JPS61170387A - Novel maltase and production thereof - Google Patents

Abstract

PURPOSE:To produce the titled enzyme useful as a reagent for the determination of amylase in blood, etc., economically on an industrial scale, by culturing a microbial strain belonging to Bacillus genus and capable of producing a novel maltase having a specific molecular weight and specific properties, and collecting the enzyme accumulated in the microbial cell. CONSTITUTION:A microbial strain belonging to Bacillus genus and capable of producing novel maltase is cultured, and the novel maltase accumulated in the microbial cell is separated therefrom. The maltase has the following properties. Optimum temperature, about 24 deg.C when 0.1ml of the enzyme is made to contact with 0.05ml of 1% maltose for 30min; activity, reactive to maltose, maltotriose, maltotetraose, p-nitrophenyl-alpha(beta)-maltoside, and isopanose, and inert to soluble starch, amylopectin, amylose and pullullan; optimum condition, 30 deg.C and 7.0pH; stale at 6.0-7.0pH; deactivated by leaving at >=8.0pH and 4 deg.C for 24hr, or heating at 35 deg.C for 30min; molecular weight, 57,000.

Description

[Detailed description of the invention] The present invention relates to a novel maltase and a method for producing the same.

In recent years, malto-oligosaccharides, which lie between monosaccharides and polysaccharides and have been delayed in development and utilization, have attracted attention, and their uses include blood amylase measurement substrates in clinical tests, chemical synthesis raw materials 1 food materials, and biochemical uses. Examples include reagents. Therefore, maltooligosaccharides are produced using various maltooligosaccharide producing enzymes. One of these, maltopentaose, is produced by hydrolysis of starch or amylose using various amylases, but the decomposition products contain a considerable amount of maltooligosaccharides other than maltopentaose, making it extremely difficult to purify maltopentaose. It is. Maltopentaose has recently been used as a substrate for measuring serum amylase, and its range of use has expanded, and mass production is expected, but as mentioned above, the production cost is relatively high. Therefore, as a method for efficiently producing maltopentaose, malto-oligo I other than maltopentaose, which is a by-product of starch or amylose hydrolysis, has been proposed. One possible method is to perform column chromatography after decomposing maltotetraose, maltotriose, and maltose (for example, maltotetraose, maltotriose, and maltose) into glucose. Maltase may be used in combination. However, all maltases derived from microorganisms that have been purified and whose properties have been clarified have an optimal temperature of 35.
℃ or higher.

Therefore, if the optimal temperature and stable temperature of the maltooligosaccharide producing enzyme is below 35°C, conventional maltase cannot fully meet the requirements (for example, in JP-A No. 5
No. 8-142330).

Therefore, as a result of repeated research to search for maltase that has sufficient activity even below 30°C, the present inventors discovered Bacillus cereus (Bacillus cereus), which the present inventors isolated from soil.
By aerobically cultivating N Y -14, it can be grown even at temperatures below 30°C.

They discovered that maltase, which is active in microbial cells, is produced and accumulated within microbial cells, and based on this knowledge, they completed the present invention.

The present invention first relates to a novel maltase having the following properties.

(1) 0.05 ml of 1% maltose and this enzyme [50
5mM EDTA in mM phosphate buffer (pH 6,86)
The optimum temperature is around 24°C when 0.1ml (containing) is applied for 30 minutes.

(2) This enzyme is maltose and maltotriose.

Acts on maltotetraose, paranitrophenyl-α-maltoside, paranitrophenyl-β-maltoside, isopanose, and maltohexaose.

It has no effect on maltoheptaose, soluble starch, amylopectin, amylose, and pullulan.

(3) The optimal pH of this enzyme is around 7.0 at 30°C, and it is stable around pH 6.0 to 7.0.

(4) This enzyme is inactivated when left at pH 8.0 or higher and 4°C for 24 hours. Moreover, it is deactivated by heating for 30 minutes at 35°C.

(5) The molecular weight of this enzyme is approximately 57,000 (SDS
- by acrylamide gel electrophoresis).

A second aspect of the present invention is the production of a novel maltase, which is characterized by culturing a microorganism that belongs to the genus Bacillus and has the ability to produce a novel maltase having the above-mentioned properties, accumulating the maltase in the bacterial body, and collecting the maltase. It is the law.

The microorganism used as the novel maltase-producing microorganism of the present invention belongs to the genus Bacillus and may be any microorganism that has the ability to produce maltase having the above-mentioned properties.For example, Bacillus cereus isolated from soil by the present inventors N
Y-14 is mentioned. The mycological properties of this bacterium are as follows.

■ Morphological properties ■Cell shape and size 30°C in 0.5% sodium chloride broth medium.

