Mutant microbial .alpha.-amylases with increased thermal, acid a

Details Mutant microbial .alpha.-amylases with increased thermal, acid a Mutant microbial .alpha.-amylases with increased thermal, acid a

1990-11-29

1994-11-15

Furman, Keith C.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

4352523, 435263, 435275, 4353201, 536 232, C12N 928, C12N 1556, C12N 121, D06M 1600

Patent

active

053647823

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to the field of genetic engineering and provides new DNA molecules comprising DNA sequences coding for enzymes with .alpha.-amylase activity. Specifically, mutant microbial .alpha.-amylases are disclosed having improved characteristics for use in the degradation of starch, in the desizing of textile and in other industrial processes. The disclosed .alpha.-amylases show increased thermal, acid and alkaline stability which makes them ideally suited for performing their activity under process conditions which could hitherto not be used.


BACKGROUND OF THE INVENTION

Starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose consists of linear chains of .alpha.-1,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000. Amylopectin is a branched polymer containing .alpha.-1,6 branch points every 24-30 glucose units, its MW may be as high as 100 million.
Sugars from starch, in the form of concentrated dextrose syrups, are currently produced by an enzyme catalyzed process involving: (1) liquefaction (or thinning) of solid starch with an .alpha.-amylase into dextrins having an average degree of polymerization of about 7-10, and (2) saccharification of the resulting liquefied starch (i.e. starch hydrolysate) with amyloglucosidase (also called glucoamylase or AG). The resulting syrup has a high glucose content. Much of the glucose syrup which is commercially produced is subsequently enzymatically isomerized to a dextrose/fructose mixture known as isosyrup.
.alpha.-Amylase (EC 3.2.1.1) hydrolyzes starch, glycogen and related polysaccharides by cleaving internal .alpha.-1,4-glucosidic bonds at random. This enzyme has a number of important commercial applications in, for example the sugar, brewing, alcohol and textile industry. .alpha.-Amylases are isolated from a wide variety of bacterial, fungal, plant and animal sources. The industrially most important .alpha.-amylases are those isolated from Bacilli.
In the first step of the starch degradation process, starch slurry is gelatinized by heating at relatively high temperature (up to 110.degree. C.). The gelatinized starch is liquefied and dextrinized by a thermostable .alpha.-amylase in a continuous two stage process. The major process variables are starch concentration, .alpha.-amylase dose, temperature and pH. During the liquefaction-dextrinization reaction the process variables must be maintained within narrow limits to achieve good conversion ratios, since serious filtration problems may arise otherwise. See, for example, L. E. Coker and K. Venkatasubramanian, in: Biotechnology, p. 165-171, Ed. P. N. Cheremisinoff, P. B. Quellette, Technicom Publ. Corp. Lancaster Renn. 1985. One of the problems which frequently arises is the proper regulation of the temperature in the initial stage of the degradation process: overheating often causes denaturation of the .alpha.-amylase so that the final thinning is not sufficient. One way to avoid this is the use of more thermostable .alpha.-amylases.
To that end it has been proposed to add calcium ions or an amphiphile (see e.g. EP-A-0189838), but this solution appeared to be unsatisfactory.
There is, therefore, still substantial interest to provide .alpha.-amylases with increased thermostability.


Relevant Literature

EP-A-057976 describes the isolation of a thermostable .alpha.-amylase coding gene from B. stearothermophilus the gene is cloned into a plasmid containing either a Bacillus or an E. coli origin of replication. The so obtained chimeric plasmid is used for producing .alpha.-amylase. The .alpha.-amylase gene was isolated and used without any further modification.
EP-A-0134048 describes a method for increased commercial production inter alia of .alpha.-amylase, by cloning and expression of one or more .alpha.-amylase genes in industrial Bacillus strains.
EP-A-252666 describes a chimeric .alpha.-amylase with the general formula Q-R-L in which Q is a N-terminal polypeptide of 55 to 60 amino acid residues which

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