HUT53934A - Process for producing thermostable alpha-amilase - Google Patents

Abstract

A process for the production of thermally stable alpha-amylase comprising the steps of: cultivating B. licheniformis ATCC 53,722 in a nutrient medium which contains as a primary carbon source a carbohydrate which normally represses alpha-amylase synthesis, selected from the group consisting of dextrose, starch hydrolysate, glycerol, xylose, mannose, sucrose and mixtures thereof; feeding said primary carbon source to said medium during cultivation; maintaining the level of carbon dioxide evolution from the medium during cultivation between about 4.5 % and 6.5 % of the off-gas when the air flow is 0.5 to 1.0 vvm 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.

Description

The present invention relates to an improved process for the preparation of the alpha-amylase enzyme.

and alpha-amylase trade is a well-known enzyme used in detergents for detergents such as detergents and dishwashers. It is also suitable for the conversion of starch into soluble carbohydrates, in particular for its various viscosities into starch syrups, chemical or enzymatic conversion of glucose and concentrated fructose syrups, and alpha-amylase is microbially reconstituted .16 bteonyo. 8.8111. " llahmlfoad .. tx. ». k · This procedure and strains are described by the 1 296 839 British hay liberated! Other methods and strains are described in U.S. Patent No. 4,473,645. By known methods, about 50-2000 Liquefones / ml can be obtained with animate activity. Muscle processes generally use mono-, dl- and polysaccharides such as plruvate, gloconate, arabinose · ribose, galactose, maltose, lactose, soluble starch, chemical mannitol, but the preferred source of carbon is lactose · Although relatively expensive and difficult to convert, treat lactose because it does not inhibit the synthesis of ss-alpha-amylase · On the other hand, crystallization of glucose, starch, hard!

Various products remaining after r, such as DL-70 / 8taley-Continantal Foods brand name / are relatively inexpensive and rapidly transformed but have not yet been used as a carbon source in the microbiological production of alpha-amylase because of known inhibitors of alpha-amylase synthesis / see Fukumoto I · Et al., Natúre 180 »4? 8/1957 // ·

The present invention overcomes the drawbacks of the prior art by employing a novel process for the production of algal amylase. The present invention provides a heat-stable alpha-amylase by culturing Baoillus licheniformis ATCC 53722 in a culture medium containing a carbohydrate normally suppressing alpha-amylase synthesis as a primary carbon source; adding the primary carbon source to the medium during cultivation; controlling the rate of dosing of the primary carbon source to maintain C0 _{2 from} the medium in the range of 4 to 5 to 6.5 percent of the effluent gas during culture at an air flow rate of 0.5 to 1.0 air volume / medium volume / cartilage; and maintaining the pH of the medium at 6.5-7.0.

The process according to the invention provides f

- 4 rendered alpha-amylase having the same thermal stability as those obtained in thousands of U.S. Patent 4,473,645. The resulting enzyme is stable in the presence of a low concentration of Ca ² * -ion and is functional at lower operating pH values (5.2) than commercially available conventional alpha-amylases. All of the functional advantages of the alpha-amylase produced by the process of the invention are equal to or superior to the amylase disclosed in United States Patent 4,473,645. The preferred method of the invention produces at least three times the amount of alpha-amylase per ml of nutrient as compared to the method disclosed in U.S. Patent No. 4,473,645, over a comparable culture time.

The process of the present inventionB. It is performed using a strain of Heheniformis cultured in cultures deposited at Amerioan Type Culture Collection (ATCC) under accession number 53772.

The process of the invention, the fermentation is continued at ^this temperature 38 -44 ° C, preferably 42 ° C for 3-6 days. Fermentation is carried out under aerobic conditions using aeration of 0.5-1 air / medium volume / min * ···· · * ··

Suitable primary carbon sources ab are rapidly transformed and generally inhibit alpha-amylase synthesis | these include glucose, xylose, mannose, sucrose, glycerol and cumin hydrolyzate. The initial amount of carbohydrate is 0-20 percent of the medium / wt / vol ·

In addition to the primary carbon source, the medium contains a source of nitrogen and other essential nutrients | which are known to the person skilled in the art (see, for example, U.S. Patent No. 4,473 ^ 5). , cotton nuts, and other conventional nitrogen sources known to the skilled person ·

In a preferred embodiment of the invention, the primary carbon source glucose, the nitrogen source corn jam, and the gas content of the effluent gas during fermentation are maintained at 5-6 percent by controlling the rate of glucose delivery at 0.3-1.0 cubic volume / feed volume / per aeration. when washing · The pH is preferably maintained at 6.7 by addition of ammonia / anhydrous or aqueous / or caustic soda / aqueous sodium hydroxide solution.

