JPH04173095A - Method for saccharifying starch - Google Patents

Abstract

PURPOSE:To efficiently saccharify starch by immersing a carrier in a substrate aqueous solution, immobilizing amylase on the carrier and hydrolyzing the starch with the immobilized amylase. CONSTITUTION:An immobilization carrier such as celite or swine bone crushed product is subjected to a defatting treatment, immersed in a substrate aqueous solution containing 1-50wt.% of starch, etc., at room temperature for 12hr, and subsequently subjected to an excessive water-removing treatment to prepare a starch-infiltrated carrier (A). The component A is added to a 0.001-100wt.% amylase aqueous solution, shaken at a temperature of >=25 deg.C for approximately 15min for immersing the carrier in the solution, and subsequently subjected to a crosslinking treatment using glutaraldehyde to prepare an immobilized amylase (B). A 1-50wt.% starch aqueous solution having a pH of approximately 4.5 is mixed with the component B in an amount of 0.01-50wt.% based on the substrate (starch) and hydrolyzed at the optimal temperature and pH for the component B to saccharify the starch.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to an improvement in a method for saccharifying starch in the presence of an immobilized enzyme.

(b) Prior Art Since enzymes have many functions as biocatalysts, they are used in a wide range of fields, and their applications are likely to further expand in the future. However, since enzymes are generally expensive, it is necessary to immobilize them on some kind of water-insoluble carrier to recover and reuse the enzymes after use. To date, various methods have been developed for this purpose, including adsorption methods, entrapment methods, and crosslinking methods.

Glucoamylase, one of the amylases, has the ability to hydrolyze starch from its non-reducing end into glucose units to produce glucose, and is currently used industrially to saccharify starch. Methods for immobilizing glucoamylase include physical adsorption using activated carbon, chitin, etc. as a carrier, covalent bonding using CM Nacellous, etc., crosslinking using porous glass, and entrapping method using polyacrylamide. However, in most of the immobilization methods developed so far, the activity of glucoamylase after immobilization is extremely low, and is only about 10% of the activity of glucoamylase before immobilization. The current situation is that it cannot be expressed.

For this reason, the reaction rate is slow, and in reality, 50 ~
This process requires a long time of 70 hours, and in industrial applications, it has been difficult to implement a continuous process using immobilized enzymes from an economic standpoint.

(C) Problems to be Solved by the Invention In view of the above-mentioned current situation, the present invention aims to provide a method for increasing the saccharification reaction efficiency in a starch hydrolysis reaction using an immobilized enzyme.

(Means for solving the problem d1, that is, the present invention is characterized in that, in a starch hydrolysis reaction using immobilized amylase, an immobilized amylase obtained by immersing a carrier in a substrate before immobilization is used. This is a method for saccharification of starch.More specifically, the immobilization carrier is immersed in advance in an aqueous substrate solution to infiltrate the substrate into the carrier, which is then immersed in an aqueous amylase solution and cross-linked with glutaraldehyde or the like. This is a method of increasing the efficiency of the saccharification reaction by using the immobilized product in the starch hydrolysis reaction.

In the present invention, the raw material origin of starch serving as a substrate can be used without any restriction, and examples of amylase include liquefied α-amylase, glucoamylase, etc., and these include soybean,
Those derived from plants such as wheat, Aspergillus oryzae (A
spergillus oryzae).

Aspergillus Rhizopus niveus
) and other molds such as Bacillus subtilis.
In addition to those derived from bacteria such as S. lus sub-tilis), those derived from animals and other microorganisms can be used.

For these, commercially available enzyme agents with high activity can usually be used.

Also, as a carrier for immobilization, animal bone or celite can be used.

Inorganic materials such as alumina, activated carbon, porous glass, cation or anion exchange resins, chitinous adsorbents, and organic materials such as alginates can be used alone or in combination with fibers, etc., but preferably celite, pork, etc. These include pulverized bone material and non-woven fabric sheets formed from this material.

Immobilization of amylase onto such an immobilization carrier is performed by the following method. That is, a carrier from which excess organic matter has been removed by a conventional method (degreasing treatment, etc.) is immersed in a 1 to 50% by weight starch aqueous solution at room temperature for 12 hours or more, separated and, if necessary, dried under reduced pressure or in a desiccator. Dry inside. The infiltrated carrier is added to an amylase aqueous solution containing 0.001 to 100% by weight of the starch infiltrated carrier, and after immersion with gentle shaking at 25°C or higher for about 15 minutes or at 4°C or lower for about 60 minutes, Amylase is immobilized by crosslinking with glutaraldehyde using a conventional method. After immobilization,
The carrier is washed with 0.01-IM acetate buffer (pH 4.5) until no protein is eluted from the carrier, and if necessary, this is dried.

