JPH0365759B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing starch sugar, and more specifically, the present invention relates to a method for producing starch sugar, and more specifically, a starch liquefied liquid is supplied to a reactor filled with various amylases immobilized on specific porous chitosan. The present invention relates to a method for producing starch sugars such as corresponding glucose, maltose, and alto-oligosaccharides. [Prior art and problems to be solved by the invention] Various methods have been proposed for producing starch sugar using immobilized enzymes. The manufacturing method is published in Japanese Patent Publication No. 57-17517,
No. 58-60989, etc. However, in the former method, the glucose concentration produced is insufficient, and in the latter method, high concentration glucose is achieved using potato starch as a raw material, and when using cornstarch, etc. as a raw material, which is generally difficult to produce high concentration glucose. is not described. Further, although the use of immobilized β-amylase in the production of maltose has been shown in Japanese Patent Application Laid-open No. 198977/1983, it is desirable to further improve the maintenance of enzyme activity. Furthermore, the present inventors have already developed a method for producing maltooligosaccharides by immobilizing amylase that produces maltooligosaccharides having a degree of polymerization higher than that of maltotriose (Japanese Patent Application No. 125093/1983 (JP Patent Application No. 125093/1983). No. 285998)). Although this technique can be fully implemented industrially, there is room for improvement in making more effective use of expensive enzymes. [Means for Solving the Problems] As a result of repeated studies on carriers for immobilizing amylase, the present inventors have found that when a specific porous chitosan is used, the liquid passing rate can be increased compared to the conventional method. The present invention was achieved based on the discovery that the enzyme activity can be stably maintained. We have also discovered that the yield of the desired starch sugar can be increased by combining immobilized enzymes to form a complex system. First, the present invention is based on deacetylated natural polymer chitin, which is then crosslinked with dicarboxylic acid, dialdehyde, diisocyanate, etc. to impart acid resistance. The present invention relates to a method for producing starch sugar, which is characterized in that amylase immobilized on porous chitosan into which a group has been introduced is made to act on a starch liquefaction solution, and secondly, it is characterized in that amylase immobilized on porous chitosan into which a group has been introduced is made to act on a starch liquefied solution. The present invention relates to a method for producing starch sugar, which is characterized in that it is made to act on a starch liquefaction liquid together with a cutting enzyme. There are various types of amylases used in the present invention, and glucoamylases derived from fungi such as Rhizopus, Aspergillus, Mucor, and Pyricalaria are mainly used, and those derived from Rhizopus deremer are particularly preferred. It is. In addition, yeasts such as Endomyces, Trichoderma, and Satucharomyces and bacteria such as Clostridium acetobutylicum are known.
β-Amylases include those derived from plants such as soybeans and malt, as well as from Bacillus polymyxa [D.
French, Arch.Biochem.Biophys., 104 , 338
(1964)], Bacillus cereus [Y.Takasaki,
Agric.Biol.Chem., 40 , 1515, 1523 (1976)], Pseudomonas bacteria [S.Sinke et al., J.Ferment.
Technol., 53 , 693, 698 (1975)], Streptomyces hygroscopicus [Y. Hidaka et al.
Sta¨rke, 26 , 413 (1974)], Streptomyces
Plecoticus [Katsuo Wakao et al., Starch Chemistry, 25 , 155
(1978)] are of microbial origin. Furthermore, the following amylases are known to produce oligosaccharides having a degree of polymerization higher than maltotriose. Maltotriose-producing amylase [Katsuo Wakao et al.: Starch Chemistry, 26 , 175 (1979), originating from Streptomyces griseus ; Yoshiyuki Takasaki: Abstracts of the 1983 Japan Agricultural Chemistry Conference, P169 (1983) , originating from the genus Bacillus ] maltotetraose-producing amylase [JF
Robyt and RJ Ackerman: Arch.Biochem.
Biophys., 145 , 105 (1971), Pseudomonas
Originated from Pseudomonas stutzeri ] Maltopentaose-producing amylase [N.
Saito: Arch.Biochem.Biophys., 155 . 290
(1973), Bacillus licheniformis
licheniformis) origin; Shoichi Kobayashi et al.; Showa period
Abstracts of the 1958 Japanese Starch Society Conference, P301 (1983);
Naohiro Yoshigi et al.: Abstracts of the 1984 Japan Agricultural Chemistry Conference, P584 (1984)] Maltohexaose-producing amylase [K.
