Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-02-20
1999-09-28
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435201, 435202, 435203, 435204, 4352523, 43525231, 43525411, 4353201, 435325, 435410, 510226, 510300, 510305, 510320, 510374, 510392, 536 232, C12P 1914, C12N 926, C12N 928, C07H 2104
Patent
active
059587393
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention is directed to .alpha.-amylases having altered performance characteristics. The present invention is also directed to novel mutant .alpha.-amylase enzymes having at least an asparagine residue which is substituted with a different amino acid or deleted, wherein the resultant .alpha.-amylase exhibits altered low pH starch hydrolysis performance, altered stability and altered activity profiles.
BACKGROUND OF THE INVENTION
.alpha.-Amylases (.alpha.-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) hydrolyze internal .alpha.-1,4-glucosidic linkages in starch, largely at random, to produce smaller molecular weight malto-dextrins. .alpha.-Amylases are of considerable commercial value, being used in the initial stages (liquefaction) of starch processing; in alcohol production; as cleaning agents in detergent matrices; and in the textile industry for starch desizing. .alpha.-Amylases are produced by a wide variety of microorganisms including Bacillus and Aspergillus, with most commercial amylases being produced from bacterial sources such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, or Bacillus stearothermophilus. In recent years, the preferred enzymes in commercial use have been those from Bacillus licheniformis because of their heat stability and performance, at least at neutral and mildly alkaline pH's.
In general, starch to fructose processing consists of four steps: liquefaction of granular starch, saccharification of the liquefied starch into dextrose, purification, and isomerization to fructose. The object of a starch liquefaction process is to convert a concentrated suspension of starch polymer granules into a solution of soluble shorter chain length dextrins of low viscosity. This step is essential for convenient handling with standard equipment and for efficient conversion to glucose or other sugars. To liquefy granular starch, it is necessary to gelatinize the granules by raising the temperature of the granular starch to over about 72.degree. C. The heating process instantaneously disrupts the insoluble starch granules to produce a water soluble starch solution. The solubilized starch solution is then liquefied by .alpha.-amylase (EC 3.2.1.1.).
A common enzymatic liquefaction process involves adjusting the pH of a granular starch slurry to between 6.0 and 6.5, the pH optimum of .alpha.-amylase derived from Bacillus licheniformis, with the addition of calcium hydroxide, sodium hydroxide or sodium carbonate. The addition of calcium hydroxide has the advantage of also providing calcium ions which are known to stabilize the .alpha.-amylases against inactivation. Upon addition of .alpha.-amylases, the suspension is pumped through a steam jet to instantaneously raise the temperature to between 80-115.degree. C. The starch is immediately gelatinized and, due to the presence of .alpha.-amylases, depolymerized through random hydrolysis of .alpha.(1-4) glycosidic bonds to a fluid mass which is easily pumped.
In a second variation to the liquefaction process, .alpha.-amylase is added to the starch suspension, the suspension is held at a temperature of 80-100.degree. C. to partially hydrolyze the starch granules, and the partially hydrolyzed starch suspension is pumped through a jet at temperatures in excess of about 105.degree. C. to thoroughly gelatinize any remaining granular structure. After cooling the gelatinized starch, a second addition of .alpha.-amylase can be made to further hydrolyze the starch.
A third variation of this process is called the dry milling process. In dry milling, whole grain is ground and combined with water. The germ is optionally removed by flotation separation or equivalent techniques. The resulting mixture, which contains starch, fiber, protein and other components of the grain, is liquefied using .alpha.-amylase. The general practice in the art is to undertake enzymatic liquefaction at a lower temperature when using the dry milling process. Generally, low temperature liquefaction is believed to be less efficient
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Day Anthony
Mitchinson Colin
Requadt Carol
Ringer Christopher
Ropp Traci
Genencor International Inc.
Stole Einar
Stone Christopher L.
Wax Robert A.
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