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Field Of The Invention. This invention relates to an improved process for production of the enzyme alpha-amylase . Description Of The Prior Art. Alpha-amylase is a well known enzyme useful in commercial cleaning products such as laundry and dishwashing detergents. It is also useful in the conversion of starch to water-soluble carbohydrates,
particularly suited for eventual conversion, either chemical or enzymatic, to corn syrups having various D.E. values, dextrose and high fructose corn syrup.
Alpha-amylase has been produced by microbiological processes which involve cultivating certain strains of
Bacillus licheniformlε . One such process and strains is disclosed in British patent specification No. 1,296,839.
Another process and strains is disclosed in U.S. Patent No. 4,473,645 to Horwath. Known processes result in fermented medium having enzyme activity of about 50 to about 2,000 liquefons/ml. In these processes, mono-, di- and
polysaccharides such as pyruvate, gluconate, arabinose, ribose, galactose, maltose, lactose, soluble starch, or mannitol are conventionally used; however lactose is the preferred carbon source. Even though it iε relatively expensive and is poorly metabolized, lactose is preferred because it does not repress alpha-amylase synthesis.
Dextrose, starch, starch hydrolysate and various products remaining after crystalization of dextrose such as "DL-70" (trade name of Staley-Continental Foods) on the other hand are relatively inexpensive and rapidly metabolized, but have not heretofore been used as a carbon source in
microbiological alpha-amylase production because they are well known repressors of alpha-amylase synthesis. See
Fukumoto, I., et al., 180 Nature 438 (1957).
The present invention overcomes the disadvantages of the prior art by providing a novel process for production of alpha-amylas . In accordance with the method of the present invention, thermally stable alpha-amylase is produced by cultivating the B. licheniformis ATCC 53,722 microorganism in a nutrient medium which contains as a primary carbon source a carbohydrate which normally represses alpha-amylase synthesis; feeding said primary carbon source to said medium during cultivation; maintaining the level of carbon dioxide evolution from the medium during cultivation between 4.5% and 6.5% of the off-gas when the air flow is between 0.5 to 1.0 volume of air per volume of medium per minute by controlling the rate at which, the primary carbon source is fed to said medium during cultivation; and maintaining the pH of the medium between 6.5 and 7.0.
The alpha-amylase produced by the method of the present invention is of the same order of thermal stability as that produced in accordance with the process disclosed in U.S. Patent No. 4,473,645. It is stable in the presence of low concentrations of calcium ion and is functional at a lower operational pH (5.2) than most conventional commercially available alpha amylases . The alpha amylase produced by the method of the instant invention has or exceeds all functional advantages of the amylase claimed in the specification of

U.S. Patent No. 4,473,645. The preferred method of the present invention yields at least about three times the amount of alpha-amylase per ml of medium as compared to the maximum yield of the process disclosed in U.S. Patent No.
4,473,645 in a comparable time of cultivation.
The process of this invention is carried out using a strain of Bacillus licheniformiε, viable cultures of which have been deposited with the American Type Culture Collection as number 53 , 772.
In the process of this invention, the fermentation is carried out for three to six days at a temperature ranging from 38°C to 44°C, preferably at 42 °C. The fermentation is aerobic with air flow ranging from 0.5 to 1.0 volume of air per volume of medium and preferably at 0.6 volume of air per volume of medium per minute.
Suitable primary carbon sources which are rapidly metabolized and which normally repress alpha-amylase
synthesis include dextrose, xylose, mannose, sucrose,
glycerol and starch hydrolysate. The initial amount of carbohydrate can vary from 0 to 20% w/v of the culture medium.