Kainuma et al.: FEBS Lett., 26 . 281 (1972), Aerobacter aerogenes
aerogenes) origin; JFKennedy and C.
A. White: Sta¨rke, 31 , 93 (1979); Hajime Taniguchi et al.: Starch Chemistry, 29 . 107 (1982); Y. Takasaki: Agric.
Biol.Chem., 47 . 2193 (1983)] Next, as a porous chitosan used as a carrier for the above-mentioned amylase, there is, for example, the trade name: Chitopal (manufactured by Fujibo Co., Ltd.), which is made by deacetylating the natural polymer chitin and then adding dicarboxylic acid, dicarboxylic acid, These are porous beads that have been cross-linked with dialdehyde, diisocyanate, etc. to give them acid resistance, and then have a functional group such as an aliphatic or aromatic group introduced as a spacer, resulting in PH stability, chemical resistance, and thermal stability. Excellent. This “Chitopearl” has a particle size of 0.1~
3.0mm, pore diameter 3.0μm or less, specific surface area 15-230m ² /g
However, the present invention is not limited to this value. Any method can be used to immobilize various amylases on chitosan, and for example, a method of contacting both amylases in a buffer solution can be adopted. As an example, 100 mg of "Chitopearl" is sufficiently equilibrated with various buffer solutions (PH4.0 to 8.0) with a molar concentration of 0.01 to 0.20, and then 5 to 500 units of various amylases are dissolved in 2 ml of the buffer solution. Add and mix thoroughly. Then, after being left at room temperature for 0.5 to 24 hours or subjected to reciprocating shaking treatment (120 strokes/min) for 0.5 to 5.0 hours,
Pass through a glass filter, followed by various buffers.
Wash with 50ml. The immobilized enzyme thus obtained has an apparent immobilization rate of 90% or more, and the expression activity of the immobilized enzyme is 40 to 2000 units per gram of wet weight of the carrier. Note that the apparent immobilization rate is a value calculated using the following formula. Supplied enzyme activity - Enzyme activity in washing solution / Supplied enzyme activity x 100 (%) In addition to the method described above, enzyme immobilization methods include filling a column with a carrier and then passing the enzyme solution through it by a descending method or an ascending method. This method can also be applied. The immobilized amylase used in the present invention is extremely easy to immobilize on a carrier, and the enzyme expression activity is sufficient for practical use. Next, the immobilized debranching enzyme used in the second invention will be explained. As a branch cutting enzyme, pullulanase originating from microorganisms such as Bacillus acidopluriticus and Glebsiella pneumonia, and isoamylase produced by microorganisms of the genus Pseudomonas amylodermosa and Cytophaga can be used, but most glucose-producing amylases are PH4. Since most maltooligosaccharide-producing amylases have an optimum pH in the range of 5.0 to 8.5, it is desirable to use a debranching enzyme that is also stable and has a similar optimum pH range. Regarding the carrier for immobilizing the debranching enzyme, if it shows high expression activity by immobilization operation,
Although any carrier may be used, it is particularly desirable to use the following carriers. That is, as a result of our study to select carriers that can effectively immobilize various debranching enzymes from a large number of carriers, we found that, in particular, weakly acidic porous adsorption resins, weakly acidic cationic resins,
We have found that phenolic adsorption resins, granular porous chitosan, and the like are suitable carriers. More specifically, the product names of Duolite resin (manufactured by Diamond Shamlok Co., Ltd.) are "S-761", "S-762",
"ES-771", "C-464", "A-7", "S-587"
,
Examples include "A-562" and the above-mentioned "Chitopearl". Note that the method of immobilizing the debranching enzyme is not limited;
For example, the method described above can be applied. In addition, the method for measuring the activity of native debranching enzymes and immobilized debranching enzymes uses pullulan (manufactured by Hayashibara Biochemical Research Institute) or amylopectin as a substrate, and the reaction is performed at their optimum pH. is the same as Various raw material starches can be used in the present invention, but potato starch, sweet potato starch, corn starch, waxy corn starch, cabbage mackerel starch, etc. are usually used. In addition, the glucose equivalent (DE) of the starch liquefaction liquid passed through the reactor is usually 1 to 1.
