Title of Invention	METHOD OF PURIFYING LANTIBIOTICS
Abstract	Disclosed is 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 dictate 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 optimized for selective proteolytic activity.

Title of Invention

METHOD OF PURIFYING LANTIBIOTICS

Abstract

Disclosed is 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 dictate 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 optimized for selective proteolytic activity.

Full Text	WO 2004/033704 PCT/US2003/031986 METHOD OF PURIFYING LANTIBIOTICS Background of the Invention [0001] 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. [0002] 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 (3-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. [0003] 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. [0004] 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. WO 2004/033704 PCT/US2003/031986 -2- Therefore, a method is needed that improves lantibiotic purity and that is practical to employ on a commercial scale. [0005] 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. [0006] 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 [0007] 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. WO 2004/033704 PCT/US2003/031986 -3- Detailed Description of the Invention [0008] Lantibiotics are a group of ribosomally synthesized, post-translationally modified peptides containing unusual amino acids. Such amino acids include the thioether amino acids lanthionine (Lan) and/or MeLan, in addition to a number of modified residues, such as 2,3-didehydroalanine (Dha) and 2,3-didehydrobutyrine (Dhb). The presence and influence of these residues on the structure and activity of lantibiotics has been the subject of significant research efforts. [0009] It has been observed, for example, that the sequence-specific dehydration of serine (to Dha) and threonine (to Dhb) results in modified amino acids with electrophilic centers which can react with neighboring nucleophilic groups. The thioether lanthionine is formed when the double bond in Dha is attacked by the thiol (SH) group of a neighboring cysteine residue. As a consequence of the presence of these intramolecular bridges, lantibiotics are polycyclic structures containing a number of lanthionine rings. The presence - of these lanthionine rings is thought to be essential for a number of important lantibiotic properties including, for example, maintenance of peptide rigidity and resistance to thermal inactivation. [0010] The present invention is based on Applicants' surprising discovery that a crude or partially purified preparation of a lantibiotic can be subjected to protease treatment under conditions which result in selective proteolytic activity against protein or polypeptide impurities, without measurable proteolysis of the lantibiotic. The use of such conditions represents an effective method for the purification of a lantibiotic. [0011 ] The prior art teaching which relates to the sensitivity of lantibiotics to protease digestion is mixed. Many of these reports relate specifically to the well-studied lantibiotic, nisln. For example, Gross et al. (J. Am. Chem. Soc. 93(18) (1971) 4634-4635) reported that nisin was trypsin-sensitive. A contradictory report was later published by Wilimowska-Pelc (Acts Microbiol. Pol A 8(1) (1976) 71-77). More recently, Chan et al. (Int. Food Microbiol. 390 (2001) 267-281) reported that nisin is subject to tryptic digestion, but at a reduced rate. In summary, one of skill in the art, familiar with relevant teaching of the prior art, could not have predicted with any degree of certainty that it would be possible to identify conditions under which non-iantibiotic protein, polypeptide and peptide impurities present in a crude or partially purified lantibiotic containing solution could be selectively degraded by the action of a proteolytic enzyme, without substantially degrading the lantibiotic present in the solution. WO 2004/033704 PCT/US2003/031986 -4- This discovery is particularly important given the long-felt need for an improved, cost- effective, high purity method for lantibiotic purification which yields a lantibiotic preparation suitable for pharmaceutical use. [0012] The conditions for protease digestion determined to be effective in connection with the present invention can be characterized as selective. That is, the conditions established for the incubation that includes the impure lantibiotic and a proteoiytic enzyme are conditions under which the proteoiytic enzyme operates selectively to degrade non- lantibiotic impurities. Parameters which can be varied to modulate these selective conditions include altered pH ranges, reduced protease to lantibiotic ratios, reduced temperatures, etc. This listing of parameters which can be altered to optimize the selective operation of the proteoiytic enzyme is not intended to be comprehensive as those skilled in the art will recognize other parameters that can be altered to effect the desired goal. [0013] One of skill in the. art can readily determine optimal conditions for a particular proteolytic enzyme. If produced commercially, the manufacturer typically provides such Information in packaging materials shipped with the enzyme. Alternatively, it is a matter of routine experimentation to empirically determine such conditions. [0014] Optimal conditions, however, are not necessarily selective conditions. Under incubation conditions which are optimal for a given proteoiytic enzyme, both the lantibiotic, as well as protein or polypeptide impurities, may be degraded. Generally speaking, selective conditions are empirically determined. A starting point for such a determination is the manufacturer's recommended optimal condition. If, under these conditions, both lantibiotic and impurities are degraded, the conditions may be "de-tuned" arrive at the selective conditions. For example, as discussed below, the reported optimal pH for the enzyme trypsin has been reported to be about 8.0. However, the optimized selective conditions described in the Specification include, under otherwise identical incubation conditions, a pH within the range of