidic side, and the enzymatic chemistry of the immobilized enzyme is considerably improved, and the reaction is performed under extremely advantageous conditions compared to when using the original enzyme. Can be done.

When the maltooligosaccharide is produced by the method of the present invention, various production methods can be adopted depending on the desired purity of the maltooligosaccharide. For example, a maltooligosaccharide having a purity of about 20 to 60% can be obtained by supplying the starch liquefaction solution to the reactor filled with the immobilized maltooligosaccharide amylase described above under the conditions described above. Further, a higher-purity (60 to 100%) maltooligosaccharide can be obtained by further purifying and separating the product obtained from the reactor. In this case, the purification / separation means is not particularly limited and various methods can be adopted, but, for example, means such as ultrafiltration, gel filtration, cation exchange resin column chromatography, carbon column chromatography are effective. Further, a part or all of the undegraded product obtained when the above-mentioned purification and separation is recirculated to a reactor filled with an immobilized maltooligosaccharide-forming enzyme to form a part of the feedstock, thereby liquefying the raw material starch. The yield of maltooligosaccharide per unit can be increased. Furthermore, a part or all of the undegraded material can be used as it is as a limit dextrin.

〔Example〕

 Next, the present invention will be described in detail with reference to Examples.

Example 1 Maltotetraose-forming amylase (originating from Pseudomonas stotteri, specific activity 80.8 IU / mg as enzyme)
-Immobilized enzyme was obtained by using DIAION-based adsorption resin SP-206 as a carrier. That is,
Immobilize 20000 IU of enzyme to 150 ml of 50 mM phosphate buffer of pH 7.0 for 20 g of carrier and put it in a 500 ml Erlenmeyer flask, and immobilize the enzyme on the carrier while shaking it at 120 strose, 4 cm width for 1 hour at room temperature. did. Then, this was filtered to remove the filtrate, and further washed with 10 mM phosphate buffer (pH 7.0) until the protein did not flow out, and the expression activity was 137I.
U / g of immobilized enzyme was obtained.

20 g of the obtained immobilized enzyme was packed in a column having a diameter of 27 mm and a length of 130 mm. 20w / w as raw material for the column reactor
% Waxy cornstarch liquefied liquid (DE7.5) 40g / hr,
That is, weight-based space velocity per unit activity 2.92 × 10 ^-3
It was supplied under the conditions of hr ^-1 (IU / g) ^-1 and temperature of 45 ° C. Table 1 shows the composition of the reaction product after 360 hours. The half-life of the enzyme in the reaction was 1680 hours.

Example 2 10 g of immobilized maltotetraose-forming amylase (expression activity 139 IU / g) obtained in the same manner as in Example 1 using a Duolite-based adsorption resin ES-8610 as a carrier was filled in a reactor. 16.7w / w% soluble starch (manufactured by Wako Pure Chemical Industries, Ltd.) as a raw material 10.7g / hr (solid content 1.79g / hr), temperature
It was supplied at 40 ° C. (weight-based space velocity per unit activity: 1.3 × 10 ⁻³ hr ⁻¹ (IU / g) ⁻¹ ). The operation result is the first
Shown in the figure.

As is clear from the figure, the operation has continued extremely stably over 1500 hours.

Further, the reaction product at the outlet of the column reactor was separated into a product 1 containing maltotetraose as a main component and a product 2 which was a separation residue by using an ultrafiltration device having a molecular weight cutoff of 1000. Table 1 shows the composition of Product 1 after 1500 hours of operation.

Comparative Example 1 Using a diaion-based adsorption resin HP-50 as a carrier, 7 g of immobilized maltotetraose-producing amylase (expression activity of 167 IU / g) obtained in the same manner as in Example 1 was charged into a reactor. Liquefied liquid (DE8) of 4.76 w / w% waxy corn starch was supplied to this at 1.21 g / hr at a temperature of 40 ° C (weight standard space velocity per unit activity 4.9 × 10 ^-5 hr ^-1 (IU / g) ^{- 1} ) was carried out to obtain a reaction product. The composition of this product is shown in Table 1.

Comparative Example 2 Using a diaion-based adsorption resin HP-50 as a carrier, 7 g of immobilized maltotetraose-forming amylase (expression activity 49 IU / g) obtained in the same manner as in Example 1 was charged into a reactor.
23.1w / w% waxy cornstarch liquefied liquid (DE
8) was supplied at a temperature of 40 ° C at 56.0 g / hr (weight-based space temperature per unit activity: 3.8 × 10 ^-2 hr ^-1 (IU / g) ^-1 ) to obtain a reaction product. The composition of this product is shown in Table 1.

