JPS63185380A - Novel alpha-1,6-glucosidase and production thereof - Google Patents

Abstract

NEW MATERIAL:An alpha-1,6-glucosidase having the following enzymatic and chemical properties. Enzymatic action, breaks alpha-1,6-glucoside bond and forms straight- chain amylose; substrate specificity, relative activities to polysaccharides as shown in the table; optimum pH, 5.0-5.5; pH stability, stable up to pH 4.5-6.5 by the treatment at 40 deg.C for 30min and up to 5.0-6.0 at 50 deg.C for 30min; optimum temperature, 55 deg.C; temperature stability, maintains 80% of the activity by the treatment at pH 4.5 and 40 deg.C for 30min and loses 90% of the activity by the treatment at 60 deg.C for 30min; effect of metal salt, hardly influenced with Ni<2+>, Ba<2+>, Zn<2+>, Cd<2+> and Mn<2+>, inhibited with Fe<3+> and inactivated with Hg<2+> and Ag<2+>; molecular weight, 95,000 (by SDS electrophoresis); etc. USE:Enzyme for the production of straight-chain amylose. PREPARATION:The enzyme can be produced by culturing Bacillus sectorramus (FERM P-8973).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel α-1,6 glucosidase and a method for producing the same. More specifically, α-1,6 glucosidase-producing bacteria succumbing to the new bacterial species Bacillus sectorramus was cultured, and α-1,6 glucosidase was obtained from the culture and reached at an optimum pH of around 5.0 to 5.5. ! ? The present invention relates to a novel α-1,6 glucosidase whose temperature is around 55° C. and a novel method for producing α-1,6 glucosidase characterized by collecting α-1,6 glucosidase from a culture of the strain.

α-1,6 glucosidase is an enzyme that cleaves α-1,6 glucosidic bonds in starch, etc. and produces linear amylose, and is widely used in the saccharification industry to produce maltose, glucose, high-fructose sugar, etc. ing.

[Prior art]

α-1,6 glucosidase has long been found in higher plants or microbial yeasts, but in recent years its production has been reported in various bacteria. For example, Aerobacter (Δerobacter ae)
rogenes), Escherichia intermedia (Escherichia inLermedia)
, Pseudomonas amyloderamosa
onasamy]oderamosa), Streptococcus mites
1tes), Cytophaga (Cytophaga)
) genus, Streptomyces (Strepto-Hyce)
s) 5 and Flavochromogenes (Flavochr)
o-mogenes), etc.

On the other hand, α-
As a 1,6 glucosidase, Bacillus cereus (B
acillus cereus) IPo 30 inches and Bacillus ferm
us) IFO3330 and Bacillus acidopluriticus (Bacillus acidopu-11ul)
yticus) etc. are known.

[Problems to be solved]

Most of the previously known α-1,6 glucosidases have a neutral or slightly alkaline pH, and it is disadvantageous to use these α-1,6 glucosidases in the saccharification industry, which performs saccharification on the acidic side. there were.

On the other hand, Pseudomonas amyloderamosa (Pseud
omonas amyloderamosa) and Bacillus ac.
idopullulyticus), but the former has no temperature resistance and is not suitable for practical use, and the latter has a long culture time of 60 to 73 hours, which is a long time for grouper terriers, and is economically problematic.

[Failure method to solve the problem]

Therefore, the present inventors have developed a method that has temperature resistance and reaches 4p on the acidic side.
++ and a higher productivity of α-1,6 glucosidase.

Bacillus newly isolated from the soil
The present invention was completed by discovering that a new bacterial species belonging to the genus S. lus produces a large amount of α-1,6 glucosidase in the culture after a very short cultivation period of about 10 to 15 hours, and by collecting this, the present invention was completed. .