35, preferably in the range of 5 to 20. Here, the DE of the starch liquefied liquid is less than 1 in the case of waxy corn starch, and in the case of other starches.
Those with a DE of 5 or less are subject to severe aging and require careful handling during the process. On the other hand, when DE exceeds 35, retrosynthesis is promoted for glucose production by glucoamylase, and isomaltose,
The production of panose etc. increases and the yield of glucose decreases. In addition, the production of various maltooligosaccharides increases the production of low-molecular sugars such as glucose and maltose, and the yield of maltooligosaccharides decreases, so it is not suitable. Although there are no particular limitations on the method of liquefying various starches, the method is usually treated with liquefied α-amylase or an acid such as hydrochloric acid. Next, malto-oligosaccharide means maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and the like. The present inventors have conducted various studies on conditions for efficiently producing various starch sugars using immobilized enzymes, and have found that the following factors have a large influence. In other words, the conditions include the type of substrate used, its concentration and its supply amount, the type (physical properties) of the immobilized carrier, the amount of immobilized enzyme, the amount of the enzyme packed into the packed column, the temperature of the reaction system, the pH, etc. As a result of a systematic study of these factors, it has been found that each starch sugar can be efficiently produced within the range of the expressions and conditions shown below. That is, in the method of the present invention, the immobilized amylase is packed into a reactor, and the above-mentioned starch liquefaction liquid is heated to a mass superficial velocity of 1×10 ⁻⁴ to 2×10 ⁻¹ hr ⁻ per unit activity of the immobilized enzyme. ¹ (IU/g)
By supplying under the conditions of ^-1 , various starch sugars can be efficiently produced. More preferably 1×10 ⁻⁴ to 3× for glucoamylase
10 ^-3 hr ^-1 (IU/g) ^-1 , 1 x 10 ^-4 to 4 x 10 ^-3 hr ^-1 (IU/g) ^-1 for β-amylase, for various maltooligosaccharide-producing amylases 3×10 ^-4 ~2
The condition of ×10 ^-1 hr ^-1 (IU/g) ^-1 should be applied. Here, the weight-based superficial velocity per unit activity of the immobilized enzyme is a value determined as follows.
First, add 10 mg (wet) of the same immobilized amylase that will be filled into the reactor to 0.5 ml of 10 mM various buffers (PH7.0) (in a 50 ml Erlenmeyer flask), mix well, and then supply to the reactor. Add 5.0 ml of the same substrate (same type of starch, same concentration, etc.) and perform the enzymatic reaction at the same temperature as the reactor while shaking with a reciprocating shaker at 120 strokes/min. and a width of 4 cm. Reducing sugars are measured by the Somogyi-Nelson method, or starch sugars produced directly with an analytical instrument such as high-performance liquid chromatography are measured to determine the expressed activity (this expressed activity is expressed as A IU/
g-Carrier. ). The enzyme activity is expressed as 1 unit (1 international unit IU), which is the amount of enzyme that cleaves 1 μmol of glycosidic bonds per minute under each reaction condition. In addition, when the immobilized amylase to be filled in the reactor is Bg (wet) and the amount of starch liquefaction liquid supplied to the reactor is solid content as Cg - solid content/hr, the weight-based superficial velocity per unit activity is C /(A×B)hr ^-1 (IU/g) ^-1 . In addition, when DE is large, the target starch sugar and sugars smaller than it are included in the raw material, so it is more practical to use the solid content excluding these as the value of C. The weight-based superficial velocity per unit activity is 2 x 10 ^-1 hr ^-1 (IU/g) ^-1 , and in the case of glucoamylase it is 3 x 10 ^-3 hr ^-1 (IU/g) ^-1 , β- 4 x 10 ^-3 hr ^-1 (IU/g) ^-1 in the case of amylase, 2 x 10 ^-1 hr ^-1 in the case of malto-oligosaccharide producing amylase with a degree of polymerization higher than maltotriose.
If it is larger than (IU/g) ^-1 , that is, if the reaction time in the reactor is short, the hydrolysis reaction will not be carried out sufficiently, resulting in a poor yield of each starch sugar, which is not preferable. In addition, in the case of producing malto-oligosaccharides, when the weight-based superficial velocity per unit activity becomes smaller than 1×10 ^-4 hr ^-1 (IU/g) ^-1 , the reaction time in the reactor becomes longer. Then, as revealed in the following publication, the maltooligosaccharide produced is further overdecomposed, resulting in the production of low-molecular sugars such as glucose and maltose.
