Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1989-09-25
1994-01-11
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
536 232, 435198, 4353201, C12N 121, C12N 1555
Patent
active
052780660
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to the preparation, by recombinant DNA technology, of enzymes for the enzymatic degradation of fatty materials, specifically lipolytic enzymes which have characteristics which make them suitable for use as detergent additives.
BACKGROUND
A special problem associated with laundry cleaning is the removal of stains of a fatty nature. Currently, fat-containing dirt is emulsified and removed using a combination of elevated temperature and high alkalinity. However, there is a recent strong tendency toward the use of relatively low washing temperatures, namely about 40.degree. C. or lower, conditions particularly unsuited for fatty stain removal. There is therefore a need for detergent additives which are effective at the lower washing temperatures, stable in high alkaline detergent solutions and stable under storing conditions in both solid and liquid detergent compositions. A group of enzymes which hydrolize triglycerides are lipases (E.C. 3.1.1.3). Lipases are widely distributed, occurring in many different prokaryotic organisms, as well as eukaryotic cells. Depending upon the source of the enzyme, substrate specificity as well as other characteristics including stability under various conditions, varies widely. Lipases have been used in detergent compositions, however those used exhibited low cleaning efficiency under washing conditions and in addition, did not meet the stability requirements for detergent use.
Lipolytic enzymes which are capable of exhibiting lipase activity under modern washing conditions, i.e., they are stable and effective at high detergent concentrations, at high pH and at low washing temperatures, are produced by certain strains belonging to the species of Pseudomonas pseudoalcaligenes, Pseudomonas stutzeri and Acinetobacter calcoaceticus (see European Patent Application EP-A-0218272). However, these species are potentially pathogenic for plants and animals, and few data are available on the fermentation conditions required for an effective production process for lipases using these microorganisms.
It is therefore desirable to develop an efficient and safe way to produce lipolytic enzymes having the desired characteristics for detergent additives. Moreover, it is desirable that the lipases be secreted by the host organism so that the enzyme may be recovered directly from the extracellular fluid of the fermentation mixture.
RELEVANT LITERATURE
For lipases produced by Pseudomonas species it is known that the culture conditions strongly influence the final localization of these enzymes (Sugiura, In: "Lipases", eds. B. Bergstrom and H. L. Brockman (1984) pp. 505-523, Elsevier, Amsterdam). Problems are often encountered in obtaining efficient expression of heterologous genes in microorganisms, including incorrect folding of the proteins formed, protein degradation and improper localization of the products. Harris, In: "Genetic Engineering", Vol. 4 (1983), Academic Press, New York. In E. coli the use of secretion-cloning vectors generally enables the transport of heterologous gene products into the periplasmic space and products are only occasionally found in the culture medium; Lunn et al., Current Topics in Microbiol. and Immunol. 125 (1986) 59-74. When using E. coli as the host cell, cloned microbial lipases, described by Gotz et al., Nucleic Acids Res. 13 (1985) 5895-5906, Kugimiya et al., Biochem. Biophys. Res. Commun. 141 (1986) 185-190 and Odera et al., J. Ferment. Technol. 64 (1986) 363-371, are poorly secreted into the culture medium.
Wohlfarth and Winkler, J. Gen. Microbiol. 134 (1988) 433-440, report on the physiological characterization of newly isolated lipase-deficient mutants from Pseudomonas aeruginosa strain PAO 2302 and on the chromosomal mapping and cloning of the corresponding gene.
Bacillus species, in particular Bacillus subtilis strains have been used with varying degrees of success as host strains for the expression of both foreign and endogenous genes and for the secretion of the encoded protein products. For a review, see
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Andreoli Peter M.
Cox Maria M. J.
Farin Farrokh
Gist-Brocades NV
Mosher Mary E.
Rae-Venter Barbara
Schwartz Richard A.
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