HU191134B - Process for preparing heat stable alpha amylase - Google Patents

Abstract

A process for the preparation of thermostable alpha amylase which comprises cultivating B. licheniformis ATCC No. 39326 in a nutrient medium therefore to produce the enzyme product. - (5pp)

Description

The present invention relates to a process for the preparation of a thermostable alpha-amylase enzyme.

Alpha-amylases are well-known enzymes used to make commercially available cleaning agents and to convert starch to water-soluble carbohydrates, and these enzymes are particularly suitable for the chemical or enzymatic conversion of starch to a commercially available product.

Amylase enzymes have previously been produced by microbiological methods, for example by culturing strains of Bacillus species. British Patent No. 1,296,839 discloses a process for the preparation of heat-stable alpha-amylase which comprises the production of Bacillus licheniformis. The thermal stability of the enzyme thus obtained is much higher than that obtained by culturing Bacillus subtilis strains, but the enzyme activity values given in the said British Patent are not high and therefore this process is unsatisfactory for the industrial production of the thermostable amylase enzyme.

It has been found that one strain of Bacillus licheniformis is particularly suitable for the industrial production of the heat-stable alpha-amylase enzyme, since this strain can be produced in high yield. Generally, this strain of Bacillus licheniformis has a yield of at least about 10 times greater than that described in the above-mentioned British Patent. A viable culture of this microorganism was deposited in the American Type Culture Collection, which was registered under ATCC 39,326.

The thermal stability of the alpha-amylase enzyme produced by the process of the present invention is of the same order of magnitude as that of the similar preparation of the process described in British Patent No. 1,296,839, and is relatively stable even in the presence of high concentrations of calcium ion binding agents. The enzyme produced by the process of the present invention has all the advantageous properties set forth in the aforementioned British Patent and can also be produced in high yield.

The desired results of the process of the present invention can be achieved by culturing the indicated strains of Bacillus licheniformis in a suitable medium under submerged aeration until a significant amount of enzyme is accumulated in the medium. The fermentation is usually carried out for 2 to 6 days at a temperature between 25 ° C and 55 ° C using conventional sources of carbon and nitrogen and other essential nutrients well known to those skilled in the art. Suitable sources of carbon include carbohydrates, lactose, glucose and starch, as well as carbohydrate-containing materials such as soy, corn meal, and mixtures thereof. The amount of carbohydrate can be varied within a wide range, for example between 1% and 25% by weight, and preferably between 5% and 15% by weight.

In the most preferred embodiment of the process according to the invention, lactose is used as the carbon source, since this provides the highest yield of the enzyme. The amount of lactose in the medium is generally from 1 to 20% by weight, and preferably from 5 to 15% by weight. As the carbon source, lactose may be used alone or in combination with other carbon sources, such as whey or soy flour, the latter being generally not more than 5% by weight.

Nitrogen sources include organic or inorganic compounds such as soluble nitrates or ammonium salts. Organic nitrogen sources include, for example, yeast extract, corn jam, earthmeal flour.

Said microorganism is cultured under aeration in the medium, for this purpose, the fermentation vessel is aerated, usually with about one volume of air per minute of the volume of the medium.

Once it has been determined that the desired amount of enzyme has accumulated in the medium, the enzyme is isolated from the medium. Methods of isolation are known to those skilled in the art, such as precipitation of the enzyme with known precipitants such as sodium sulfate or water-miscible organic solvents such as acetone or ethanol. The precipitated material is centrifuged or filtered and dried to give a purified solid enzyme.

In addition to the use of the above-mentioned microorganism, the present invention also encompasses the use of mutants and variants of this microorganism, as well as genetically engineered variants obtainable by introducing the enzyme genes into said microorganisms.

The process of the present invention is illustrated by the following, without limiting the scope of the invention. The percentages in the examples are by weight.

Example 1

the step

The slant agar culture of Bacillus licheniformis ATCC 39326 Difco starch agar was plated on a Difco starch agar plate and cultured at 40 ° C ± 1 ° C for 18 ± 1 hours.

b) tearing

One loop of cells is inoculated into the following medium:

Aqueous inoculum (inoculum) culture weight%

Yeast extract 0.5%

Tripton (casein hydrolysed with trypsin) 0.5%

EC potassium hydrogen phosphate 0.1%

D-Glucose 0.1%

350 ml of this medium were placed in a 1 liter flask.

The flask is shaken for 1-2 days at 40 ° C ± 1 ° C on a New Brunswick G-50 shaker at 200 rpm.

191 134 'step c)

The entire volume of the above shake flask culture is used as inoculum to inoculate a final volume of 3150 ml of fermentation broth with the following composition:

Wet fermentation media% by weight

Lactose* 10 % Dikáhum hydrogen phosphate * 1.4 % Potassium dihydrogen phosphate * 0.6 % Ammonium sulfate 0.6 % Sodium citrate 0.3 % Whole soybean meal 3.0 % Calcium chloride dihydrate 0.05% Mazu DF 6000 (Antifoam) 1.0 ml Initial pH = 6.8 - 7.0 * Autoclave separately from other components

The fermenter was maintained for 1 hour at 121 ° C, 1.03 · 10 ⁵ Pa overpressure.

For optimum production of the alpha-amylase enzyme, the pH of the medium should be below 8.0 but above 5.5.

To prevent foaming that may occur during the growth of the microorganism, a Mazu DF 6000 antifoam agent is added to the culture medium using standard equipment.

Fermentation conditions:

Speed: 600 rpm

Temperature: 40 'C

Aeration rate: 1 liter / l medium per minute.

