Cleaning compositions for solid surfaces – auxiliary compositions – Cleaning compositions or processes of preparing – Enzyme component of specific activity or source
Reexamination Certificate
1997-12-09
2001-04-03
Fries, Kery (Department: 1751)
Cleaning compositions for solid surfaces, auxiliary compositions
Cleaning compositions or processes of preparing
Enzyme component of specific activity or source
C510S321000, C510S330000, C510S226000
Reexamination Certificate
active
06211134
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to &agr;-amylases having altered performance characteristics. The present invention is also directed to novel mutant &agr;-amylase enzymes having a mutation, wherein the resultant &agr;-amylase exhibits improved specific activity and starch hydrolysis performance.
BACKGROUND OF THE INVENTION
&agr;-Amylases (&agr;-1,4-glucan4-glucanohydrolase, EC 3.2.1.1) hydrolyze internal &agr;-1,4-glucosidic linkages in starch, largely at random, to produce smaller molecular weight malto-dextrins. &agr;-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. &agr;-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° 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 &agr;-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 &agr;-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 &agr;-amylases against inactivation. Upon addition of &agr;-amylases, the suspension is pumped through a steam jet to instantaneously raise the temperature to between 80-115° C. The starch is immediately gelatinized and, due to the presence of &agr;-amylases, depolymerized through random hydrolysis of &agr;(1-4) glycosidic bonds to a fluid mass which is easily pumped.
In a second variation to the liquefaction process, &agr;-amylase is added to the starch suspension, the suspension is held at a temperature of 80-100° 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° C. to thoroughly gelatinize any remaining granular structure. After cooling the gelatinized starch, a second addition of &agr;-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 &agr;-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 than high temperature liquefaction in converting starch to soluble dextrins.
Typically, after gelatinization the starch solution is held at an elevated temperature in the presence of &agr;-amylase until a DE of 10-20 is achieved, usually a period of 1-3 hours. Dextrose equivalent (DE) is the industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE of virtually zero, whereas the DE of D-glucose is defined as 100.
The maximum temperature at which the starch solution containing &agr;-amylase can be held depends upon the microbial source from which the enzyme was obtained and the molecular structure of the &agr;-amylase molecule. &agr;-Amylases produced by wild type strains of
Bacillus subtilis
or
Bacillus amyloliquefaciens
are typically used at temperatures no greater than about 90° C. due to excessively rapid thermal inactivation above that temperature, whereas &agr;-amylases produced by wild type strains of
Bacillus licheniformis
can be used at temperatures up to about 110° C. The presence of starch and calcium ion are known to stabilize &agr;-amylases against inactivation. Nonetheless, &agr;-amylases are used at pH values above 6 to protect against rapid inactivation. At low temperatures, &agr;-amylase from
Bacillus licheniformis
is known to display hydrolyzing activity on starch substrate at pH values as low as 5. However, when the enzyme is used for starch hydrolysis at common jet temperatures, e.g., between 102° C. and 109° C., the pH must be maintained above at least pH 5.7 to avoid excessively rapid inactivation. The pH requirement unfortunately provides a narrow window of processing opportunity because pH values above 6.0 result in undesirable by-products, e.g., maltulose. Therefore, in reality, liquefaction pH is generally maintained between 5.9 and 6.0 to attain a satisfactory yield of hydrolyzed starch.
Another problem relating to pH of liquefaction is the need to raise the pH of the starch suspension from about 4, the pH of a corn starch suspension as it comes from the wet milling stage, to 5.9-6.0. This pH adjustment requires the costly addition of acid neutralizing chemicals and also requires additional ion-exchange refining of the final starch conversion product to remove the chemical. Moreover, the next process step after liquefaction, typically saccharification of the liquefied starch into glucose with glucoamylase, requires a pH of 4-4.5; therefore, the pH must be adjusted down from 5.0-6.0 to 4-4.5; requiring additional chemical addition and refining steps.
Subsequent to liquefaction, the processed starch is saccharified to glucose with glucoamylase. A problem with present processes occurs when residual starch is present in the saccharification mixture due to an incomplete liquefaction of the starch, e.g., inefficient amylose hydrolysis by amylase. Residual starch is highly resistant to glucoamylase hydrolysis. It represents a yield loss and interferes with downstream filtration of the syrups.
Additionally, many &agr;-amylases are known to require the addition of calcium ion for stability. This further increases the cost of liquefaction.
In U.S. Pat. No. 5,322,778, liquefaction between pH 4.0 and 6.0 was achieved by adding an antioxidant such as bisulfite or a salt thereof, ascorbic acid or a salt thereof, erythorbic acid, or phenolic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, or &agr;-tocopherol to the liquefaction slurry. According to this patent, sodium bisulfite must be added in a concentration of greater than 5 mM.
In U.S. Pat. No. 5,180,669, liquefaction between a pH of 5.0 to 6.0 was achieved by the addition of carbonate ion in excess of the amount needed to buffer the solution to the ground starch slurry. Due to an increased pH effect which occurs with addition of carbonate ion, the slurry is generally neutralized by adding a source of hydrogen ion, for example, an inorganic acid such as hydrochloric acid or sulfuric acid.
In PCT Publication No. WO 94/02597, a mutant &agr;-amylase having improved oxidative stability is described wherein one or more methionines are replaced b
Caldwell Robert M.
Mitchinson Colin
Ropp Traci H
Fries Kery
Genecor International, Inc.
Stone Christopher L.
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