JPS6027513B2 - Method for producing heat-stable α-1,6-glucosidase A - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing heat-stable Q-1,6-glucosidase using Bacillus bacteria.

H-1,6-glucosidase is a general name for an enzyme that hydrolyzes Q-1,6-glucoside bonds present in amylobectin, glycogen, pullulan, etc., and is also called isoamylase, pullulanase, etc.

Q-1,6-glucosigase can be used in combination with 8-amylase to produce maltose from starch in high yields, in combination with glucoamylase to produce glucose from starch in high yields, or in combination with 8-amylase to produce various types of glucose from starch. It can be used as an enzyme to produce oligosaccharides, dextrins, amylose-like substances, etc. in good yields. Q-1,6-glucosidase is derived from subordinate Bacillus, Lactobacillus,
It has been known that various bacteria such as Pseudomonas genus and actinobacteria such as Streptomyces genus produce it, but some microorganisms, such as Bacillus stea. Thermonoillus (Japan Agricultural Chemistry Conference 1971 Abstracts, page 1) and Streptomyces (Ueda et al., J. Fehada. Tec
h side 1. Most Q-1, except for Q-1,6-glucosidase produced by No. 4 Mangai, p. 552 (1971), etc.
, 6-glucosidase's optimal working temperature is 40-50 pm ○
The drawback was that it was located nearby and had poor thermal stability. For this reason, the production of maltose using 8-amylase and the production of glucose using glucoamylase must be carried out within the thermostable range of Q-1,6-glucosidase, so the production of maltose using 8-amylase and glucoamylase must be carried out within the thermostable range of Q-1,6-glucosidase. There were problems such as contamination. The present inventor has demonstrated that Q-1,6-glucosidase produced by Bacillus subtilis, an amylase G3-producing bacterium that produces maltotriose from starch, produces Q-1,6-glucosidase with extremely excellent thermostability. In addition, this Q-1,6-glucosidase acts over a very wide range of pHs, from acidic to alkaline, from 4 to 10, and has a maximum effect at two pH levels: around 5 and around 7 to 7.3. It turned out to be an interesting enzyme.

That is, this enzyme is an enzyme that can efficiently react in an acidic state around pH 5 and in a neutral to slightly alkaline region of pH 7 to 8. The pH stability of this enzyme is also shown in Figure 1d.
As is clear from the above, it is stable over an extremely wide pH range of about 4.5 to about 11. Furthermore, the molecular weight of this enzyme is
While the molecular weight of conventionally known Q-1,6-glucosidase is in the range of 90,000 to 150,000, it is a protein with an extremely high molecular weight of approximately 550,000, which makes it difficult for this enzyme to withstand heat and coexistence. This is thought to contribute to stability.

Thus, the Q-1,6-glucosidase of the present invention is considered to be novel, and this enzyme was named Q-1,6-glucosidase A. The present invention has been made based on these findings. That is, the present invention provides heat resistance Q-
The present invention relates to a method for producing Bacillus Q-1,6-glucosidase, which comprises culturing Bacillus subtilis that produces 1,6-glucosidase.

The enzymatic properties of Q-1,6-glucosidase of the present invention will be described below. '1} Action: Decomposes the Q-1,6-glucoside bond that acts on pullulan and produces maltotriose.

It also breaks down Q-1,6-glucoside bonds in starch, amylobectin, glycogen or derivatives thereof. ■
Working temperature range and optimum working temperature: works up to about 70 qo, and the optimum working temperature is about 6000 (1% pullulan, 0.08
Reaction for 30 minutes under M Tris buffer (Figure 1b)'3
} Working pH range and optimum working pH; pH about 4 to about 10
It acts on a wide pH range of around pH 5 and pH 7 to 7.5.
An action maximum is observed in .

{1% pullulan, 0.08M buffer (acetate buffer pH 3
~3.5 under phosphate buffer pH5.5~10)
React at 0°C.

Figure 1a}'4} Thermal stability: After heating at each temperature for 10 minutes in 0.08M Tris buffer (pH 7.0), residual activity was measured.

As a result, heating at 50° C. for 10 minutes caused almost no inactivation, but heating at 60 qo for 1 minute resulted in approximately 70% inactivation. (Figure 1 c white circle) Leg - Stability; Stable at pH about 4 to about 10 {
After standing at room temperature (25°C) in a 0.1M buffer, the remaining activity was measured (Fig. 1d) ■ Inhibitor;
-3M of HgCso2, AgN03, CuS04, etc. inhibited about 98%, about 98%, and about 87%, respectively.

'7} Stabilization: The presence of calcium ions increases the thermostability of the enzyme (Fig. 1c, black circle).