The pH of the medium was measured after 72 hours of culture; the pH is generally between 6.5 and 7.0.

The activity of the alpha-amylase enzyme is measured by methods known per se. Such a method is a modified version of the Wohlgemuth method, wherein the degradation time required to determine the degree of dextralisation of the starch is determined to determine the activity of the enzyme. The method is set up so that the amylase activity can be directly calculated in units of ixphone / g as described below.

The activity of the alpha-amylase enzyme in units of lyconone / g is determined as follows:

Prepare a 0.025 M solution of calcium chloride as a solvent and diluent in the sample so that a 5 ml aliquot of the final diluted sample has a dextrinization time of about 10 minutes. If the activity of the enzyme preparation is not known at all, the appropriate sample size and / or dilution is determined by testing.

5.0 ml of dilute iodine solution was filled into 13 x 200 mm test tubes and placed in a 30 ° C water bath. 10-12 such tubes are used.

ml of Lintner's starch solution (solylated starch, commonly used for the analysis of enzymatic, mainly amylase activity) is poured into a 23 x 200 mm test tube and placed in a 30 ° C water bath.

- 30 ml of dilute alpha-amylase solution of unknown activity are placed in 23 x 200 mm test tubes and placed in a 30 ° C water bath.

A 5 ml aliquot of the diluted enzyme sample of unknown activity is poured into 10 ml of the killing solution while the stopwatch is started when half of the alpha-amylase solution of unknown activity is added and the other half of the solution is then added to the starch solution.

At appropriate intervals, place 1 ml portions of the hydrolyzing mixture containing starch and alpha-amylase solution into a dilute iodine solution maintained at the appropriate temperature with a 1 ml pipette. The mixture is shaken each time, poured into a 13 mm precision square cuvette and compared in a Hellige comparator to a standard alpha-amylase dye. The contents of the 1 ml pipette are blown into the iodine solution to more accurately measure the end point of the hydrolysis time. When the hydrolysis end point is near, samples should be taken at 0.1 minute intervals (6-12 seconds).

When the reaction is complete, the time and dilution are recorded and the activity of the enzyme is calculated in units of Liquidone / g or Liquidone / ml.

The enzyme activity is calculated using the formula:

Likphone / g / or / ml) = _____1140____

Dilution x actual weight (or volume) of sample x dextrinization time (minutes)

The solutions used in the above activity determination method are prepared as follows:

Starch solution Lintner starch g is mixed with 35 ml of distilled water in a flask, the mixture is boiled and refluxed for 5 minutes with constant stirring. After cooling to room temperature, 125 ml of acetate buffer solution are added, water is added to make up to 500 ml, and finally 1 ml of toluene is added. Stability of the solution is checked using a standard sample of enzyme alpha amylase of known activity.

Cetate buffer solution

One mole (82.0 g) of sodium acetate was weighed and dissolved in 800 ml of water. The pH of the solution was adjusted to 6.2 ± 0.1 with acetic acid and the solution was diluted to 1 liter. To make 1 liter of starch substrate, use 20 ml aliquots of the above buffer solution.

191,134

0.025M solution of calcium chloride

11.1 g of chemically pure anhydrous calcium chloride are dissolved in 4 liters of water.

Buffer solution

Dissolve 25.3 g of particulate sodium hydroxide and 340 g of potassium dihydrogen phosphate in distilled water and make up to 2 liters in a volumetric flask. When the buffer solution reaches room temperature, it is diluted to its final volume (pH 6.2 ± 0.1).

Iodine stock solution

5.50 g of chemically pure crystalline iodine and 11.0 g of potassium iodide are dissolved in water and the solution is 250 ml. make up to volume. Store the solution in a dark glass vial.

Diluted iodine solution

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

The activity of the enzyme produced by the method of the present invention is greater than 1000 lyconone / ml and in most cases reaches 1500-2000 lyconone / ml in order to determine the activity of the enzyme expressed in units of lyconone / ml. The Bacillus licheniformist described in the above-mentioned British Patent was cultivated in the fermentation medium described above and the activity of the enzyme was also determined as described above, the activity of the enzyme was on average 125 lyconone / ml while the same culture was grown in the medium described in tb, resulted in activity below 50 likonphone / ml.

The activity of the alpha-amylase enzyme is modified by the modified SKB method described in the above-mentioned British patent [SK.B method see Cerial Chemistry 16; 712-723 (1939)]. Using this method, the cultures described in the aforementioned British Patent produced 500 SKB Units / ml enzyme activity, while the cultures of the present invention had an enzyme activity of about 10,000 SKB Units / ml.

Claims (6)

A process for the preparation of a thermostable alpha-amylase enzyme, wherein the strain Bacillus licheniformis ATCC 39326 is present in an amount of from 1 to 25% by weight of carbon sources such as lactose, glucose, starch, soy, corn flour or mixtures thereof and nitrogen sources such as soluble nitrates, culturing in a culture medium containing yeast extract, corn jam, peanut flour, and isolating the alpha-amylase enzyme from the culture medium if desired; and the pH of the medium is preferably maintained between 8 and 5.5. 2. The l. The method of claim 1, wherein the alpha-amylase enzyme is isolated from the culture medium. 3. The method of claim 1, wherein the strain Bacillus licheniformis ATCC 39326 is cultured in a medium containing lactose as a carbon source. The process according to claim 3, wherein the amount of lactose in the medium is between 5% and 15% by weight. The method of claim 3, wherein the alpha-amylase enzyme is isolated from the medium. 6. The method of claim 3, wherein said medium is maintained at a pH below 8 but above 5.5.