Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Treating animal or plant material or micro-organism
Reexamination Certificate
2002-10-09
2004-09-21
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Treating animal or plant material or micro-organism
C435S068100, C435S071300
Reexamination Certificate
active
06794181
ABSTRACT:
BACKGROUND OF THE INVENTION
Resistance of bacteria to conventional antibiotics used to treat human disease has risen to an international crisis level. A contributing factor has been the widespread use of antibiotics to treat non-life-threatening infections. In recent years, there has been much focus on a promising new class of bacteriocins known as lantibiotics. At present, lantibiotics are being used extensively by the food industry. Lantibiotics have significant commercial value and broad applicability, and practical methods for their production would have a significant economic impact.
Bacteriocins are antimicrobial proteins produced by bacteria that display growth-inhibitory activity against a range of related bacteria. Lantibiotics are polypeptide antimicrobial agents that are produced by certain bacteria and are distinguishable from other antibiotics because of their polypeptide nature and bioactive properties. For example nisin, which is used as a preservative for certain foods, has the unusual amino acid residues, lanthionine and &bgr;-methyl-lanthionine. Nisin, a lysine-rich lantibiotic, is non-toxic to humans and animals, is resistant to high temperatures and is bacteriostatic at very low concentrations. Unfortunately, although lantibiotics are versatile and have unique and advantageous properties, the lack of commercially viable methods for isolation at high purity has limited their utility.
Analysis of the opportunity for the use of nisin by the dairy industry is illustrative of the impact of the lack of cost-effective purification methods. Recently, the potential value of nisin for the milk industry has been recognized, in particular, in connection with the ability of nisin to help fight mastitis infection in cows. The advantage offered by nisin stems, in large part, from its potential to reduce or eliminate “withhold period” rules. The withhold period is a time established, during treatment of mastitis infection of cows, when milk from the infected cow must be discarded. Thus, milk from cow treated for mastitis infection with nisin may enter the fluid milk stream much sooner than traditional antibiotic treatment.
Unfortunately, nisin that is commercially produced by currently available methods of production and purification is considered food grade quality and is not of sufficient purity for pharmaceutical applications. It contains peptide impurities that cause inflammatory reactions when administered to cows. Therefore, due to the lack of efficient alternative methods of purification which could selectively remove such impurities, the value derived from treatment of mastitis with nisin is insufficient to counterbalance current practices. Therefore, a method is needed that improves lantibiotic purity and that is practical to employ on a commercial scale.
To be commercially practical, a purification scheme must be relatively high yield and low cost. With regard to lantibiotic production, it is relatively simple, inexpensive, and routine to culture an organism that either naturally expresses, or has been engineered to express, the desired bacteriocin. Invariably, however, the lantibiotic must be separated from the myriad of proteins that are co-expressed by the organism and that represent contaminating impurities in the initial preparation. Because lantibiotics are polypeptides, and hence share biochemical features with other proteins, the challenge has been in designing a practical protocol that is able to discriminate between the lantibiotic and the other protein or polypeptide impurities.
With few exceptions, the use of proteases or enzymes having protease-like activity is avoided in protein purification protocols in light of the fact that the protein to be purified is typically sensitive to such treatment. For example, trypsin can not be used in a method for purifying a peptide that contains internal lysine or arginine residues because trypsin recognizes such residues and cleaves a protein or polypeptide containing such residues at their location. Moreover, purity and recovery of a protein, using a protease-based purification scheme, can be inversely related. This means that, under conditions that favor increased recovery of the protein of interest, impurities tend to remain insufficiently digested.
SUMMARY OF THE INVENTION
The present invention relates to a method for purifying a lantibiotic from a crude or partially purified solution containing the lantibiotic. In preferred embodiments, the lantibiotic is nisin, although the common structural features of lantibiotics govern the effectiveness of the disclosed purification methods for other members of the lantibiotic genus. The method includes the step of forming an incubation mixture comprising the solution containing the lantibiotic and a proteolytic enzyme, and incubating the mixture under conditions which are optimized for the selective proteolytic activity of the enzyme, thereby digesting non-lantibiotic protein, polypeptide and peptide components of the solution containing the lantibiotic, while leaving the lantibiotic substantially undigested.
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Coughlin Richard T.
Crabb Joseph H.
Atwood Pierce
Beisner William H.
Farrell Kevin M.
ImmuCell Corporation
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