Cells cultured aerobically for 24 hours are 1μ x 3.0-4.0
Mu long rods exist singly or in chains of 2 to 5 or 6.

■ Presence or absence of motility There is no mobility. There are no flagella either.

■ Spores were formed. It has an oval shape of 0.7-0.8μ and is located at or near the center. There are no sporangia or vesicles.

■ Durham stainability It is positive.

■ Growth status on various media ■ Juice agar plate culture (30℃, 48 hours) Colonies are expansive, the surface is flat and rough, and the periphery is ear-shaped and grayish white.

■ Juicy agar slant culture (30℃, 48 hours) Growth is abundant, coarse, opaque, and expansive, with lobed peripheral growth. There is no stickiness.

■ Juicy puncture culture (30°C, 5 days) Growth is thick on the surface and grows only along the puncture line.

■ Meat juice liquid culture (30℃, 5 days) Growth is good, the liquid is transparent and sediment is formed, no bacterial membrane is produced, and a closed ring that easily collapses is formed.

No pigment is produced.

■ 0.5% sodium chloride broth agar plate culture (30℃
, 48 hours) The shape of the colony is irregular, the surface is flat and rough, and the periphery is ear-shaped and grayish white. There are no comma-shaped villages.

■ 0.5% sodium chloride broth agar slant culture (30℃
, 48 hours) The growth is abundant, coarse, opaque, expansive, and the marginal growth is lobed.

There is no stickiness.

■ 0.5% sodium chloride broth puncture culture (30℃, 5
(days) The growth is thick on the surface and grows only along the puncture line.

■ 0.5% sodium chloride broth liquid culture (30°C1
(5 days) Growth is good, but the liquid becomes cloudy and sediment is formed. Bacterial membrane 1 pigment is not produced.

■ Milk agar plate culture (30℃, 24 hours) Casein has a wide thawing zone.

[Phase] Meat juice gelatin puncture culture (20°C, 7 days) quickly liquefies in eruptive or layered form.

■ Physiological properties ■ Reduction of nitrate: Reducing ■ VP test: Produces niacetylmethylcarbinol ■ MR test: Positive ■ Utilization of citric acid: Utilize ■ Formation of indole: Not produced ■ Formation of hydrogen sulfide: Produce ■ Ammonia production: Produce ■ Effect on milk: Coagulate ■ Catalase: Positive [phase] Growth range: pH 5.2 to 10.2
.

Temperature: 7-37°C ■ Attitude towards oxygen: Grows on glucose under aerobic and anaerobic conditions.

@O-F test: Anaerobically I7! (glucose) to produce acid.

■ Growth in Sabouraud dextrose culture solution/agar slant culture: Grows ■ Presence or absence of acid and gas production from sugars: D-xylose, L-arabinose, L-rhamnose, mannitol, D-raffinose, glycerol, D-galactose , D-mannose, lactose, and sucrose.

Soluble starch, D-glucose, D-fulc)-4,l-
It grows on levalose, salicin, and i-blast sugars and produces acid, but does not produce gas.

■ Growth in 0.001% lysozyme: Grow 6
Growth in 0.02% azide: Grow 0 7
% growth in salt: The properties shown above are as described in Purge Aids Manual of Determinative Bacteriology, 8th edition (1974) (Be
rgey's Manual of Determin
Active Bacteriology 8th
ed, 1974), the native species belongs to Bacillus cereus, and the present inventors named the native species Bacillus cereus NY-14. Its home country has been deposited as FERM BP-329 at the Institute of Microbial Technology, Agency of Industrial Science and Technology. As mentioned above, the microorganisms used in the present invention may belong to the genus Bacillus and have the ability to produce maltase having the above-mentioned properties, and are limited to Bacillus cereus NY-14 and its variants and mutants. It's not something you can do.

Cultivation of microorganisms for producing the novel maltase of the present invention is carried out in a medium containing nutrients that can be utilized by common microorganisms. Specifically, for the production of novel maltase,
Polysancharides or oligosaccharides containing α-1,4-glucosidic bonds, such as various starches, amylopectin, amylose, dextrin, maltohexaose, etc., are required. Therefore, Bacillus cereus N
The medium for Y-14 contains one or more of these polysaccharides or oligosaccharides and other components necessary for bacterial growth, such as organic and inorganic nitrogen sources; organic and inorganic salts; vitamins, etc. as appropriate. Those that do are used.

The culture conditions for the microorganism used in the present invention vary depending on the bacteria used, but should be selected so as to maximize the production of the desired new maltase. Specifically, Bacillus cereus NY-14 strain In this case, the pH of the medium was set to 8.0. The appropriate culture temperature is 30°C, the culture time is approximately 24 hours, and aerobic culture is performed. For example, in the case of soluble starch, an effective amount of polysaccharide or oligosaccharide added to the medium is in the range of 0.5 to 10%.