The α-1,6 glucosidase producing bacterium used in the present invention belongs to a new bacterial species, and the present inventors have identified it as Bacillus sectorramus.
) was named. Its microorganism storage accession number at the Institute of Microbial Technology, Agency of Industrial Science and Technology is FERM-Pli, 8973, and is hereinafter referred to as this strain.

Next, the mycological properties of this strain will be described.

A. Morphology (1) Cell shape Bacillus (2) Cell size 1.0-1.3 X 1.5-
5.2μ (3) Presence of motility Absent (4) Presence of spores Presence (5) Durham staining Positive (6) Acid-fast staining Negative B, Growth status in each medium (1) Standard agar plate culture Colonies are round, peripheral It has a gold edge and a smooth, semi-transparent grayish white surface.

(2) Standard agar slant culture Straight, smooth surface, buttery, shiny, medium growth.

(3) Standard liquid culture It is slightly cloudy and has a powdery precipitate. No pigment generation.

(4) Standard gelatin puncture culture Straight, not liquefied.

(5) Ridmus milk No change.

C0 physiological properties (1) Nitrate reducing property positive (2)
Denitrification reaction Positive (3) Methyl red test Positive (4) Production of acetylmethylcarbinol Negative (5) Production of indole
Negative (6) Generation of hydrogen sulfide
Negative (7) Hydrolysis of fine powder
Positive (8) Use of citric acid Positive (9) Use of inorganic nitrogen sources (bitrates and ammonium salts) Positive (10) Formation of pigment
Negative (11) Urease activity
Negative (12) Oxidase activity
Negative (13) Kafrase activity
Positive (14) Growth range pH 4.0-7.1 Temperature 45.5°C-15.0°C (15) Attitude towards oxygen Facultative anaerobic (16) Production of dioxyacetone Negative (17) Decomposition of hippuric acid
Negative (18) Decomposition of arginine
Negative (19) Phenylalanine removal Negative (20) Temperature resistance (tolerance for 110 minutes at 85°C) Positive (21) Sodium chloride resistance 7%
Negative 3.5% Positive (22) Growth on Sabouraud agar medium Positive (2
3) Growth on 0.001% lysozyme medium Positive (
24) Decomposition of 1,000 rosin Positive (25
) Alkali production in citric acid/ammonium agar
Positive (26) Degradability of casein
Negative (27) Degradability of gelatin
Growth in negative (28) anaerobic medium
Positive (29) Pine Conkey medium growth
Negative (30) Lecithinase reaction Negative (31) Alkali production ability in VP medium Negative (32) Sugar utilization and biointoxicity (al l-arabinose Positive (b)
l D-xylose positive Ic)
D-glucose positive (di D-
Mannose positive (141D-fructose positive (fl D-galactose positive (g) maltose
positive lhl sucrose
Positive + 11 lactose
Negative (j) Trehalose positive (kl D-Sorvit negative T
lID-Mannitol Negative ([+1
1 inosite negative (nl glycerin positive (0) starch
positive (p) melibiose
Positive (q+ salicin
negative tr) ethanol
If the mycological properties are positive or higher, please refer to Verge's Manual of Determinative Bacteriology (B).
ergey's Manual of Determi
native Bacteriology) 8th edition and the International Journal of Systemic Bacteriology (Internationala
l Journal of Systematic B
The strain was identified as described in P. acterio-1ogy) 1974-1986.

In other words, it is clear that this strain belongs to the genus Bacillus because of its positive Durham staining, ability to attach spores, and aerobic growth.

Next, although this strain was not similar to any known bacterial species within the genus Bacillus, the vegetative cell size was relatively large at 1.0 to 1.3μ. to B (U species include Bacillus cereus (B
acillus cereus) and Bacillus megaterium
can be given.

However, these strains can be distinguished from the strains used in the present invention in the following points.

That is, Bacillus cereus
eus) grows in 7% salt and is positive for egg yolk reaction, casein decomposition reaction, and gelatin decomposition reaction, but this strain cannot grow in 7% salt and is positive for egg yolk reaction, casein decomposition reaction, and gelatin decomposition reaction. All are negative.

