Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1999-02-26
2001-04-10
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S222000, C435S220000, C435S221000
Reexamination Certificate
active
06214576
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to bacterial strains useful for degrading keratin. More particularly, the invention relates to isolating from wild birds bacterial strains capable of degrading feathers. The invention also relates to the keratin-degrading enzymes produced by these bacterial strains. Another aspect of the invention is processes in which the bacterial strains and enzymes can be used, such as the disposal of waste feathers and dead birds; the production of animal feed, natural gas, or fertilizer from poultry waste; and the cleaning of fabrics.
BACKGROUND OF THE INVENTION
Keratins are a major class of structural proteins that are highly resistant to biological degradation. Common enzymes which break down protein, such as trypsin, do not affect keratin. Keratins are insoluble in water. Keratins, like other proteins, are made of a long string of various amino acids, which fold into a final 3-dimensional form. Alpha helix and beta sheet are common parts of such a 3-dimensional form. Long, thin biological structures often contain alpha helixes, while flat structures are often built from beta sheets. Some keratins are rich in alpha helix structures, while others are mostly beta sheet. These two major types of keratin are known as alpha-keratin and beta-keratin. Wool and hair are largely composed of alpha-keratin. Feathers are largely composed of beta-keratin.
The poultry processing industry produces huge quantities of turkey and chicken feathers as a by-product. These feathers constitute a sizable waste disposal problem. Several different approaches have been used for disposing of feather waste, including landfilling, burning, natural gas production, and treatment for animal feed. Most feather waste in the United States is landfilled or burned, which involves expense and can cause contamination of air, soil and water.
Natural gas production from waste materials is rarely used in the U.S., due to the low price of U.S. natural gas and the availability of land for landfilling. Thermophilic anaerobic poultry waste digestors are known which can produce significant amounts of natural gas from poultry manure and other waste. Bacteria are combined in these digestors with the poultry waste, fermented, and the natural gas produced is collected. The effluent remaining after fermentation can be used as animal feed or fertilizer.
Animal feed typically includes a carbohydrate source and a protein source. The protein source provides needed amino acids to the animal. Common protein sources used in animal feed include soy meal; fish meal; blood meal; meat or poultry by-products; and meat and poultry meal. These protein sources are generally expensive. Feather waste is high in protein and very inexpensive, but cannot be used directly in animal feed, as it is difficult for animals to digest. A small percentage of feather waste is steamed, chemically treated, ground, or some combination of these to form feather meal. Feather meal is used as a dietary protein supplement for animals. Typical treatments to form feather meal are expensive. These treatments also tend to destroy some amino acids, such as heat-sensitive amino acids when steam treatment is used. This lowers the quality of the protein in the feed. Due to these problems, feather meal is not extensively used in feed, despite the expense of other sources of dietary protein.
Bacterial strains are known which are capable of degrading feathers. These bacterial strains produce enzymes which selectively degrade the beta-keratin found in feathers. These enzymes make it possible for the bacteria to obtain carbon, sulfur, and energy for their growth and maintenance from the degradation of beta-keratin. An enzyme capable of degrading protein is known as a protease, and is described as having proteolytic activity. An enzyme which degrades keratin is a keratinase, while a beta-keratinase is an enzyme capable of degrading beta-keratin. An enzyme which degrades keratin can also be described as having keratinolytic activity. The same enzyme can be referred to as a protease, a keratinase, and a beta-keratinase if it has all three properties.
Prior feather-degrading bacterial strains, such as
Bacillus licheniformis
PWD-1 (ATCC 53757), have been isolated from chicken waste and poultry waste digestors. See generally Williams, C. M. et al, Isolation, Identification, and Characterization of a Feather-Degrading Bacterium,
Applied and Environmental Microbiology
, Vol. 56, No. 6, June 1990, pp. 1509-1515. Specific primers, probes, and polymerase chain reaction (PCR) techniques for the keratinase gene of
B. licheniformis
PWD-1 are known. Keratinolytic microorganisms have been found in soil and agricultural compost. U.S. Pat. Nos. 5,063,161 and 4,959,311 describe methods of degrading keratinaceous material and bacteria useful for such degradation. U.S. Pat. No. 5,171,682 describes a purified
B. licheniformis
PWD-1 keratinase capable of degrading feathers. U.S. Pat. No. 5,186,961 describes a method and composition for maintaining animals on a keratin-containing diet. U.S. Pat. No. 4,908,220 describes a hydrolyzed feather feed ingredient (feather-lysate) and animal feeds containing feather-lysate.
Large numbers of dead, unsaleable birds are another waste product produced by commercial poultry processing. Since the feathers are resistant to degradation, the feathers often remain after the soft parts of a dead bird have decomposed. Other wastes from poultry processing, such as poultry manure, often contain significant amounts of difficult-to-degrade feathers. The feathers in these waste products make disposal more difficult and expensive.
Proteins make up many common stains that are found on clothing. When the proteins involved are insoluble in water, the stain is often difficult to remove. Blood stains are an example. Addition of enzymes capable of degrading proteins to detergents for cleaning clothes is well known in the art, U.S. Pat. No. 5,079,154. Since keratin is a protein which is insoluble in water, enzymes with keratin-degrading activity are known to be effective detergent additives.
B. licheniformis
is a rod-shaped, endospore-forming bacteria. The rods are often in chains. Colonies on agar are opaque with dull to rough surface. Wrinkled outgrowths are common. The colonies are usually attached strongly to the agar, and mounds and lobes consisting largely of slime often accumulate on the colony, especially on glucose agar or glutamate-glyceral agar. Glutamyl polypeptide is formed as an extracellular amorphous slime. Levan is produced extracellularly from sucrose and raffinose. Red pigment is formed by many strains on carbohydrate media containing sufficient iron Aged cultures may become brown. Freshly isolated strains grow with ammonia as the sole source of nitrogen in the absence of growth factors. Spores are known to occur in soil. Many spores survive severe heat treatment. Refer generally to
Bergey's Manual of Systematic Bacteriology
, Vol. 2, Section 13, P.H.A. Sneath, ed., Williams & Wilkins, L.A.
SUMMARY OF THE INVENTION
The present invention includes three methods of screening a wild bird for the presence of feather-degrading bacteria. The first method involves as a first step capturing a wild bird and rubbing the feathers of the bird with a sterile applicator dipped in a sterile saline solution. Next, the applicator is placed in a nutrient broth of sodium chloride content ranging from 7.0% to 8.0% weight/volume and a pH ranging from 7.0 to 7.7. Preferably the nutrient broth contains 7.5% weight/volume sodium chloride and is of a pH ranging from 7.5 to 7.7. Next the broth is incubated at a temperature between 45° C. and 60° C. for a period of 2 to 14 days. Preferably the broth is incubated for 7 days at a temperature ranging from 50° C. to 55° C. Next, any surviving bacteria are grown on trypticase soy agar until colonies are formed, and bacteria from an individual colony are transferred to a physiologically balanced solution containing feather. The solution is incubated at a temperature between 35° C. a
Burtt, Jr. Edward H.
Ichida Jann M.
Angres Issac A.
Marx Irene
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