After completion of the culture, the novel maltase can be collected and purified from the bacterial cells by a combination of known appropriate methods.

For example, microbial cells are obtained from the culture by appropriate means such as filtration and centrifugation, and after extracting remaltase from the microbial cells, a purified product of maltase can be obtained by applying chromatography such as gel filtration or ion exchange.

The maltase thus obtained has the following properties.

(11 Substrate specificity This enzyme is maltose, maltotriose, maltotetraose, paranitrophenyl-α-maltoside, paranitrophenyl-β-maltoside.

Acts on isopanose, maltohexaose, maltoheptaose, soluble starch, amylopectin.

It has no effect on amylose and pullulan.

(2) Optimal pH and stable pH This enzyme has an optimal pH of 7.0 at 30°C;
It is stable between 6.0 and 7.0. The relationship between pH value and amylase activity is as shown in FIG. 1, and the activity was measured according to the titer measurement method shown in 3 below under the following conditions.

Measurement conditions: temperature 30°C, 0.5 M acetate buffer at pH 5, 0.5 M phosphate buffer at pH 6-8.

p) (0.5M in 9) Lys buffer was used.

(3) Titer measurement method Enzyme solution 0.1 mA for 1% maltose 0.05 mβ
5mM in C30mM phosphate buffer (pH 6,86)
EDTA-containing] was added and allowed to react at 30°C for an appropriate time, then heated in a boiling water bath for 3 minutes, and the resulting glucose was processed using the method of Papadopoulas et al.
The amount of enzyme was determined by quantification method of reducing sugar, P139 (Gakkai Publishing Center), and the amount of enzyme that cleaves 1 μmol of glucoside bond per minute was defined as 1 unit.

(4) Range of optimal temperature for action The optimal temperature for this enzyme is as shown in Figure 2, at pH 6.8.
6 and around 24°C. The relationship between temperature and maltase activity was measured according to the measurement method described in (3) above, except that the reaction temperature was varied from 15 to 60°C.

(51 pH, conditions for inactivation due to temperature, etc. This enzyme has a pH of 8.
If left at 4°C for 24 hours at a temperature of 0 or higher, it will be inactivated. Moreover, at 35°C, it is deactivated by heating for 30 minutes.

(6) Inhibition and activation of various metal ions of this enzyme (final concentration I
) is shown in Table 1. As is clear from the table, iron, cobalt, copper, mercury,
An inhibitory effect was observed in zinc. In addition, there were no particularly activated metal ions.

Table 1 □100 MgCEz 84 Fe SOa 8 Co C1z 44 CuCβ20 gCe2Q BaC121Q4 ZnS0. 11 Ca C! lz 93 (7) Purification method 2ON The culture solution obtained by liquid culture using a jar fermenter was filtered and collected, and 5mM E was extracted from the bacterial cells.
0.05M phosphate buffer containing DTA (pH 6,86)
The solution from which the enzyme was extracted was used as a crude enzyme solution. The crude enzyme solution was diluted with 50mM'J acid buffer (pH 6, 86, 5mM
DEAE-5ep equilibrated with EDTA)
hadex A-50 column (column volume: 2.5c
After washing with the same buffer containing 0.1 MNa C1, 0.3 MNa C7! Elute the enzyme with the same buffer containing Next, the active fraction is loaded onto a TOYOPEARL HW40S column (column volume: 5.0 cm x 40 cm) equilibrated with 50 mM phosphate buffer (pH 6, 86, containing 5 mM EDTA), and eluted with the same buffer. Then, the active fraction was D E A E
-3 ephadex A-50 column (column volume: 1.
5cm x 15.5cm) and 0.2M NaCl
After washing with the same buffer containing 0.2M NaCl to 0.
Elute with the same buffer containing 3M NaCl. Next, the active fraction was dissolved in 0005M phosphate buffer (pH 6,86,5
TOYOPE equilibrated with mM EDTA)
ARLHW40S column (column volume: 5.Ocmx4
0 cm) and elute with the same buffer. Next, the active fraction was applied to a DEAE-Biogel A column (
Column volume = 1.0 ctax x 8.5 cm) and eluted with the same buffer containing 0.2M NaClt.

Next, the active fraction was added to 50mM phosphate buffer (pH 6,86
, containing 5mM EDTA).
PEARLHW50F (column volume: 1.5CIIIX
48CI11) and elute with the same buffer. The active fraction thus obtained is powdered by lyophilization.

The enzyme purified by the above method was found to be single by polyacrylamide gel electrophoresis.