Also, Bacillus megaterium (Bacillus me
ga-terium) grows even in 7% salt and is positive for both casein and gelatin decomposition reactions, whereas this strain can survive in 7% salt and has positive casein and gelatin decomposition reactions. All are negative.

Furthermore, JP-A Shogaku-4, which is also a new bacterial species of the genus Bacillus.
Bacillus aci 1-pluriticus (B
acillus acidopullyticus
) and this strain, they can be distinguished as shown in Table 1.

Table 1 (blank below) Based on the above, it was recognized that this bacterial strain is a new bacterial species, unlike known bacterial species of the genus Bacillus, and was named as described above.

Next, conditions for culturing this strain to produce and accumulate α-1,6 glucosidase will be described.

First, in addition to inorganic salts suitable as medium components, carbon sources include starches such as soluble starch, waxy starch, and potato starch, starch hydrolysates such as maltose and dextrin, and nitrogen sources include polypeptone and meat. Appropriate combinations of extracts, yeast extracts, cornstarch liquor, ammonium phosphate, and protein hydrolysates such as casein and soybean protein produce high titer pullulanase activity.

The culture conditions are as follows: pH of the medium as described above is 4.0.
This bacteria was inoculated into the temperature adjusted to ~7.0, and the temperature was adjusted to 20-45℃.
Culture at a temperature of 1 to 2 days (best for 10 to 20 hours) under conditions such as standing still, shaking, or aeration.

After the culture was completed, the bacterial cells were removed from the culture solution and the resulting supernatant was concentrated to obtain a high-unit pullulanase standard. Furthermore, this enzyme preparation was subjected to affinity chromatography using T-zylodextrin-Sepharose 6B (manufactured by Pharmacia) after being fractionated with ammonium sulfate.
It was purified to a single band by 3-polyacrylamide gel electrophoresis.

The enzymatic chemical properties of the purified α-1,6 glucosidase are detailed below.

1. Action: This enzyme acts on α-1,6 glucosidic bonds to produce linear amylose.

2 Km value and Vmax for the substrate Km value and Vmax for pullulan, potato-derived amylopectin, and corn-derived amylopectin of the dual enzyme are as shown in Table 2. The unit of Km value is (■/mf!), and the unit of Vmax is (p mol / min /
nv protein).

(Left below) Table 2 Relative activity towards the 3 polysaccharides: Corn soluble amylopectin, soluble starch, and pullulan activity of this enzyme
The relative activities of oyster glycogen, rabbit glycogen, corn amylopectin, and potato amylopectin are shown in Table 3.

(Margins below) Table 3 4 Optimum pH: Using a 25mM citric acid-50mM disodium phosphate buffer, the optimum pH is around pH 5.0 to 5.5 (as shown in Figure 1).

5 pi stability: 25mM citric acid-50mM disodium phosphate slow i! It is stable up to pH 4.s ~ 6.5 when treated at 40°C for 30 minutes using I solution, and at 50°C,
It is stable up to pH 5.0 to 6.0 in a 30 minute treatment (as shown in Figure 2).

6 Optimum temperature: 50mM acetic acid mild III solution pl+4.5
The optimal temperature is found around 55°C (as shown in Figure 3).

7 Temperature stability: 80% stable when treated at 40°C for 30 minutes using 50mM acetate buffer pH 4.5,
90% inactivation occurs upon treatment at 60°C for 30 minutes (as shown in Figure 4).

8 Activity measurement method: 0.5% pullulan solution (pH 4, 5)
Add enzyme solution 1- to 4- and react for 130 minutes at 40'C, then add Somogyi solution 2-, heat for 20 minutes, and cool. After cooling, add ammonium arsenic molybdate solution 1-,
Then water is added to make 25-.