This is not preferred because it not only significantly reduces the purity of the product, but also impairs the efficiency of subsequent purification and separation. Regarding the overdecomposition of malto-oligosaccharides having a degree of polymerization higher than maltotriose, the malto-oligosaccharide-producing amylase is capable of producing various oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, etc.) in the early stage of the reaction. ), but it has been revealed that the product itself is decomposed in the later stages of the reaction [T.Nakakuki et al; Carbohydr.
Res., 128 , 297 (1984)]. In addition, in the case of glucoamylase, the weight-based superficial velocity per unit activity is 1×10 ^-3 hr ^-1 (IU/g).
From around ^-1 , the reverse reaction that produces panose, isomaltose, etc. proceeds, and the yield of the desired glucose decreases, making it less efficient. On the other hand, realistically, when the weight-based superficial velocity per unit activity becomes 1×10 ^-4 hr ^-1 (IU/g) ^-1 or less, the residence time in the reactor becomes longer and the reactor The size becomes too large, making it economically ineffective, and the target starch sugar content decreases due to aging of the raw starch liquefaction liquid, which may also cause operational troubles. According to the present invention, by using immobilized amylase, it is expected that the reaction conditions can be expanded compared to the original amylase that is not immobilized. For example, in the case of immobilized maltotetraose-generating amylase immobilized on chitosan, the temperature stability extends to the higher temperature side by about 10°C, and the PH stability is also improved over a wide range. We also found that the optimal temperature increases by 10 to 15 degrees Celsius due to immobilization, and the optimal PH curve also expands to the acidic side.
The enzymatic chemistry of the immobilized enzyme was significantly improved;
The reaction can be carried out under much more favorable conditions than when using the original enzyme. When producing maltooligosaccharides, for example, by the method of the present invention, various production methods can be used depending on the desired purity of the maltooligosaccharides. For example, maltooligosaccharide with a purity of 20-60% is 40%
It is obtained by supplying (w/w) starch liquefied liquid to the reactor filled with the above-mentioned immobilized malto-oligosaccharide amylase under the above-mentioned conditions. Furthermore, maltooligosaccharides of higher purity (60 to 100%) can be obtained by further purifying and separating the product obtained from the above reactor. Purification and separation means in this case are not particularly limited and various methods can be used, but for example, means such as ultrafiltration, gel filtration, cation exchange resin column chromatography, and carbon column chromatography are effective. In addition, some or all of the undegraded products obtained during the purification and separation described above can be recycled to a reactor filled with an immobilized maltooligosaccharide-forming enzyme and used as part of the feedstock. The maltooligosaccharide yield per starch liquefaction solution can be increased. Furthermore, part or all of the undecomposed product can be used as it is as limitodextrin. Next, a second invention in which an immobilized debranching enzyme is used in combination with immobilized amylase will be described. Regarding the ratio of immobilized debranching enzyme used in combination with immobilized amylase, the concentration (yield) of starch sugar can be increased as the amount of the latter increases, but usually the former is 1 to 0.1 to the latter on an expression activity basis. 5, preferably in the range of 0.2 to 2. Even if the ratio of the immobilized debranching enzyme is increased above the upper limit, the corresponding effect will not be achieved, and the size of the reactor will increase proportionately, which is not economically preferable. Here, when a debranching enzyme is used in combination, the weight-based superficial velocity per unit activity is determined by the expression activity (A
IU/g) may be determined by considering only the expression activity of the amylase used without considering the expression activity of the debranching enzyme. In the case of a composite enzyme system in which both enzymes are used together, various forms of reactor and filling method can be considered. For example, two types of immobilized enzymes may be filled in separate containers, two types of immobilized enzymes may be mixed and then filled into the same container, or two types of native enzymes may be mixed at a certain ratio and then mixed. , a method of simultaneously immobilizing and filling a container, etc. [Effects of the Invention] According to the method of the present invention, when producing maltooligosaccharides having a degree of polymerization higher than that of glucose, maltose, or maltotriose, the weight-based superficial velocity per immobilized enzyme unit activity can be made larger than before. Moreover, the enzyme activity is stably maintained over a long period of time. Therefore, the desired starch sugar can be produced efficiently and with a high yield. In particular, when a debranching enzyme is used together with amylase to form an immobilized composite enzyme system, the yield of the target substance is significantly improved. Furthermore, even when corn starch is used as the raw material starch, starch sugar (especially glucose) can be obtained at a high concentration comparable to that of native enzymes. [Examples] Next, the present invention will be explained in detail by examples. Example 1 Using porous chitosan (trade name: Chitopal BCW3505, manufactured by Fujibo Co., Ltd.) as a carrier, glucoamylase originating from Rhizopus deremer (manufactured by Shin Nihon Kagaku Co., Ltd.) was immobilized. That is, after fully equilibrating chitosan as a carrier with 20mM acetate buffer (PH5.5), 10g of carrier (wet weight) was weighed into a 100ml Erlenmeyer flask, and glucoamylase was added to the carrier in an amount of 1.
