JPH0568233B2 - - Google Patents

Description

[Detailed Description of the Invention] Industrial Application Field This invention relates to polysaccharide pullulan and starch α-
The novel enzyme neopululanase has the ability to cleave 1.4 and α-1.6 glucosidic bonds.

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION Pullulan, which is one of the substrates on which this enzyme acts, consists entirely of maltotriose, except for the rare presence of maltotetraose.
It is a polysaccharide in which such structural units are linked through α-1.6 glucosidic bonds. In addition, starch is made of amylose, which is glucose bound with α-1.4 glucoid bonds,
A part of it is composed of amylopectin containing branches due to α-1,6-glucoside bonds.

Enzymes that act on pullulan include glucoamylase, which acts on glucose units from the end of pullulan, pullulanase, which cleaves only the α-1.6 glucoside bond, isopullulanase, which cleaves the α-1.4 glucoside bond in pullulan to give isopanose, and panose ( Thermoactinomyces vulgaricus amylase (TVA), which produces 6 ² -α-glucosyl maltose, is known.

In contrast to these conventionally known enzymes, the enzyme of the present invention acts on starches other than pullulan, provides panose, glucose, maltose, etc., and has excellent heat resistance, so it is widely used in various food processing industries including the starch saccharification industry. It can be used, and new applications can be expected in the future.

Means and Effects for Solving the Problems The present invention is directed to a group of bacteria selected from a group of bacteria that grows at 60°C from samples such as compost and has the ability to decompose pullulan. This is done by culturing bacteria belonging to the virus in a medium containing soluble starch, etc., and collecting neopululanase from the culture solution. Among stearothermophiles, especially Bacillus stearothermophilus
Strain TRS40 (Feikoken Bacteria Deposit No. 9609) has a high ability to produce neopullulanase and is preferably used. This strain was isolated by the inventors from compost in Tsurumi Ward, Osaka City, and has the following mycological properties.

(1) Morphological properties (1) Cell shape and size...(0.6×1.0)×(2
~3.5) μ bacilli. There is no linkage and there are few capsules.
The sweetened meat juice agar medium shows the same rod shape as the meat juice agar medium.

(2) Motility...Yes.

(3) Spores: (0.4-0.5) x (0.5 x 0.6) μ oval shape, present near the end, and the membrane wall is thin. 60
Many forms in 16 hours at °C. Swelling of the sporangium is observed.

(4) Gram staining...Positive.

2. Growth status (1) Juice agar plate culture...good growth. The surface is slightly dry and almost white.

(2) Meat juice agar slant culture...The result is spread-like and the surface is not glossy.

(3) Meat juice liquid culture...Good growth at pH 7.0. The liquid becomes cloudy.

(4) Gelatin puncture culture: liquefy gelatin.

(5) Salt broth liquid culture...Does not grow in a saline solution with a concentration of 7%.

(6) Glucose/asparagine agar medium...Poor growth.

(7) Glucose nitrate agar medium... Slight growth.

(iii) Physiological properties (1) Reduction of nitrate...Positive.

(2) Starch hydrolysis...Iodine coloration is negative when grown on a shear plate containing starch.

(3) Fermentability of citric acid...Positive.

(4) Catalase production...negative.

(5) Range of growth...Trypsin, yeast extract,
Grows up to 65℃ in medium containing salt. The optimal growth temperature is 60℃.

(6) Sugar fermentability...glucose, lactose,
Forms acid from sucrose. However, it does not generate gas.

(7) Formation of acetylmethylcarbinol...Positive.

(8) Decomposition of gelatin...Positive.

The above results are summarized in Bergier's Manual of
Bergey's Manual of Systematic Determination Volume 2
Bac-teriodetermination vol. 2. (1986)), the bacteria was identified as Bacillus stearothermophilus.

Next, the enzyme produced by this strain will be explained in detail.

1. Action and substrate specificity This enzyme acts on pullulan and ultimately produces panose, glucose, and maltose. In terms of quantity, panose is the most abundant.

When acting on starch, mainly maltose and glucose are produced, but in the late stage of the reaction, panose and isomaltosyl maltose (IMM) are produced as branched dextrins.
generate. It is considered to be a type of α-amylase because the iodine color decreases more quickly than the production of reducing sugars. In general, α-amylase is broadly classified into a saccharifying type that hydrolyzes starch relatively well, and a liquefying type that has a high starch liquefaction rate but has a low degree of saccharification. When the former is applied to starch, the minimum low molecular weight limit dextrin obtained is
IMM. The enzyme of the present invention produces glucose and maltose from the beginning when it acts on starch, and belongs to the saccharification type group, but the smallest limit dextrin is panose, not IMM. In this respect, it is an extremely unique α-amylase. Furthermore, when this enzyme is allowed to act on pullulan and starch at the same concentration and under the same conditions, it acts on pullulan as well as on starch or more effectively. TVA has a significantly greater effect on starch than on pullulan. in this sense
TVA is considered to be a type of α-amylase, whereas this enzyme is a type of pullulan-degrading enzyme, which is why it was named neopululanase.

When this enzyme is applied to maltosyl-β-cyclodextrin, in which maltose is bound to β-cyclodextrin via an α-1.6 glucosyl bond, the α-1.6 bond is first cleaved, producing maltose and β-cyclodextrin. Then β-cyclodextrin also undergoes re-degradation,
Provides glucose, maltol and maltotriose.

In other words, this enzyme can be considered to be an enzyme with a new action specificity that mainly recognizes maltose and cleaves maltol whether it is bound through an α-1.4 bond or an α-1.6 bond. can.