-Λ- In addition to the primary carbon source, the medium contains a nitrogen source and other essential nutrients, all of which are known to those skilled in the art. See, for example, U.S. Patent No. 4,473,645. Suitable nitrogen sources can be inorganic such as soluble nitrates or ammonium salts, or organic such as yeast extract, corn steep liquor, peanut meal, monosodium glutamate, soy flour, cottonseed flour, cottonseed meal and other common organic nitrogen sources familiar to those skilled in the art.
In the preferred embodiment of this invention the primary carbon source is dextrose, the nitrogen source is corn steep liquor and the carbon dioxide evolution is
preferably maintained between 5% and 6% of the off-gas when the aeration rate is 0.5 to 1.0 wm by controlling the rate at which dextrose is fed to the medium during
fermentation. The pH is desirably maintained at 6.7 by the addition of ammonia (anhydrous or aqueous) or caustic soda (aqueous sodium hydroxide).
The enzyme is recovered from the medium after desired levels of enzyme activity are attained. The methods of recovery are well known, including, for example,
precipitation by sodium sulfate, ammonium sulfate, acetone or ethanol followed by centrifugation or filtering and then drying the precipitate. The most commonly available
commercial preparations of alpha-amylase are recovered as liquid products in which the B. licheniformiε microorganism has been removed from the whole broth by filtration, the filtrate then being concentrated to a specified alpha-amylase potency by evaporation or ultrafiltration.

The following example further illustrates the invention.
Example 1
A. Slant Preparation. A fresh slant of B.
licheniformis ATCC 53,772 was prepared from a suitable working stock culture using standard microbiological
techniques. Suitable working stock cultures may be
maintained in the frozen state as lyophilized cultures or as a separate slant culture. The slant medium typically consists of Difco Antibiotic Medium No. 3 containing 0.5% (w/v) corn starch. The fresh slant was grown for 25 to 30 hours at 37°C prior to use for inoculating the seed
B. Seed Fermentor. The growth of the fresh slant was suspended in a small amount of Difco Antibiotic Medium No. 3 and then immediately transferred to a seed fermentor
2% Corn Starch
1.5% Dextrose
8% Corn Steep Liquor
0.1% Mazu DF-37C
0.3% H2P04
The seed fermentation was operated for 25 to 30 hours at 42°C with an aeration rate of 0.6 wm. The agitation rate will vary depending upon the size of the seed fermentor, but typically falls in the range of 150 to 1000 rpm. In this example the agitation rate was 750 rpm in a 20 liter vessel containing 14 liters of medium. The pH was maintained at or above 6.5 using ammonia gas as needed.

C. Production. The inoculation size for the
production fermentor is 3% of the initial inoculated volume.

For example, if the desired inoculated fermentor volume is

100,000 liters, then 3000 liters of seed culture would be transferred to 97,000 liters of production medium. In this example, 360 ml of seed culture was transferred to 11,640 ml of production medium to give 12,000 ml total in a 20 liter vessel. The production medium consisted of:
4% corn starch
6% corn steep liquor
0.03% CaCl2-2H20
0.1% MgS04.7H20
0.25% Mazu DF-37C
trace MnSO.
0.67% K2HP04
0.5% KH2 P04
2.4% dextrose
The potassium phosphate salts were sterilized
separately and asepticly added to the remaining components of the sterile production medium. After the initial
carbohydrate was completely depleted as indicated by an abrupt drop in carbon dioxide evolution and rise in pH, the continuous addition of 70% dextrose solution (as
DL-70, starch hydrolysate or a combination thereof) is begun. Here, DL-70 was the carbohydrate source used. The aeration rate was 0.6 wm. The dextrose solution was fed at a rate to maintain the carbon dioxide evolution at 5 to 6% of the off-gas. Generally the glucose feed causes the pH of the medium to decrease, and the pH was maintained at or above 6.7 by the addition of ammonia gas as needed.
Fermentation was continued for 116 hours. At 76
hours the activity of alpha-amylase in the medium was 6064 liquifons/ml (as measured by the assay procedure described in U.S. Patent No. 4,473,645 to Horwath) . At 116 hours the activity was 7965 liquifons/ml.
In addition to the ATCC 53,772 microorganism, the preferred embodiment of the present invention contemplates the use of mutants and variants of ATCC 53,772 as well as genetically transformed microorganisms derived therefrom.
While the foregoing has been described with respect to the preferred embodiment and alternatives thereto, one skilled in the art should realize that modifications and changes may be made to the foregoing while still falling within the intent and scope of the present invention. All such modifications and changes should be considered a part hereof.