1050 units per g (liquid volume 10 ml) were added. Then, shake back and forth for 1 hour at room temperature (120 strokes/
(min) and fixed. Furthermore, it was thoroughly washed with 20 mM acetate buffer (PH5.0) until no protein was eluted, to obtain an immobilized glucoamylase preparation. When the expression activity of this preparation was measured by the method described above, it was found to be 435 IU/g of carrier. Fill a glass column (diameter 10 mm, length 20 mm) with 10 ml of this immobilized glucoamylase and use it as a substrate.
30% (w/w) cornstarch liquefied liquid (DE=
11, PH5.5), temperature 50℃, superficial velocity 0.25,
0.5 and 1.0 hr ^-1 (weight superficial velocity per unit activity is 2.48 × 10 ^-4 , 4.95 × 10 ^-4 and
The solution was continuously passed under various conditions of 9.89×10 ⁻⁴ hr ⁻¹ (IU/g) ⁻¹ ). Note that the superficial velocity was calculated using the following formula. The results are shown in Table 1. Superficial velocity (hr ^-1 )=Liquid flow rate (ml/hr)/bed volume (ml) Comparative Example 1 Porous weakly basic anion exchange resin Duolite A-7 (manufactured by Diamond Shamlok Co., Ltd.) was used as a carrier. The same procedure as in Example 1 was carried out except that immobilization was carried out by the method described in Example 1 of JP-A No. 58-60989. Furthermore, the expression activity of the immobilized enzyme is
157 IU/g of carrier. Using this immobilized enzyme, an experiment was conducted in the same manner as in Example 1 (the weight-based superficial velocity per unit activity was 6.85×
10 ^-4 , 1.37×10 ^-3 and 2.74×10 ^-3 hr ^-1 (IU/g)
^-1 ). The results are shown in Table 1. Table 1 Glucose concentration in reaction solution (%) Superficial velocity (hr ^-1 ) Example 1 Comparative example 1 0.25 95.2 94.2 0.5 96.7 93.8 1.0 95.2 87.4 Example 2 Porous chitosan (trade name: Chitopal BCW3505, β-amylase (originated from soybean, manufactured by Nagase Sangyo Co., Ltd.) was immobilized using a conventional method. The expression activity of the obtained immobilized β-amylase was 230 IU/g-carrier. This immobilized β-amylase was packed into a glass column (diameter 27 mm, length 130 mm), and 25%
(w/w) of starch liquefied liquid (DE=7, PH6.0) at a temperature of 50℃, superficial velocity of 0.2, 0.5, 1.0 and
1.5hr ^-1 (weight superficial velocity per unit activity is 3.09×10 ^-4 , 7.73×10 ^-4 , 1.55×10 ^-3 and
The solution was continuously passed under various conditions of 2.32×10 ⁻³ hr ⁻¹ (IU/g) ⁻¹ ). The results are shown in Table 2. Also, the sky velocity
Figure 1 shows the change in maltose content in the reaction solution over time when the solution was continuously passed through the reactor at 1.5 hr ^-1 . Comparative Example 2 β-amylase was immobilized in the same manner as in Example 2, using Duolite adsorption resin S-761 (manufactured by Diamond Shamlok Co., Ltd.) as a carrier. The expression activity of immobilized β-amylase is 198IU/
g-carrier. 10 ml of this immobilized enzyme was packed into a glass column in the same manner as in Example 2, and a 25% (w/w) starch liquefaction solution (DE=7, PH6.0) was used as a substrate at a temperature of 50°C.