2. Effect PH PH5.0-7.5. Measurement was carried out using 0.05M sodium acetate.
Using an acetate buffer (PH4-6) or a disodium phosphate-monopotassium phosphate buffer (PH6-8), react in 0.5% pullulan at 60°C for 15 minutes. The reducing sugar produced was measured using the DNS method.

(3) Optimal pH for action Approximately PH6.0 under the above two conditions.

4. Temperature of action: 75°C when reacted for 15 minutes at various temperatures in 0.1M phosphate buffer (PH6.0) and 0.5% pullulan.
I reacted to.

(5) Optimum temperature for action The temperature was 60-65°C under the above conditions.

6. Thermal stability 60 at various temperatures in 0.2M acetate buffer (PH6.0)
When the residual activity was measured after heating for a minute, 60% of the activity remained at 65°C. Furthermore, when 1mM EDTA was added to this system, more than 90% of the activity remained.

7. PH stability Stable between PH5.5 and 9.0 (using various buffers)
After adjusting the pH and leaving it at 60°C for 60 minutes, the pH was adjusted to 6.0 and the residual activity was measured).

8. Purification method After ammonium sulfate fractionation of the culture supernatant (35-60% saturation), the precipitate was dissolved in a small amount of water, dialyzed, and the PH
8.0 and adsorbed onto DEAE-cellulose. The enzyme was then eluted by gradually increasing the salt concentration to 0.6M. Furthermore, after repeating column chromatography using Sephadex G-200 twice, α-
Sepharose combined with cyclodextrin
Affinity chromatography was performed on a 6B (Sepharose 6B) column.

The protein absorption of the eluate showed one peak, which completely coincided with the enzyme activity peak. Electrophoresis of this active fraction by SDS-PAGE showed a single band.

(9) Molecular weight: 62,000 daltons (SDS-PAGE method) (10) Products: In addition to panose, glucose and maltose were given from pullulan. We also fed glucose, maltol, panose, and IMM from starch.

11. Titer measurement method: Add 0.25 ml of enzyme solution to 0.25 ml of 1% pullulan (dissolved in 0.2 M acetate buffer (PH6.0)) and mix at 60°C for 15
The reaction was allowed to proceed for minutes, and the resulting increase in reducing power was quantified by the DNS method. 1 μmol per minute in the above reaction
The enzyme power to produce reducing sugar was defined as one unit.

From the above physicochemical properties, it can be said that the enzyme of the present invention is a novel pullulanase that has never been reported before.

In the present invention, such Bacillus stearothermophilus bacteria or Bacillus stearothermophilus
The TRS-40 strain is cultured and neopullulanase is collected from it.

As a culture method, for example, 2.5% starch, 1% starch
Tryptone, 0.5% yeast extract, and 0.5% salt may be used as the basic medium. It is not necessary to add pullulan, but in addition to starch, glucose, maltose, dextrin, sucrose, etc. can be used as a carbon source, and polypeptone, casamino acids, corn staple liquor, yeast extract, protein hydrolyzate, etc. can be used as a nitrogen source. Use about 0.5 to 2%.

It seems best to maintain the culture temperature at about 40 to 60°C, especially around 60°C.

Although the culture time is not specified depending on the amount of inoculated bacteria and other conditions, it is usually about 8 to 30 hours, and usually about 16 hours.

To collect neopululanase from the culture cultured in this way, since the enzyme secretes and accumulates outside the bacterial cells, first remove insoluble substances such as bacterial cells by centrifugation, filtration, etc., and make a transparent culture medium. get. Then, it is concentrated and purified by conventional purification means such as fractional precipitation with ammonium sulfate, alcohol precipitation, and membrane concentration.

Examples Example 1 A medium containing 2.5% starch, 1% tryptone, 0.5% yeast extract, and 0.5% salt was adjusted to pH 7.0, and after sterilization, Bacillus stearothermophilus strain TRS40 was inoculated and incubated at 60°C and 16°C. Cultured with shaking for hours. After culturing,
The supernatant from which bacterial cells were removed by centrifugation contained 0.2 u/ml of neopululanase. It was salted out with ammonium sulfate, and the desired enzyme was precipitated in the 0.35 to 0.6 saturated precipitate range. The precipitate was separated and the powder obtained by drying contained a small amount of α-glucosidase in addition to neopululanase.

50 g of dry enzyme powder was obtained from culture solution 10. This enzyme showed an activity of 32u/g. The recovery rate of the enzyme was 80%.

Example 2 The enzyme powder obtained in Example 1 was dissolved in a small amount of water and after dialysis, DEAE cellulose and Cephadex G-
Purified enzyme was obtained by affinity chromatography using 200 and α-cyclodextrin as a ligand. The enzyme recovery rate calculated from this enzyme activity was 4%, and a single band was obtained by SDS-PAGE.

Effects of the present invention The enzyme of the present invention acts on pullulan, panose,
It is a new enzyme that produces maltose and glucose. In addition, this enzyme acts on starch to produce glucose, maltose, and panose. Panose is said to be a carbohydrate that not only prevents glucan synthesis by caries-forming bacteria, but also activates Bacteria bifidus in the human intestine, and is expected to be widely used in a society with a long life expectancy. This enzyme also makes it possible to produce starch syrup containing panose.

Claims (1)

[Claims] 1 Bacillus stearothermophilus is a bacterium that produces an enzyme (hereinafter referred to as neopululanase) that has the ability to produce panose, maltose, etc. from pullulan.
A method for producing a new heat-resistant pullulanase, which is characterized by culturing TRS40 (Feikoken Bacteria Deposit No. 9609) and collecting neopululanase from it.