Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-09-29
2000-08-08
Prats, Francisco
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 96, 435105, 435202, 435203, 435204, C12P 1914, C12P 1920, C12N 932, C12N 930
Patent
active
061000736
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to novel acid-stable and thermo-stable enzymes having an .alpha.-1,4 hydrolytic activity and which are derived from strains of the genus Sulfolobus, and in particular strains of the species Sulfolobus acidocaldarius, and the use of these novel enzymes in starch degradation.
.alpha.-amylases (E.C. 3.2.1.1) are hydrolyzing enzymes having .alpha.-1,4 hydrolytic activity in starch, amylopectin and starch. These enzymes are utilized for a wide variety of industrial applications. Such industrial applications can require that the .alpha.-amylase used be highly acid-stable and/or highly thermo-stable.
An important industrial application for such acid-stable and thermo-stable enzymes having such .alpha.-1,4 hydrolytic activity (such as .alpha.-amylases) is the enzymatic degradation (hydrolysis) of starch for the production of sugars, such as glucose. Because starch is composed of glucose units joined by both .alpha.-1,4 and .alpha.-1,6 [inkages, to complete the hydrolysis thereof, the use of a number of enzymes with differing substrate specificities are required. This enzymatic degradation process involves two enzymatic steps: liquefaction and saccharification.
In liquefaction, typically starch granules are slurried in water in the presence of an .alpha.-amylase. This slurry is naturally acidic, able to have a pH of about 4.0. The slurried granules/.alpha.-amylase are then gelatinized with heat by passage through a jet cooker, which rapidly raises the temperature to about 105.degree. C.-110.degree. C. After a few minutes, the temperature of the slurry is then decreased to 90.degree. C.-95.degree. C. and held at that temperature for at least one hour.
The .alpha.-amylases conventionally-used in liquefaction are those derived from B. licheniformis and B. stearothermophilus. These .alpha.-amylases hydrolyze the starch and solubilize the dextrins, producing low viscosity hydrolysates suitable for further processing (in saccharification) to sugar syrups, such as glucose syrups. Unfortunately, the use of those .alpha.-amylases requires that two major adjustments be made in liquefaction.
The first adjustment necessary to permit use of the conventional .alpha.-amylases in liquefaction is to raise the pH of the naturally-acidic pH of the starch slurry from about 4.0 to between about 5.5 and about 6.5. Such an adjustment is necessary to permit the maximum enzymatic activity of the .alpha.-amylase to be expressed (indeed, the pH optimum of the .alpha.-amylase from B. licheniformis is about 6.0). Such an adjustment is also necessary for the enzyme since, in liquefaction conditions, that enzyme is relatively unstable in pHs lower than 6.
However, increasing the pH of the starch slurry in liquefaction has drawbacks. These drawbacks include an increase of color formation, the risk of generating filtration problems and the spontaneous formation of maltulose which, because it cannot be hydrolysed to dextrose in (subsequent) saccharification, leads to a loss in yield. Thus, while performing liquefaction at the higher pH is generally considered as advantageous for the .alpha.-amylase, it is nonetheless considered disadvantageous for the process.
The second adjustment necessary to permit use of the conventional .alpha.-amylases in liquefaction is the use of calcium ions to stabilize the enzyme. Indeed, the .alpha.-amnylase of B. licheniformis requires that at least fifty (50) ppm (parts per million) calcium be used for stabilization thereof in liquefaction conditions. As the calcium ion concentration is increased, the stability of the .alpha.-amylase is increased, thereby permitting the liquefaction medium to have a lower pH. Unfortunately, however, the presence of these calcium ions interferes with the refining step in the production of high fructose syrups. A low concentration of calcium ions in liquefaction is therefore preferred.
A further drawback presented by the use of the conventional .alpha.-amylases is that, at temperatures higher than about 110.degree. C., in liquefaction conditions, the conven
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Amory Antione
Deweer Philippe
Genencor International Inc.
Prats Francisco
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