JPH0472506B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an improved method for producing thermostable alpha-amylase.

BACKGROUND OF THE INVENTION Alpha-amylase is well known as a component of commercial detergents and as an enzyme for converting starch to water-soluble carbohydrates, particularly glucose.

Conventionally, amylase has been produced by culturing strains of the genus Bacillus. UK patent no.
No. 1296839 describes that thermostable alpha-amylase can be produced by culturing a strain of Bacillus licheniforms.

[Problems to be Solved by the Invention] The enzyme produced by the above method exhibits significantly higher thermostability than that produced by culturing a strain of Bacillus subtilis. The yield of enzyme activity reported in the British patent specification is not very good, and improvements in this respect are required for commercial production of the thermostable amylase.

[Means for Solving the Problem] The present inventors have discovered the fact that certain strains of Bacillus ritsieniformis can be used for commercial production of thermostable alpha-amylase due to their high yield of enzymatic activity. I found out.

[Operations and Effects of the Invention] Generally, the yield of oxygen activity by the specific strain of Bacillus ritsieniformis used in the present invention is as follows:
The yield is at least about 10 times higher than that reported in the British patent specification. The standard strain of Bacillus lithueniformis used in the present invention is stored in the American Type Culture Collection under deposit number ATCC39326.

The alpha-amylase produced according to the present invention exhibits a thermal stability comparable to that obtained by the method of the above-mentioned British patent specification and is relatively stable even in the presence of high concentrations of calcium ion precipitants. It is. In addition to all the functional advantages described in the British patent specification, high yields are achieved with the process of the invention.

In order to obtain the preferred results of the present invention, a specific strain of Bacillus ritsieniformis may be used and cultured in a nutrient medium until sufficient enzymatic activity is obtained. Fermentation typically uses assimilable carbon and nitrogen sources as well as essential nutrients, and uses approximately 25 to
It is carried out over a period of about 2 to 6 days at a temperature of 55°C. Such fermentation conditions are themselves conventional and well known to those skilled in the art. Examples of suitable carbon sources include carbohydrates (eg, lactose, glucose, starch), carbohydrate-containing substances (eg, soybean, cornmeal), or mixtures thereof. The amount of carbohydrate can vary widely, but is usually about 1-25% (w/v), preferably about 5-15% (w/v).

In a preferred embodiment of the invention, good enzyme activity yields are achieved by using lactose as a carbon source. Usually in a nutrient medium,
Lactose is used at a concentration of about 1-20% (w/v), preferably about 5-15% (w/v). Lactose may be used as the sole carbon source,
It may also be used in conjunction with other carbon sources such as whey or soy flour. The carbon source used in combination is usually about 5% (w/v)
It is as follows.

The nitrogen source may be an organic or inorganic substance;
Examples include soluble nitrates, ammonium salts, yeast extract, cornstarch liquor,
Examples include peanut meal.

Cultivation may be carried out in a nutrient medium under aerobic conditions. Aerobic conditions are usually achieved by introducing about 1 volume of air per volume of medium into the fermentation vessel.

After the enzyme activity in the medium reaches the desired level,
Obtain the enzyme. The enzyme may be obtained by a conventional method, for example by precipitating the enzyme using a known precipitant such as sodium sulfate or using a water-miscible organic solvent such as acetone or ethanol. . The precipitated enzyme can be recovered by centrifugation or filtration, and then dried to obtain a solid enzyme with good purity.

Instead of the above-mentioned specific bacterial strain itself, mutant strains thereof and genetically modified strains obtained by introducing enzyme genes can also be used in the present invention, and such cases also fall within the scope of the present invention. Included.

[Example] The present invention will be described in further detail with reference to Examples below.

Example 1 (A) Bacillus ritsieniformis ATCC39326
The culture on the Difco starch agar slant is inoculated onto a Difco starch agar plate and cultured for 18±1 hours at 40±1°C.

(B) Inoculate one loopful of the bacterial cells obtained in the above culture into the following medium: Aqueous inoculation medium 0.5% yeast extract, 0.5% tryptone, 0.1
%K ₂ HPO ₄ , 0.1% D-glucose 350ml/1
Flask The above flask was taken from New Brunswick G.
-200 rpm on 50 shaker (2” orbit)
Culture at 40±1°C for 1 to 2 days.

(C) Inoculate the entire contents of _the flask into 3150 ml of the following fermentation medium: Aqueous fermentation medium 10% Lactose ^* , 1.4% _K2HPO4 ^* , 0.6%
_{KH2PO4 *} , 0.6% _{( NH4} ) _{2SO4 ,} 0.3% sodium citrate, 3.0% ^soy flour, 0.05% _CaCl22H2O ,
1.0ml “MazuDF6000”, initial pH = 6.8-7.0 (*marked is sterilized separately from other ingredients) Fermentation vessel is sterilized at 121°C for 60 minutes at 15lbs.

In order to facilitate optimal production of alpha-amylase, the pH of the medium is adjusted to below 8.0 and above 5.5.

