Mutant .alpha.-amylase

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

Patent

Rate now

  [ 0.00 ] – not rated yet Voters 0   Comments 0

Details

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

REFERENCES:
patent: Re34606 (1994-05-01), Estell et al.
patent: 4261868 (1981-04-01), Hora et al.
patent: 4284722 (1981-08-01), Tamuri et al.
patent: 4493893 (1985-01-01), Mielenz et al.
patent: 4620936 (1986-11-01), Kielman et al.
patent: 4634551 (1987-01-01), Burns et al.
patent: 4732973 (1988-03-01), Barr et al.
patent: 4752585 (1988-06-01), Koths et al.
patent: 4760025 (1988-07-01), Estell et al.
patent: 4863626 (1989-09-01), Coyne et al.
patent: 5118623 (1992-06-01), Boguslawski et al.
patent: 5322778 (1994-06-01), Antrim et al.
patent: 5346823 (1994-09-01), Estell et al.
patent: 5364782 (1994-11-01), Quax et al.
patent: 5736499 (1998-04-01), Mitchinson et al.
patent: 5763385 (1998-06-01), Bott et al.
Joyet et al. (Dec. 1992 Hyperthermostable variants of a highly thermostable alpha-amylase. Biotechnology 10: 1579-1583.
Declerck et al. (Jun. 1995) Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: multiple amino acid replacement and molecular modeling. Protein Engineering 8(10): 1029-1037.
Declerck et al. (Jun. 1990) Use of amber suppressors to investigate the thermostability of Bacillus licheniformis alpha-amylase. J. Biol. Chem. 265(26): 15481-15488.
Ngo et al. (Jun. 1994) Computational Complexity, Protein Structure Prediction, and the Levinthal Paradox. In: The Protein Folding Problem and Tertiary Structure Prediction. Eds. Merz et al. Birkhauser. Boston, MA. pp. 491-495.
Rudinger (Jun. 1976) Characteristics of the amino acids as components of a peptide hormone sequence. In: Peptide Hormones. Ed. J. A. Parsons. University Park Press. Baltimore, MD. pp. 1-7, Jun. 1976.
Bealin-Kelly et al., "Studies on the Thermostability of the .alpha.-amylase of Bacillus caldovelox," Appl Microbiol Biotechnol (1991) 36:332-336.
Bierbaum et al., "Production of protease with Bacillus licheniformis mutants insensitive to repression of exoenzyme biosynthesis," Appl Microbiol Biotechnol (1994) 40:611-617.
Boel et al., "Calcium Binding in .alpha.-Amylases: An X-ray Diffraction Study at 2.1-.ANG. Resolution of Two Enzymes from Aspergillus," Biochemistry (1990) 29:6244-6249.
Bott et al., "the Three-dimensional Structure of Bacillus amyloliquefaciens Subtilisin at 1.8 .ANG. and an Analysis of the Structural Consequences of Peroxide Inactivation," J Biol Chem (1988) 263:7895-7906.
Brady et al., "Solution of the Structure of Aspergillus niger Acid .alpha.-Amylase by Combined Molecular Replacement and Multiple Isomorphous Replacement Methods," Acta Cryst (1991) 47:527-535.
Brayer et al., "The structure of human pancreatic .alpha.-amylase at 1.8 .ANG. resolution and comparisons with related enzymes," Prot Science (1995) 4:1730-1742.
Brosnan et al. "Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus .alpha.-amylase," Eur J Biochem (1992) 203:225-231.
Burley et al., Aromatic-Aromatic Interaction: A Mechanism of Protein Structure Stabilization,: Science (1985) 229:23-28.
Byrne et al., "Energetic Contribution of side Chain Hydrogen Bonding to the Stability of Staphylococcal Nuclease," Biochemistry (1995) 34:13949-13960.
Chang et al., "Cyrstallization and Preliminary X-ray Crystallographic Analysis of .alpha.-Amylase from Bacillus subtilis," J Mol Biol (1993) 229:235-238.
Chen et al., "Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamore glucoamylase," Protein Engineering (1995) 8:575-582.
Clarke et al., "Engineered Disulfide Bonds as Probes of the Folding Pathway of Barnase: Increasing the Stability of Proteins against the Rate of Denaturation," Biochemistry (1993) 32:4322-4329.
Dao-pin et al., "Contributions of Engineered Surface Salt Bridges to the Stability of T4 Lysozyme Determined by Directed Mutagenesis," Biochemistry (1991) 30:7142-7153.
Delboni et al., "Crystal structure of recombinant triosephosphate isomerase from Bicillus stearothermophils. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures poin

LandOfFree

Say what you really think

Search LandOfFree.com for the USA inventors and patents. Rate them and share your experience with other people.

Rating

Mutant .alpha.-amylase does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Mutant .alpha.-amylase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant .alpha.-amylase will most certainly appreciate the feedback.

Rate now

     

Profile ID: LFUS-PAI-O-702308

  Search
All data on this website is collected from public sources. Our data reflects the most accurate information available at the time of publication.