The absorbance of this liquid at 500 nm is measured with a layer length of 1 cm. One unit was defined as the production of reducing sugar equivalent to 1 μmol of glucose per minute under these conditions.

9 Effect of inhibitors: p-Mercuribenzoic acid (hereinafter referred to as P-C
MB) and sodium dodecyl sulfate (SD
It is inhibited by more than 70% by orthophenanthroline and potassium ferricyanide. The effects of various inhibitors are as follows at a final concentration of 1mM.
Table 4 shows the residual activity after treatment with 0mM acetate buffer pH 4 and 5 at 40°C for 130 minutes.

Table-4 Effects of 9 metal salts: Ni'', Ba'', Zn2+, Cd2
+ and Mn2+, inhibited by Fe3+, and inactivated by 11g2+ and Ag2+. The effects of various metal salts are shown in Table 5 in terms of residual activity after treatment at 50 ml of acetate buffer TlH4,5, 40°C for 30 minutes at a final concentration of the metal salt of 5mM.

Table 5 10 Molecular weight: Approximately 95,500 (by SDS electrophoresis method) About the enzyme obtained by the present invention, JP-A-57
- Branch cutting enzyme described in No. 174089 (hereinafter referred to as A)
When compared with the properties of , they can be clearly distinguished as follows.

1 Regarding the production strain, the present invention is Bacillus ceftramus, which is a new strain of the genus Bacillus;
It is acid bullistics.

2 Regarding the optimum temperature, the present invention is 55°C, but the former is 60°C.

3 Regarding the enzyme activity at each pu and temperature, as shown in FIG. 5, the enzyme activity of this enzyme has a very large difference with respect to the temperature difference. Furthermore, regarding the pattern of enzyme activity with respect to pu, the activity peaks at pH 5.0, but the activity decreases rapidly whether the p1 is high or low. These patterns on the instep are all different.

4 Regarding the optimum p11, the present invention
．． It has a narrow pH of 5, and has very high specificity compared to the pH of the enzyme in the former, which has a pH of 3.5 to 5.5.

It can be said that the α-1,6 glucosidase obtained by the present invention is a new enzyme different from that of A.

The novel α-1,6 glucosidase obtained by the present invention can be purified from starch using, for example, glucoamylase.
- It can be added to increase the yield when producing sugar, or it can be used in combination with β-amylase to produce high-yield maltose from starch.

The present invention will be specifically explained below with reference to Examples.

Example 1 Soluble starch 1.5% Meat extract 0.5% (NlI4)2 llPO40, 3%K 2HPO4
0.1% MgSO4・71420 0.1% Initial pH 5.5 Add Bacillus sectoramas to the medium with the above composition.
lus sectorramus) FERM-Phh
8973 and cultured with shaking in a Sakalo flask at 37°C for 20 hours, a pullulanase activity of 8.1 u/+nE could be obtained from the culture solution. This culture solution is centrifuged to remove bacterial cells, the resulting supernatant is concentrated using an ultrafiltration membrane, cold alcohol is added to a concentration of 80% to precipitate the enzyme, which is centrifuged, and the obtained Pullulanase powder was obtained by freeze-drying the precipitate.

Example 2 Maltose 2% Yeast extract 0.5% 2HPO40.2% MgSO4.71120 0.1% initial pl+
4.5 307! Fill a jar fermenter with all the medium of the above composition, and add Bacillus ceftramus (Bacillus ceftramus).
Ctorramus) FERM-PNa 8973 was inoculated and cultured with aeration and stirring at 37°C for 16 hours. A pullulanase activity of 13.6 u/me was obtained from the culture solution. This culture solution was centrifuged to remove bacterial cells, and the resulting supernatant was concentrated approximately 20 times using an ultrafiltration membrane to 200 u/me (pH
A crude enzyme liquid preparation of burulanase (4, 5) was obtained. After fractionating this with ammonium sulfate, affinity chromatography was performed using T-cyclodextrin-Sepharose 6B (manufactured by Pharmacia) to obtain a purified enzyme preparation of 16.1 u/mg protein.