superficial velocity 0.2, 0.5, 1.0 and 1.5hr ^-1 (weight based superficial velocity per unit activity is 3.59 x 10 ^-4 , respectively)
8.98×10 ^-4 , 1.80×10 ^-3 and 2.70×10 ^-3 hr ^-1
(IU/g) ⁻¹ ) The solution was continuously passed under each condition. The results are shown in Table 2. Table 2 Maltotate concentration in reaction solution (%) Superficial velocity (hr ^-1 ) Example 2 Comparative example 2 0.2 52.5 52.4 0.5 52.5 44.8 1.0 52.3 37.5 1.5 52.1 30.3 As is clear from Table 2 and Figure 1. According to the present invention, a higher amount of maltose can be produced at a higher superficial velocity than when using a conventional immobilized enzyme,
Moreover, almost no decrease in activity was observed even after 30 days, and the half-life was over 1 year. Example 3 Maltotetraose-producing amylase (originated from Pseudomonas stotzeri, specific activity
80.8IU/mg protein), and chitosan beads (product name: Chitopearl) as the immobilization carrier.
An immobilized enzyme was obtained using BCW3505 (manufactured by Fujibo Co., Ltd.). That is, 20g of carrier was mixed with 50mM Tris.
−20000 IU dissolved in 100 ml of the same buffer after thorough equilibration with HCl buffer (PH7.0)
of enzyme and shake back and forth for 1 hour at room temperature (300°C).
ml Erlenmeyer flask (120 strokes/min, 4cm width)
At the same time, the enzyme was immobilized on the carrier. Next, the mixture was filtered through paper and thoroughly washed with 10 mM Tris-HCl buffer (PH 7.0) until no protein was eluted, to obtain an immobilized maltotetraose-generating enzyme with an expression activity of 350 IU/g of carrier. Next, 10 ml of the maltotetraose-producing immobilized enzyme was filled into a glass column with a diameter of 27 mm and a length of 130 mm. Using 26.2% (w/w) starch liquefaction liquid (DE=7, PH7.2) as the substrate, temperature 45℃, superficial velocity
2.0, 5.0 and 10.0 hr ^-1 (weight based superficial velocity per unit activity is 2.42 x 10 ^-3 , 6.05 x 10 ^-3 and 1.21 x 10 ^-2 hr ^-1 (IU/g) ^-1 respectively). The liquid was continuously passed under the following conditions. The results are shown in Table 3. Further, Fig. 2 shows the change over time in the maltotetraose content in the reaction solution when the solution was continuously passed under the condition of a superficial velocity of 2.0 hr ^-1 . Comparative Example 3 10 g of immobilized maltotetraose-generating amylase (expression activity:
215 IU/g) was charged into the reactor, and 26.2% (w/w) starch liquefied liquid (DE=7, PH
7.2) at 24 ml/hr, temperature 45°C, superficial velocity 2.0, 5.0 and 10 hr ^-1 (weight based superficial velocity per unit activity is 3.48 x 10 ^-3 , 8.71 x 10 ^-3 and 1.74 x, respectively).
The liquid was continuously passed under the conditions of 10 ^-2 hr ^-1 (IU/g) ^-1 ). The results are shown in Table 3. Also, superficial velocity 2.0hr ^-1
Figure 2 shows the change in maltotetraose content in the reaction solution over time when the solution was continuously passed through the reactor. Table 3 Maltotetraose concentration superficial velocity (hr ^-1 ) in reaction solution Example 3 Comparative Example 3 2.0 44.5 42.6 5.0 44.1 37.2 10.0 41.7 31.5 As described above, according to the present invention, the conventional immobilized enzyme A higher purity maltotetraose can be obtained than when using . Comparative Example 4 Immobilized maltotetraose-producing amylase obtained in the same manner as in Example 3 (however, the expression activity
273IU/g) was charged into the reactor, and 10%
(w/w) starch liquefied liquid (DE=8.0, PH=6.8) at a temperature of 40℃ and a flow rate of 1.1ml/hr (the weight-based superficial velocity per unit activity is (6.83×10 ^-5 hr ^-1
(IU/g) ^-1 ) was continuously passed through the solution to obtain a reaction product.
