Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1990-05-29
1992-09-01
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435832, 25217412, C12N 954, C12N 100, C11D 1700
Patent
active
051438402
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel proteolytic enzyme, to the preparation thereof and to the use thereof as a detergent additive.
Proteolytic enzymes of high proteolytic activity in the alkaline range are already known; cf. U.S. Pat. No. 3,674,643, U.S. Pat. No. 4,480,037 and NL-A-72/07,050. These enzymes, however, are not particularly suitable for low-temperature detergents.
The present invention provides a proteolytic enzyme which is isolable from a Bacillus spec. which was accepted for deposit on Sep. 15, 1988, as deposit DSM 4828, at the International Depositary Authority, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-3300 Braunschweig, Germany, as established under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The proteolytic enzyme has an amino acid composition of Asx 12.1%, Thr 4.8%, Ser 7.9%, Glu 6.9%, Pro 5.2%, Gly 12.9%, Ala 12.0%, Cys 0.3%, Val 7.9%, Met 3.2%, Ile 4.2%, Leu 6.5%, Tyr 4.9%, Phe 1.4%, His 3.6%, Lys 1.9% and Arg 4.0% and an amino-terminal amino acid sequence of Gln-Thr-Val-Pro-Cys-Gly-Ile-Pro-Tyr-Ile-Tyr-Ser-Asp-Val-Val-His-Arg-Gln-Gl y-Tyr-Phe-Gly-Asn-Gly-Val, whose molecular weight is about 27,000 and whose isoelectric point is at pH 8.5.
The amino acid composition was determined by total hydrolysis of the protein by the Moore and Stein method. The molecular weight was determined by SDS gel electrophoresis.
The novel enzyme is preparable by growing the microorganism Bacillus spec. DSM 4828 in a nutrient medium and isolating the enzyme from the culture broth.
The choice of nutrient medium for growing the microorganism is not critical. It is possible to use nutrient media which contain carbon sources, nitrogen sources, inorganic salts and possibly small amounts of trace elements and vitamins. The nitrogen sources used can be inorganic or organic nitrogen compounds or materials which contain these compounds. Examples are: ammonium salts, nitrates, corn steep liquor, yeast autolyzate, soybean flour, yeast extract and potato starch. The carbon sources used can be sugars, such as glucose, sucrose or .alpha.-amylase hydrolyzed starch, polyols such as glycerol or else organic acids such as acetic acid or citric acid. Examples of inorganic salts are the salts of calcium, magnesium, manganese, potassium, zinc, copper, iron and other metals Suitable anions for the salts are in particular phosphate and carbonate ions. The mixing ratio of the nutrients mentioned depends on the nature of the fermentation and is decided from case to case. The growth conditions are optimized in respect of yield. Preferred growth temperatures are 32.degree.-40.degree. C. The pH of the medium is maintained at 7-10.5, preferably 8-10. In general, an incubation of 30-50 h is sufficient. In this time, the maximum amount of the desired product builds up in the medium.
The enzyme is separated from the culture broth in a conventional manner. The broth is centrifuged or filtered to separate off the microorganisms and insoluble material. To prepare solid enzyme products, the enzyme is precipitated for example by the addition of water-miscible organic solvents or inorganic salts such as sodium sulfate or ammonium sulfate to the filtrate. After precipitation, the enzyme is separated off by filtration or centrifuging. The filter residue obtained is then dried.
The growth of Bacillus spec. DSM 4828 succeeds under aerobic conditions.
The novel enzyme is very highly suitable for the manufacture of detergents for use at 20.degree.-40.degree. C. In addition, it is very stable, for example in the presence of bleaching agents, surfactants and chelating agents.
EXAMPLE 1
Isolation of Microorganism
The microorganism which produces the alkaline protease was isolated from a soil sample by a method for selecting alcalophilic bacilli (K. Horikoshi, T. Akiba/Alcalophilic Microorganisms/Japan Scientific Societies Press Tokyo). The bacterial strain DSM 4828 grows on media which contain either 0.1 M of pH 9-10 carbo
REFERENCES:
patent: 3674643 (1972-07-01), Aunstrup et al.
patent: 4480037 (1984-10-01), Ichishima et al.
Diessel Paul
Kreimeyer Andreas
Perner Johannes
Rettenmaier Hansjoerg
BASF - Aktiengesellschaft
Meller Mike
Robinson Douglas W.
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