Example 3 In Comparative Example 1, the substrate supply rate was 9.11 ml / hr (weight basis space velocity per unit activity 3.69 × 10 ⁻⁴ hr ⁻¹ (IU / g).
^-1 ) except that the same procedure as in Comparative Example 1 was performed. The composition of the obtained reaction product is shown in Table 2.

Example 4 In Comparative Example 2, the substrate supply rate was 2.07 ml / hr (weight-based space velocity per unit activity 1.40 × 10 ⁻³ hr ⁻¹ (IU / g).
^-1 ) except that the same procedure as in Comparative Example 2 was performed. The composition of the obtained reaction product is shown in Table 2.

Example 5 Comparative Example 1 was carried out in the same manner as Comparative Example 1 except that the substrate supply rate was set to a weight-based space velocity per unit activity of 2.21 × 10 ⁻⁴ hr ⁻¹ (IU / g) ⁻¹ . However, the ratio of maltotetraose (G ₄ ) in the obtained reaction product was 47.7%.
Met.

Example 6 In Comparative Example 2, the immobilized enzyme having an expression activity of 330 IU / g was filled and the liquefied liquid was supplied at 219 g / hr (weight-based space velocity per unit activity 2.19 × 10 ⁻² hr ⁻¹ (IU / g) ^-1 ) was carried out in the same manner as in Comparative Example 2, and the ratio of maltotetraose (G ₄ ) in the obtained reaction product was 28.1%.
The ratio of the total (G ₅ ⁺ ) of maltooligosaccharides having a degree of polymerization of maltopentaose or more and limit dextrin (G ₅ ⁺ ) was 65.9%.

[Effects of the Invention] The activity is maintained for a long time depending on the supply conditions of the starch liquefaction solution according to the method of the present invention, the target maltooligosaccharide can be efficiently produced, and a highly pure product can be obtained. In addition, a wide range of reaction temperature and pH can be applied during the operation of the reactor.

Therefore, the method of the present invention is an industrially useful method for producing maltooligosaccharides.

[Brief description of drawings]

FIG. 1 shows the elapsed time and the relative activity of maltotetraose when an immobilized enzyme was used.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Masafumi Ogata Inventor Masafumi Ogata 325 Chiyoda-Kawa Construction, Kawasaki City, Kanagawa Prefecture Shinshiro Residence Co., Ltd. (72) Inventor Haruhito Kobayashi 2-12-11 Hashido, Seya-ku, Yokohama, Kanagawa Prefecture Chiyoda Kako Construction Co., Ltd. Seya Dormitory (72) Inventor Taizo Miwa 2954 Imaizumi, Fuji City, Shizuoka Prefecture (72) Inventor Teruo Nakakuki 57 Kamo Green Hill, Kamo 57, Mishima City, Shizuoka Prefecture (72) Inventor ▲ Yoshita Masahiro 2954 Imaizumi, Fuji City, Shizuoka Prefecture (56) Reference JP 59-198977 (JP, A) JP 59-17992 (JP, A) JP 58-170492 (JP, A) JP 58 -63392 (JP, A)

Claims (1)

[Claims] 1. Malto-oligosaccharides (abbreviated as Gn, n represents an integer of 3 to 10) from maltotriose (abbreviated as G ₃ ) to maltodecaose (abbreviated as G ₁₀ ). When using malto-oligosaccharide-producing amylase (abbreviated as Gn-producing amylase), the specific surface area of pores having an affinity for the enzyme of 80% or more and a pore diameter of 100Å or more is 20 to 300 m ² Weight-based space velocity C / (A × B) hr per unit activity of immobilized enzyme determined by the following formula in a reactor filled with Gn-producing amylase immobilized on a porous carrier of / g ^-1 (IU / g) ^-1 (where A is the expression activity (IU / g), B is the immobilized Gn-producing amylase (g) packed in the reactor, and C is the starch liquefaction supplied to the reactor. Liquid volume (solid content) (g-solid content / hr) is 1
A method for producing a maltooligosaccharide, which comprises supplying under a condition of × 10 ^{-4 to} 3 × 10 ^-2 hr ^-1 (IU / g) ^-1 .