The composition of this product is shown in Table 4. Comparative Example 5 Immobilized maltotetraose-producing amylase obtained in the same manner as in Example 3 (however, the expression activity
46.7IU/g) was charged into the reactor, and 30
% (w/w) of starch liquefaction liquid (DE=8.0, PH=6.8)
using conditions of temperature 40℃ and flow rate 210ml/hr (weight-based superficial velocity per unit activity is 2.29×10 ^-1 hr ^-1
(IU/g) ^-1 ) was continuously passed through the solution to obtain a reaction product.
The composition of this product is shown in Table 4. [Table] As shown above, when the weight-based superficial velocity per unit activity is smaller than 1×10 ^-4 hr ^-1 (IU/g) ^-1 and when the superficial velocity per unit activity is smaller than 2×10 ^-1 hr ^-1 (IU/g ) ^-1 , it can be seen that the yield of maltotetraose decreases in all cases. Example 4 Same as Example 1 except that pullulanase (derived from Curvesiella pneumoniae, specific activity 50 IU/mg-protein, manufactured by Amano Pharmaceutical Co., Ltd.), which is a branching enzyme, was used and phosphoric acid buffer with a pH of 6.0 was used. It was immobilized on Chitopearl BCW3505 using the method described above. The expression activity of the obtained immobilized pullulanase was 129IU/
g-carrier. Next, glass column 2 with a diameter of 10 mm and a length of 200 ml
Using a book, 4 ml of immobilized glucoamylase and 9.0 ml of immobilized pullulanase obtained by the above method (expression activity ratio: about 3:1) were mixed and filled. on the other hand,
A 30% (w/w) cornstarch liquefied liquid (DE=11, PH5.5) was used as the substrate at a temperature of 50°C and a superficial velocity of 0.25, 0.5 and 1.0 hr ^-1 (weight based superficial velocity per unit activity). are 2.48×10 ^-4 and 4.95×10 ^-3 respectively.
and 9.89×10 ⁻⁴ hr ⁻¹ (IU/g) ⁻¹ ). The results obtained are shown in Table 5. Table 5 Glucose concentration in reaction product Superficial velocity (hr ^-1 ) Example 5 Example 5 0.25 95.8 95.2 0.5 97.3 96.7 1.0 96.1 95.2 As shown in the table, the complex immobilized enzyme system is better than the single immobilized enzyme system. The glucose concentration in the reaction product increased by about 1%. Example 5 10 ml of immobilized β-amylase obtained in the same manner as in Example 2 and 10 ml of immobilized pullulanase obtained in the same manner as in Example 4 were mixed (expression activity ratio: β-amylase: pullulanase = 2.1:1.0). and filled each. Using 25.0% (w/w) starch liquefaction liquid (DE = 7, PH 6.0) as a substrate, the temperature was 50°C and the flow rate was 15.
ml/hr (weight-based superficial velocity per unit activity is
The liquid was continuously passed under the conditions of 2.32×10 ⁻³ hr ⁻¹ (IU/g) ⁻¹ ). Table 6 shows the sugar composition of the column effluent after 0, 15, and 30 days of operation. As shown in the table, the maltose purity of the complex immobilized enzyme system is approximately 10-12% higher than that of the single enzyme system containing only β-amylase.