- 6 After reaching desired levels of enzyme activity · Isolate from culture medium · Recovery procedures are well known; such as precipitation with sodium sulfate, ammonium sulfate, acetone or ethanol followed by centrifugation or filtration followed by drying of the precipitate. The most commonly available commercial alpha-amylase formulations are isolated as liquid products; microorganism B, licheniformis is removed from the whole medium by filtration, and the filtrate is concentrated by evaporation or ultrafiltration to a defined alpha-amylase activity *

The following examples further illustrate the invention.

Preparation of * Solid slant medium

Fresh slant medium containing B. licheniformis ATCC 53772 is prepared from a suitable stock culture using standard microbiological techniques. Appropriate plant stock shoots are maintained as frozen lyophilized cultures or as a separate slant culture. The slant medium typically consists of 0.5% / w Difco Biotin Anti Medium No. 3 containing volume of ^M / corn starch. Fresh medium was cultured for 25-30 wells at 37 ° C before being used to inoculate the growth fermentor ·

3, Multiplication fermenter

The bacterial culture of fresh slant medium was suspended in a small amount of Difco Antibiotio Medina No. 3 ”and then directly transferred to the sprouting torment containing 2% cornstarch,

1.5 * glucose,

8% corn jam,

0.1% Mazu DF-37C in Oz

Contains 0.3% KHgPO ^ ·

The fermentation is carried out for 25-30 hours at 42 ° C with 0.6 air / medium volume / porous aeration. The speed of mixing depends on the size of the fermentor, but typically is in the range of 150-1000 rpm of powder per minute. In this example, the mixing speed is 750 rpm in a 20 liter dish containing 14 liters of nutrient medium · The pH is maintained at or above 6.5, if necessary with ammonia gas ·

0. I.naeliii

The inoculum corresponding to 3% of the initial inoculated volume of the producing femontary fetus is given. For example, if the desired production fexmentor volume is 100,000 liters, then 5000 liters of inoculum is added to 97,000 liters of production medium. Composition of the production medium m starch 6% corn jam 0.05% CaClg.aHgO 0.1% MgSO4 THgO 0.25% Ash W-5TC Trace MnSO ^ 0.67% ^ HF0 ₄ 0,% 2.4% Glucose ·

The potassium phosphate salts were added in separately sterilized and aseptically other ingredients of the sterile production medium · After the initial carbohydrate completely consumed - which C0 ₂ fojlődés sudden signal increase ceased and & pH - begin a 70% glucose / DL -70, starch * hydrolyzate or a combination / continuous addition. Aeration rate 0.6 air volume / medium volume / trial · Glucose solution is added at a rate such that the developing carbon dioxide represents 5 ~ 6 percent of the effluent · Generally, the glucose dose is: ·· * ··· ».»

- to reduce the pH of 9 solutions, the pH is maintained at 6 to 7 by introducing ammonia in the case of dehydration.

Fermentation is continued for 116 hours * After fermentation for 1 hour, the medium has an activity of alpha-amylase of 6064 Liquiphones / ml / as determined by US Patent 4,475,645 / / After a fermentation of 116 hours, the activity is 7965 Liquifones / ml.

In addition to ATCC 55772, mutants and variants of ATCC 55772 and the genetically transformed microorganisms derived therefrom may also be used in the preferred process of the present invention.

While the foregoing has never been described in terms of the realization of my advantage and its alternatives, it will be appreciated by those skilled in the art that the foregoing may be modified and varied without departing from the spirit and scope of the invention.

Claims (10)

A process for the preparation of a heat stable alpha-amylase, characterized in that Baolllu strain < RTI ID = 0.0 > 11 < / RTI > the primary source of slurry is added to the medium during the culture, and the level of carbon dioxide evolved during the culture in the outlet gas is maintained at 4.5-6.5 percent 0.5-1.0 air volume / volume of medium / minute Aeration controlling the rate of dosing; and maintaining the pH of the medium at between 6.5 and 7.0. Process according to claim 1, characterized in that the primary carbon source is glucose, starch hydrolyzate, glycerol, / or xylose, nannitol sucrose or a mixture thereof. 4 » - 11 A process according to claim 1, wherein the nitrogen source in the medium is soy flour, cornmeal, cottonseed meal, yeast extract, peanut meal, soluble nitrates or ammonium salts, or mixtures thereof. A process according to claim 2, wherein the nitrogen source in the medium is soy flour, cornmeal, cottonseed meal, yeast extract, peanut meal, soluble nitrates or ammonium salts, or mixtures thereof. The method according to claim 1, characterized in that the cultivation is carried out within 72-144 hours. 6. The method of claim 1, wherein the cultivation is carried out at a temperature of 58 ° to 44 ° C. Method according to claim 1, characterized in that the pH of the medium is maintained between 6.5 and 7.0 after the addition of ammonia. 8. The method of claim 2, wherein the culturing is carried out within 72-144 hours. 9 · Claim 2 »; The method of claim 1, wherein the culture is performed at 38 ° C to -44 ° C. The process according to claim 2, wherein the medium has a pH of between 6.5 and 7.0 after the addition of ammonia.