(8) Molecular weight 5DS-acrylamide gel electrophoresis (Methods
in tinzymology, Vol. 26
, pp. 3-27, 1972), this enzyme has a molecular weight of about 57,000.

(9) Polyacrylamide disk electrophoresis (3,
J, Dayi5. Ann, New York Aca.
d, Sci.

121, Art 2,404 (1964)) in Tris-glycine buffer (pH 8,3).
When electrophoresed on a standard gel for 90 minutes at 3 mA/unit and then stained with Coomassie blue, the enzyme was detected as shown in Figure 3 (one hand is shown).

According to the present invention, it is possible to produce a large amount of highly pure maltooligosaccharides from sin and soluble starch in combination with maltooligosaccharide-producing amylase, which is not heat resistant, and it is possible to produce maltooligosaccharides in large quantities from sin and soluble starch, and it is possible to produce maltooligosaccharides in large quantities from sin and soluble starch. Expansion is expected.

Next, the present invention will be explained in detail with reference to examples.

Example 1 A medium containing 2.5% soluble starch, 1% peptone, and 0.5% di-salt (pH 8,0) 101 to 207! I put it in a jar fermentor and sterilized it. On the other hand, Bacillus cereus NY-14 (FERM B
P-329) was cultured with shaking at 30°C for 24 hours, 200ml of this preculture was inoculated into the medium, and 4818
(150 rpm.

161 air/win) Shaking culture was performed.

After culturing, the culture was centrifuged to obtain 256 g of bacterial cells, and a final enzyme preparation was obtained by the method described in (7) Purification method below. The specific activity of the final enzyme preparation was 2.36 U/■,
Purified 31 times.

Example 2 A 10 N medium (pH 8,0) containing 0.5% soluble starch, 1% peptone, and 0.5% salt was placed in a 201 JAF Armenter and sterilized. On the other hand, in a medium with the same composition as this medium, Bacillus cereus NY-14 (FERM
BP-329) was cultured with shaking at 30°C for 24 hours, 200mff1 of this preculture was inoculated into the medium, and the culture was incubated at 30°C for 24 hours (150 rpm).

161 air/ml) shaking culture was performed.

After culturing, the culture was centrifuged to obtain 221 g of bacterial cells, and a final enzyme preparation was obtained by the method described in (7) Purification method below. The specific activity of the final enzyme preparation was 2.20 U/■.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between pH and activity of the maltase of the present invention, and FIG. 2 is a graph showing the relationship between temperature and activity. FIG. 3 is an acrylamide gel disk electropherogram of purified maltase.

Claims (1)

[Claims] 1. A novel maltase having the following properties. (1) 0.05 ml of 1% maltose and this enzyme [50
5mM EDT in mM phosphate buffer (pH 6.86)
When 0.1 ml of A-containing product is applied for 30 minutes, the optimum temperature is around 24°C. (2) This enzyme contains maltose, maltotriose, maltotetraose, paranitrophenyl-α-maltoside,
It acts on paranitrophenyl-β-maltoside and isopanose, but does not act on maltohexaose, maltoheptaose, soluble starch, amylopectin, amylose, and pullulan. (3) The optimum pH of this enzyme is around pH 7.0 at 30°C, and it is stable around pH 6.0 to 7.0. (4) This enzyme is inactivated when left at pH 8.0 or higher at 4°C for 24 hours. Moreover, it is deactivated by heating for 30 minutes at 35°C. (5) The molecular weight of this enzyme is approximately 57,000 (according to SDS-acrylamide gel electrophoresis). 2. A method for producing a novel maltase, which comprises culturing a microorganism belonging to the genus Bacillus and having the ability to produce a novel maltase having the following properties, accumulating the maltase in the bacterial cells, and collecting the maltase. (1) 0.05 ml of 1% maltose and this enzyme [50
5mM EDT in mM phosphate buffer (pH 6.86)
When 0.1 ml of A-containing product is applied for 30 minutes, the optimum temperature is around 24°C. (2) This enzyme contains maltose, maltotriose, maltotetraose, paranitrophenyl-α-maltoside,
It acts on paranitrophenyl-β-maltoside and isopanose, but does not act on maltohexaose, maltoheptaose, soluble starch, amylopectin, amylose, and pullulan. (3) The optimum pH of this enzyme is around pH 7.0 at 30°C, and it is stable around pH 6.0 to 7.0. (4) This enzyme is inactivated when left at pH 8.0 or higher at 4°C for 24 hours. Moreover, it is deactivated by heating for 30 minutes at 35°C. (5) The molecular weight of this enzyme is approximately 57,000 (according to SDS-acrylamide gel electrophoresis). 3. The production method according to claim 2, wherein the novel maltase-producing bacterium belonging to the genus Bacillus is Bacillus cereus NY-14 (FERMBP-329).