To suppress foaming that occurs during incubation, "MazuDF6000" antifoam is added through a conventional foam suppressor.

Fermentation conditions 600rpm 40℃, 1v/v air PH after 72 hours is usually 6.5-7.0.

Measurement of alpha-amylase enzyme activity is performed by standard methods. An example of such a method is the modified Wholegermuth method, in which activity is indicated by the time of digestion to produce a color change, which is an obvious step in the dextrinization of the starch. If this is converted into a quantitative line, the amylase strength can be expressed as follows:
It can be calculated directly in grams.

The Liquehon alpha-amylase quantitative method is carried out as follows. That is, a sample solution is prepared using a 0.025 molar CaCl ₂ solution as the solvent and diluent such that the final diluted sample of 5 ml has a dextrinization time of approximately 10 minutes. If the approximate activity of the enzyme preparation is unknown, trial chain error (trial
(and error) to find the appropriate sample volume and dilution. Dispense 5.0 ml diluted iodine solution into 13 x 200 mm test tubes and keep 10-12 test tubes in a water bath (30 °C). Transfer 10ml Lintner starch to a 23 x 200mm test tube and store at 30°C.
Keep in water bath. Transfer 25-30 ml of diluted unknown alpha-amylase solution to a 23 x 200 mm test tube and keep in water at 30°C. Add 5 ml of the unknown diluted enzyme sample to 10 ml of starch solution. When half of the amount has been added, start the stopwatch and pipet in the remaining half of the amount. At appropriate time intervals, 1 ml (1 ml pipette) of the hydrolysis mixture (starch, alpha-amylase solution) is added to the test tube containing the temperature-controlled dilute mode solution. This is mixed well, poured into a 13 mm precision rectangular test tube, and compared with a standard alpha-amylase color disc in a Hellige comparator. 1ml
By squirting the contents of the pipette into the iodine solution,
The end point time can be measured more accurately. When the end point time is almost reached, every 0.1 minute (6 to 12
Collect the sample in seconds). When the reaction is complete, record the time and dilution and calculate the Requephon unit activity per gram or ml. The following formula is adopted to calculate the enzyme activity: Liquehon/g or ml = 1140 x dilution / actual amount of sample (g or ml) x dextrinization time (min) The solutions used in the above quantitative method are as follows: Prepare as follows.

Starch Solution Soluble Lintner starch (10 g) is mixed with 35 ml of distilled water in a flask, heated to boiling and maintained at boiling point for 5 minutes with stirring. Cool to room temperature;
Add 125ml of acetic acid buffer and enough water to make 500ml.
Then add 1 ml of toluene. The stability of this solution is confirmed using a standard sample of alpha-amylase of known activity.

Acetic acid buffer Weigh 1 mol sodium acetate (82.0g) and add water
Dissolve in 300ml. Adjust the pH to 6.2±0.1 with acetic acid and dilute to 1. The 20 ml is used to prepare the starch substrate of 1.

0.025M Calcium Chloride Dilute Solution Dissolve 11.1g of CP anhydrous calcium chloride in 4 parts of water.

Buffer Solution 25.3 g of sodium hydroxide pellets and 340 g of potassium dihydrogen phosphate are dissolved in distilled water and adjusted to 2 in a volumetric flask. Adjust the volume when the buffer reaches room temperature (PH6.2±0.1).

Stock iodine solution Dissolve 5.50g of CP iodine crystals and 11.0g of potassium iodine in water to make 250ml. Store in a brown bottle.

Diluted iodine solution Dissolve 20.0g of potassium iodide in water, add 2.00ml of stock iodine solution, and dilute to 500ml.

When measured using the Liquehon method described above, the enzyme activity of the fermented mixture obtained in the above example was more than 1,000 Liquehon/ml, and in most cases 1,500 -
It reaches up to 2000 requests/ml. According to this quantitative method, the strain of Bacillus ritsieniformis described in the British patent specification gives an average enzyme activity of 125 Liquehons/ml in the fermentation medium described above, but Some give even lower enzymatic activity, ie less than 50 requesthons/ml.

Another method for measuring alpha-amylase activity is the modified SKB method described in the UK patent specification mentioned above. Under the conditions of this test method,
While the strain of the British patent specification gave an activity of less than 500 SKB units/ml, the strain of the present invention gave an activity of about 500 SKB units/ml.
It gave an activity of 10000 SKB units/ml.

Claims (1)

[Scope of Claims] 1. Bacillus lithueniformis
39326) ATCC No. 39326 in a nutrient medium to produce alpha-amylase. 2. The production method according to item 1, wherein the produced alpha-amylase is collected from a nutrient medium. 3. The method according to item 1 or 2, wherein the nutrient medium contains lactose as a carbon source. 4. The method according to item 3, wherein lactose is contained at a concentration of 5 to 15% (w/v). 5. The manufacturing method according to any one of items 1 to 4, wherein the pH of the nutrient medium is maintained at 8 or less and 5.5 or more.