Almost no decrease in activity was observed even after 30 days. Table 6 Change in maltose production amount over time Number of days over time Single enzyme system Combined enzyme system 0 53.0% 64.2% 15 51.8% 64.2% 30 50.1% 64.0% Example 6 Immobilized maltotetraose production obtained in the same manner as Example 3 Mix 10 ml of enzyme and 5 ml of immobilized pullulanase obtained in the same manner as in Example 3 (expression activity ratio, β
- amylase: pullulanase = 4.1) and filled respectively. Using 26.2% (w/w) starch liquefied liquid (DE = 7, PH 7.2) as a substrate, the temperature was 40°C and the flow rate was 24/hr (weight-based superficial velocity per unit activity was 4.06 × 10 ^-3 hr). ^-1 (IU/g) ^-1 ). Table 7 shows the sugar composition of the column effluent after 0, 15, and 30 days of operation. As shown in the table, the maltotetraose purity of the complex immobilized enzyme system is approximately 5% higher than that of the single enzyme system containing only the immobilized maltotetraose-generating enzyme, and there is almost no decrease in activity even after 30 days. I couldn't help it. Table 7 Change in the amount of maltotetraose produced over time Number of days over time Single enzyme system Combined enzyme system 0 45.2% 50.5% 15 44.0% 48.3% 30 42.9% 46.2% Reference example Maltotetraose producing amylase (originated from Pseudomonas stotzeri) as an enzyme , specific activity 80.1 IU/mg protein), and three types of carriers were used to obtain immobilized enzymes. Here, the three types of carriers are the first carrier according to the present invention, which is obtained by deacetylating natural polymer chitin, drying and pulverizing it, adding it to an aqueous solution of glutaraldehyde to crosslink it, and further using it as a spacer. Two volumes of 4,4'-diphenylmethane diisocyanate having an aromatic functional group were added, treated at 30°C for 15 hours, and then washed with acetone (hereinafter referred to as carrier-1). The second one was crosslinked in the same manner as above, and then hexamethylene diisocyanate having an aliphatic functional group was similarly introduced as a spacer (hereinafter referred to as carrier-2). Furthermore, the third one was crosslinked in the same manner as above, but without introducing a spacer (hereinafter referred to as carrier-3). 200IU of enzyme per 0.2g of carrier in 50ml of pH7.0
Add 2.0 ml of M phosphate buffer to a 50 ml Erlenmeyer flask and incubate for 1 hour at room temperature for 120 strokes.
The enzyme was immobilized on the carrier while shaking at a width of 4 cm. Next, this was filtered to remove the filtrate, and further 10
The immobilized enzyme was obtained by washing thoroughly with mM phosphate buffer (PH 7.0) until no protein flows out. Regarding this immobilized enzyme, the apparent immobilization rate and the expression activity of the immobilized enzyme were measured by the following method. The apparent immobilization rate is determined by adding 0.5% reduced soluble starch solution to 0.1 ml of the supernatant diluted 300 times.
Add 0.5ml and 0.4ml of 50mM phosphate buffer (PH7.0) and react at 40℃ for 10 minutes to remove the produced reducing sugar.
The enzyme activity in the supernatant was determined by the Somogyi-Nelson method and determined by the following formula. Added enzyme activity - enzyme activity in supernatant/added enzyme activity x 100 The results are shown in Table 8. As is clear from the table,
The immobilized enzyme using Carrier-1 and Carrier-2 according to the present invention has a high immobilization rate and extremely excellent activity. On the other hand, carrier-3, in which only crosslinking was performed, gave unsatisfactory results, demonstrating that the prediction that chitosan would be a good carrier for maltooligosaccharide-producing amylase does not hold true. 【table】

[Brief explanation of drawings]

FIG. 1 shows the change over time in the maltose content in the reaction solution in Example 2, and FIG. 2 shows the change over time in the maltotetraose content in the reaction solution in Example 3 and Comparative Example 3.

Claims (1)

[Scope of Claims] 1 Natural polymeric chitin is deacetylated and then crosslinked with dicarboxylic acid, dialdehyde, diisocyanate, etc. to impart acid resistance, and further aliphatic or aromatic functional groups are added as a spacer. A method for producing starch sugar, characterized in that amylase immobilized on porous chitosan into which a group has been introduced is allowed to act on a starch liquefaction liquid. 2. The method according to claim 1, wherein the amylase is any one of glucoamylase, β-amylase, and maltooligosaccharide-producing amylase. 3. The method according to claim 1 or 2, wherein the starch is cornstarch. 4 Porous material made by deacetylating natural polymer chitin and then crosslinking with dicarboxylic acid, dialdehyde, diisocyanate, etc. to impart acid resistance, and further introducing functional groups such as aliphatic or aromatic groups as spacers. A method for producing starch sugar, characterized in that amylase immobilized on chitosan is allowed to act on a starch liquefaction liquid together with an immobilized debranching enzyme. 5. The method according to claim 4, wherein the amylase is any one of glucoamylase, β-amylase, and maltooligosaccharide-producing amylase. 6. The method according to claim 4 or 5, wherein the starch is cornstarch. 7. The method according to any one of claims 4 to 6, wherein the debranching enzyme is pullulanase originating from Bacillus acidopluriticus or Klebsiella pneumonia, or isoamylase originating from Pseudomonas amylodermosa or a microorganism of the genus Cytophaga.