'8} Purification method: This enzyme was purified by ammonium sulfate fractionation from the supernatant of a liquid culture, DEAE-Sepharose column chromatography (gradient elution with KCIO to 0.8M), and then Sephadex G-200 column chromatography. It can be purified to near homogeneity chromatographically and haemophoretically.

{91 molecular weight; The molecular weight measured by Cephadex G-1200 gel filtration method was about 550,000.

00 Titer measurement method: Add an appropriate amount of enzyme to 0.5 skin of 1% pullulan solution (pH 7.0) dissolved in 0.1 M phosphate buffer, make the total volume 1 with water, and react at 40 oo.

Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 mg of maltotriose in 1 hour was defined as 1 unit. The mycological properties of Bacillus spYT-1 used in the present invention are as described below.
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as 004 (Choken Bacterial Deposit No. 5854).

'1) Form: Short-shaped fungus, size, medium 0.5-0.7 Buddha x
1.7 to 2.4 mu, monotrozoan, nonmotile, gram-negative or gram-variable'2) Spores; spores with hair cells bulging but very large.

Forms spherical to oval spores. '3} Gelatin; liquefies slowly. '4 - Juicy agar; Good growth, smooth surface, yellowish.

'51 glucose gravy agar; growth is poorer than gravy agar,
Light horizontal color {6} Glucose nitrate agar: Only grown '7)
Meat juice: Slightly cloudy and sedimented.

■ Salted meat juice: The presence of salt promotes growth.

It grows even under 10% salt.

{9} Citric acid agar; growth only '10 Tyrosine agar; good growth, yellow, tyrosinase negative (11) Glucose-asparagine agar; growth little (12) Indole; no production (13) Milk; slow coagulation, veptonization (1 Heart Potato; little growth (15) Acetylmethylcarpinol; produced (IQ Hydrogen sulfide: produced (17) Reduction of nitrate; negative (18) Urease; not produced (19 Catalase; produced (20) Hydrolysis of starch ;Positive/Positive (21) Utilization of carbohydrates; ○-Glucose, D-fructose, D-massonose, 0-galactose, D-xylose, D-arabinose, sucrose, lactose, maltose, starch, glycogen, etc., produce raw acids, but no gas is produced. , (22) Growth temperature; the optimum growth temperature is 38-40, the maximum growth temperature is about 50-0 or more, and Bergey, sNannualofD
ete dish iMtiveBacteriology 7th
edition and 8th edition (meWilliams & Wilk
InsCompany 957 and 1974), the bacterium was identified as Bacillus subtilis.
It seemed appropriate to consider it as a type of variety of C. cillus costilis).

For the cultivation of this bacterium to produce Q-1,6-glucosidase, commonly used organic nitrogen sources such as beptone, meat extract, yeast extract, casein, corn staple liquor, and soybean waste are used as nitrogen sources. Also, starch, dextrin, maltose, glucose, etc. are used as the carbon source.

A medium containing an inorganic nitrogen source, phosphate, magnesium salt, and various metal salts is used as a supplementary nutrient source. Cultivation is carried out aerobically at a pH of about 5 to about 9 and a temperature of 25 to 590°C. Since Q-1,6-glucosidase is an enzyme produced outside the bacterial cells, after culturing, bacteria are removed by filtration or far-D separation, and the supernatant solution is collected. Then, if necessary, concentrate and salt out with ammonium sulfate, sodium sulfate, etc.
Add an organic solvent such as acetone, isopropanol, ethanol, or methanol to collect the enzyme as a precipitate, dry it,
save. Reactions using this enzyme can be carried out either alone or in combination with various amylases such as glucoamylase, and at pH 4 to 9.
, at a temperature of 4 pi to 70 qo.

The details of the present invention will be explained below with reference to Examples.

Example 1 K2P040.3%, M
gS04・7days 20% liquid medium consisting of 200.1%, fish meat extract 3%, soluble starch 1%, and manganese sulfate 5×10-5M was added, and after sterilization by a conventional method, late cillus
spyT-1004 (FERM P-5854) was inoculated and cultured for 2 days at 3 pm.

As a result of measuring Q-1,6-glucosidase produced in the supernatant solution obtained by sterilization with a centrifuge after culturing,
It was 11.8 units per medium.

[Brief explanation of the drawing]

Figure 1 a, b, c and d are the optimal action p of Q-1,6-glucosidase produced by Bacillus subtilis, respectively.
H, optimum temperature, thermal stability and pH stability. Figure 1

Claims (1)

[Claims] 1. A method for producing Bacillus α-1,6-glucosidase A, which comprises culturing Bacillus subtilis that produces thermostable α-1,6-glucosidase.