JPS5945355B2 - Method for immobilizing bacterial α-1,6-glucosidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for recovering and insolubilizing bacterial α-1,6-glucosidase.

To obtain high yields of maltose from starch, usually
Two types of enzymes are required.

One is an enzyme that hydrolyzes maltose units from the non-reducing end of starch, and this enzyme is called β-amylase.

The other is an enzyme that hydrolyzes α-1,6-glucoside bonds, which are branched bonds in amylopectin, which make up most of the composition of starch.This enzyme includes isoamylase and pullulanase, and generally α-1,6-
It is called glucosidase.

The present inventor previously discovered that an enzyme produced by a bacterium isolated from soil produces maltose from starch in almost the theoretical yield, and that this enzyme produces maltose in a similar amount to β-amylase and a type of α-amylase.
-1,6-glucosidase was confirmed.

Therefore, the present inventor has conducted research on an enzyme insolubilization method for industrial production of this enzyme and effective use of the enzyme, and has found that α-1,6-glucosidase is extremely effective against various types of glass. It was confirmed that the enzyme was immobilized by adsorption and that the immobilized enzyme was extremely stable.

The present invention has been made based on this knowledge.

The term vitreous in the present invention refers to silicate glass powder containing silicic acid (SiO□) as a main component, porous glass made of silicate glass, volcanic glass in glass, or processed products thereof.

Adsorption and immobilization of Bacillus α-1,6-glucosidase on glass is usually carried out at a pH of 5 to 8 and a temperature of 50° C. or less.

α-1,6-glucosidase is 500~ per gram of adsorbent
2000 units are adsorbed and immobilized.

The immobilized α-1,6-glucosidase was extremely stable, with almost no decrease in activity observed for 15 days in both continuous use and repeated batch use.

The enzymatic properties of glass-immobilized α-1,6-glucosidase are almost the same as those of the original enzyme, and the optimal action p
H was found to be 6 to 6.5, and the optimum working temperature was found to be about 50°C (reaction for 30 minutes).

An interesting and specific property of α-1,6-glucosidase immobilized on glass is that its activity is significantly increased in the presence of calcium ions.

For example, the addition of about 0.02 to 0.03 mol of calcium chloride increases the activity by 1.2 to 1.5 times.

The α-1,6-glucosidase activity was measured as follows.

0.1 molar phosphate buffer (pH 7,
0) An appropriate amount of enzyme was added to 0.5 ml, the total volume was made up to 1 volume with distilled water, and the mixture was reacted at 40°C with shaking.

The amount of enzyme that produced 1 to 1 maltotriose under these conditions was defined as 1 unit.

The present invention will be explained in detail from Example ζ below.

Example 1 Bacillus, cereus, varietus, mycoites (Feikoken Bacteria No. 2391) were grown in a mixture of 4% milk casein and 0.0% starch.
5%, K2HP0.0.3%, MgS 04.7 H2
The cells were transplanted into a medium consisting of 0.1% O and 25 x 10-3 mol of CaCl, and cultured at 30°C.

The results of measuring α-1,6-glucosidase activity and β-amylase activity of the culture were 51% per d of culture medium.
．． 5 units and 1200 units.

Volcanic glass fine hollow bodies obtained from whitebait (common name, whitebait balloon, average diameter 100μ) were added to culture 1001rLl.
, manufactured by Sanyo Kansei Center Co., Ltd.) was added and stirred overnight to adsorb and immobilize α-1,6-glucosidase.

The adsorbed material was collected, washed with water, and then dried.

The yield of immobilized enzyme was as shown in Table 1.

The results of reacting the immobilized enzyme in the presence of various concentrations of calcium chloride were as shown below.

As is clear from the table, in the presence of 3 x 10-2 mol of calcium chloride, the immobilized enzyme activity increased about 1.25 times.

Example 2 Enzyme solution containing Bacillus α-1,6-glucosidase 10
TLl (α-1,6-glucylecosidase 1753 units,
2 g of calcined whitebait soil (whitebait balloon) was added to β-amylase (28,966 units), and the mixture was left overnight in a refrigerator.

The adsorbed matter was collected by filtration, washed with water, and then dried.

The yield of immobilized enzyme was as shown in Table 3.

As is clear from the table, α-1,
6-glucosidase was adsorbed and immobilized (655 units/
,F).

Note that this immobilized enzyme bound 76 units of β-amylase per I.

Example 3 Bacillus α-1,6-glucosidase-containing liquid 1077
21 (α-1,6-glucosidase 2223 units, β-
1 of commercially available glass powder (silicate glass powder, 100 to 200 mesh) was added to amylase (19631 units) and left overnight in a refrigerator.

The adsorbed material was collected by centrifugation, thoroughly washed with water, and then the activity was measured.

The results obtained are shown in Table 4.

As is clear from the table, with a yield of about 65%, 1 g of dry matter
It had 698 units of α-1,6-gelicosidase activity per sample.

The yield as activity was approximately 65%.

Example 4 About 10% starch solution (DE 6.6
) 50ml (pH 6.5, CaC1120.01M
) and reacted at 50°C.

After 24 hours, the immobilized enzyme was collected, added to a starch solution with the same composition, and reacted under the same conditions.

In this way, the immobilized enzyme was used repeatedly 10 times.

Starch was degraded as maltose with a degradation rate of 80-90%.

Claims (1)

[Claims] 1 α-1, characterized in that a culture or enzyme solution containing Bacillus α-1,6-glucosidase is adsorbed and immobilized on silicate glass in the presence of calcium ions;
Method for immobilizing 6-glucosidase.