The immobilized enzyme obtained in this manner may be used in a starch hydrolysis reaction by a conventional method, for example, a 1 to 50% by weight starch aqueous solution (0.01 to IM
acetate buffer-pfl 4.5) and 0.01 to 50% by weight of the immobilized product based on the amount of substrate. Preferably 0.1~
10% by weight to determine the optimum temperature and p of each enzyme.
The hydrolysis reaction can be carried out in H. For example, in the case of glucoamylase, pH 3 to 6, preferably pH
4-5, 45-65°C, preferably 50-60°C. In addition, in the case of liquefied α-amylase, in addition to the one at around 60°C, the suitable reaction temperature is 90 to 95°C, and even 10°C.
There are also enzymes that have good thermostability between 0 and 110°C.

(e) Examples Example 1 As an example of a carrier, a carrier made from crushed pork bones (hereinafter referred to as P
Glucoamylase was immobilized using the method of the present invention. Substrate infiltration was performed using 30% PB at room temperature.
% starch solution for 12 hours or more, and immobilization was carried out by soaking 0.5 g of starch-infiltrated PB in 1 x 1 glucoamylase 25% solution.
This was done by immersing it in water and then quickly crosslinking it with glutaraldehyde. After immobilization, the carrier was washed with 0.1M acetate buffer (pH 4.5) until no protein was eluted from the carrier.
It was dried in a silica gel desiccator. For comparison, PB without substrate infiltration treatment was also investigated. Figure 1 shows the results of the activity per unit amount of PB, adsorbed protein to PB, and specific activity for both.

In addition, in the activity comparison of the immobilized glucoamylase obtained by the above method, soluble starch (manufactured by Wako Pure Chemical Industries, Ltd., reagent grade) was used as the substrate, and this 10% aqueous solution (0.1
M acetate buffer, pH 4.5) at 37°C for 30
The amount of reducing sugar produced after the minute reaction was measured by the Somogi method, and the amount of enzyme capable of producing reducing sugar equivalent to 1 μmol of glucose per minute was defined as 1 unit.

As is clear from Figure 1, PB was added to the starch solution before immobilization.
By immersing the glucoamylase, the activity increased approximately 10 times, and the activity expression rate also increased from 5% to 60%, indicating that the method of the present invention is extremely effective in immobilizing glucoamylase.

Furthermore, the results of the study on the time required for crosslinking after immersing PB in the enzyme solution are shown in FIG. 2. 25℃
When immobilization is carried out in the enzyme solution, the activity will decrease unless crosslinking treatment is performed immediately after immersion in the enzyme solution. This is because the infiltrated starch is degraded by glucoamylase and the substrate infiltration effect is lost. However, if immobilization is performed at 4°C to prevent starch from being decomposed, if cross-linking is performed within 60 minutes after immersion in the enzyme solution, there is almost no decrease in activity and the substrate infiltration effect is exerted. I admitted that I can do it.

Example 2 PB Receipt anion exchange resin [Amberlite TRA-94J ( (manufactured by Rohm and Haas), inorganic adsorbent [Celite R-630J]
(manufactured by Jonesman Building Sales).

Chito Pearl BCW300, a porous chitosan bead
The same experiment as in Example 1 was repeated using 1J (manufactured by Fujibo Co., Ltd.). As a result, as shown in FIG. 3, this effect was observed for most carriers, and it became clear that the method of the present invention has an extremely wide range of applications. In particular, when the carrier PB made from pork bones was used, the effect was remarkable, and it was found that the method of the present invention is very suitable for PB. In addition, although FIG. 4 shows the influence of pH and temperature on the glucoamylase activity immobilized on PB according to the present invention,
Both the optimum pH and temperature did not change compared to soluble glucoamylase.

Example 3 The immobilized enzyme used in the present invention must be able to be used stably over a long period of time. Therefore, the stability of the immobilized glucoamylase obtained by the method of the present invention was examined by repeated use. The reaction was carried out repeatedly at 37°C for 48 hours per operation using starch as a substrate, and the residual activity was determined from the initial rate of each reaction. As a result, as shown in FIG. 5, the immobilized glucoamylase obtained by the method of the present invention was found to be highly stable, especially when PB was used.

(f) Effect of the invention According to the present invention, in a starch hydrolysis reaction using immobilized amylase, the rate of activity expression is increased by using a carrier in which a substrate is infiltrated in advance and amylase is immobilized. , can increase starch saccharification efficiency.

[Brief explanation of the drawing]

Figure 1 shows the infiltration effect of starch on the immobilization of glucoamylase, Figure 2 shows the effect of holding time until cross-linking on the activity of immobilized glucoamylase, and Figure 3 shows the immobilization of glucoamylase on various carriers. Figure 4 shows the effect of immobilization on pi and temperature of glucoamylase activity, and Figure 5 shows the results of repeated use of immobilized glucoamylase. Patent applicant Nisshin Oil Co., Ltd.
Joji Takahashi Yukuni Mukotaka Sohan Sotsuka R Figure 2 Activity (unit/1g of carrier) Note) 1) PB: Pulverized pork bone particles 5) Chitosan beads: [Chito Pearl BCW3001J
(Made by Fujibo ■) Figure 4

Claims (1)

[Claims] A method for saccharifying starch, which comprises using a carrier immersed in a substrate as a pre-immobilization treatment in a starch hydrolysis reaction using immobilized amylase.