Example 3 Waxy starch 2% Polypeptone 1% and 2! -IPO40.2% MgSO4・7H200.1% Initial pH' 5.0 A medium with the above composition was placed in a 1000β capacity tank, and Bacillus sectorr.
amus) FERM-pHlo, 8973,
Culture was carried out at 37° C. with aeration for 15 hours. 18. from the culture solution.
A pullulanase activity of 2 u/- was obtained. This culture solution was subjected to the same operation as shown in Example 2 to obtain a liquid pullulanase crude enzyme preparation of about 26β with a concentration of about 300 u/mf.

Experimental Example 1 Substrate for saccharification: Cornstarch (33g/d1・[)E
8.0) Saccharifying enzyme: Gluczyme NL-3 (manufactured by Ohno Pharmaceutical)
(2,5u/g D, S,) This enzyme: Example 3
Liquid pullulanase (0,1 u/g D, S
,) Saccharification temperature: 62°C Saccharification was carried out under the above conditions, single m < al), 2
The contents of saccharides (G2), trisaccharides (G3) and polysaccharides (Gn) were measured by HPLC. As a control, measurements were also carried out under saccharification conditions without the addition of this enzyme, and the results are shown in Table 6.

(Margins below) A Table-6 [Effects of the invention] The present invention is directed to the use of a new bacterial species, Bacillus ceftramus.
By short-term culturing of A. us sectorramus), a novel α-1,6 glucosidase can be produced, and a large amount of thermostable α-1,6 glucosidase can be economically produced, contributing to the starch saccharification industry. It is something.

[Brief explanation of the drawing]

Figures 1 to 5 show the enzymatic chemical properties of α-186 glucosidase of the present invention, of which Figure 1 shows the main pl+ curve, and Figure 2 shows the 10- curve at 40°C. p
1 (stability curve), --- shows the pH stability curve at 50°C, Figure 3 shows the temperature stability curve, Figure 4 shows the optimum temperature curve, and in Figure 5, 10- shows the pH stability curve at 55°C. -Rho shows the enzyme activity curve at each pH at 60°C, and -.- shows the enzyme activity curve at each pH at 65°C.

Claims (1)

[Claims] 1. A novel α-1,6 glucosidase (1) having the following enzymatic chemical properties: cleaves α-1,6 glucosidic bonds to produce linear amylose. (2) Substrate specificity: The relative activity toward polysaccharides is as follows. (3) Km value and Vmax for pullulan: Km value is 0.14 mg/ml and Vmax is 70.0 μmol/m
in/mg protein. (4) Optimum pH: around 5.0 to 5.5. (5) pH stability: pH 4 after treatment at 40°C for 30 minutes
．． It is stable up to a pH of 5 to 6.5, and stable up to a pH of 5.0 to 6.0 when treated at 50°C for 30 minutes. (6) Optimum temperature: around 55°C. (7) Temperature stability: 80% stable when treated at pH 4.5 at 40°C for 30 minutes, and 90% inactivated when treated at 60°C for 30 minutes. (8) Effects of inhibitors: inhibited by 70% or more with p-mercuribenzoic acid and sodium dodecyl sulfate, and not inhibited with orthophenanthroline and potassium ferricyanide. (9) Effects of metal salts: Ni^2^+, Ba^2^+, Z
It is hardly affected by n^2^+, Cd^2^+ and Mn^2^+, inhibited by Fe^3^+, and Hg^2^+
and is inactivated by Ag^2^+. (10) Molecular weight: Approximately 95,500 (according to SDS electrophoresis) 2 α-1,6 belonging to Bacillus sectoramas
Glucosidase-producing bacteria are cultured, and α-1,6 is added to the culture.
1. A method for producing α-1,6 glucosidase, which comprises producing glucosidase and collecting it.