about 5.50 to about 6.25. [0015] In the Exemplification section which follows, the extensively studied lantibiotic nisin was employed. However, as indicated above, representatives of the lantibiotic genus share a number of structural features, some of which have been identified as contributing to their relative resistance to proteolysis. Lanthionine rings, for example, have been specifically cited in this regard. Thus, while the Exemplification section focuses primarily on the WO 2004/033704 PCT/US2003/031986 -5- lantibiotic nisin, the principles established herein would be expected to apply to all lantibiotics due to the presence of their defining structural characteristics. Other lantibiotics include, for example, subtilin, epidermin, gallidermin, mutacin, pep5, epicidin, epilancin, lacticin, cytolysin, staphylococcin, salvaricin, lactocin, streptococcin, sublancin, carnocin, variacin, cypemycin, connamycin, duramycin, ancovenin, mersacidin and actagurdine. [0016] The methods of the present invention are particularly well-suited to the proteolytic purification of a lantibiotic present in crude or partially purified fermentation broth. In the. fermentation of nisin, for example, peptide analysis by GC/MS/MS has identified over 40 peptides that co-purify with nisin. The Exemplification section which follows clearly demonstrates the effectiveness of trypsin in the degradation of the co-purifying impurities. [0017] With respect to the proteolytic activity of trypsin, the optimal pH has been reported to be about 8.0. Under otherwise identical incubation conditions, the optimized selective conditions described in the Specification include a pH within the range of about 5.50 to about 6.25. Similar conditions fall within the selective range for other proteases including, for example, endopeptidase Arg-C, thermolysin, V8 protease, subtilisin, proteinase K, pepsin, papain, clostripain, lysyl endopeptidase, endopeptidase Asp-N, enterokinase, or Factor Xa. Preferably, conditions for selective digestion of nisin impurities by these other proteases comprise pH in the range of about 5.50 to 6.25. In the preferred embodiment, the pH is 5.80. With respect to pepsin, a pH of 3.5 is within the selective limits. [0018] The selective protease digestion conditions, as defined by the selective digestion of non-lantibiotic impurities, can be achieved in a variety of ways. This can include, for example, low protease to lantibiotic ratio, decreased temperature, altered pH, etc. The various relevant parameters can be altered independently or in combination. [0019] Following completion of proteolytic digestion, it may be desirable to remove the protease. A particularly convenient method for facilitating removal of the protease following digestion is to provide the protease attached to a solid support (e.g., an agarose or a magnetic bead). In this format, the solid support is easily separated from the incubation mixture following digestion. Alternatively, simple sizing column steps may be employed to effect removal of the protease. Other techniques 1OT removing proteases iottowing proteotyfc digestions are known in the art, including chromatography techniques such as those based on affinity, ion exchange, and hydrophobicity. * WO 2004/033704 PCT/US2003/031986 -6- [0020] It should be noted that although it may be desirable to remove the proteases after the impurities have been digested, it is not necessary to do so for all applications. For example, if the purified lantibiotic is intended to be consumed with a food product, the protease may not have to be removed in order to satisfy relevant regulations. For example, trypsin has been granted Generally Recognized As Safe (GRAS) status for certain food applications. EXEMPLIFICATION Nisin is sensitive to proteolvsis by trvpsin at neutral to slightly alkaline pH and high trypsin to nisin ratio [0021] Nisin has two internal lysine residues that are potential targets of the proteolytic enzyme trypsin. To determine whether nisin could be digested by trypsin, a digestion reaction mixture was prepared where a nisin preparation, partially purified nisin from a fermentor run, was exposed to trypsin under conditions optimal for trypsin activity. These conditions included a reaction pH of 7.0 and a nisin to trypsin ratio of about 10:1 (w/w). Samples were incubated at 30°C and allqoutes were removed for analysis every 24 hr, beginning at time zero. Coomasie blue staining of SDS-PAGE demonstrated that, under the conditions employed, nisin was degraded by trypsin. Moreover, quantitative HPLC analysis of nisin at the various time points revealed that nisin recovery began to diminish by 24 hours following exposure to trypsin. After 96 hr of treatment, more than 90% of nisin was degraded. Nisin is resistant to trypsin digestion at low pH and low trvpsin to nisin ratio [0022] Peptide analysis has identified over 40 peptides that co-purify with nisin. All of these peptides have been identified as being products of Lactococcus fermentation. Since it was determined that nisin, at neutral to slightly alkaline pH and high trypsin to nisin ratios, is subject to proteolysis by trypsin, conditions were altered in an attempt to identify conditions under which trypsin would not degrade nisin but would degrade the peptide impurities in the nisin sample. Reaction mixtures containing nisin preparations, at a concentration of 11 mg/ml, and USP grade trypsin at a concentration of 27.5 µg/ml, were incubated overnight at room temperature (400:1 w/w). The pH of the mixtures ranged from 5.25 to 6.5. While minimal hydrolysis of both nisin and impurities occurred below pH 5.50, quantitative HPLC analysis indicated that recovery of nisin was best at or below pH 6.25. These results indicated that conditions involving dilute trypsin and pH between 5.50 and 6.25 are optimal WO 2004/033704 PCT/US2003/031986 -7- for removal of peptide impurities from nisin preparations with minimum loss of nisin to tryptic digestion. [0023] To further optimize the conditions for removal of peptide impurities from nisin preparations by tryptic digestion, two liters of partially purified nisin was prepared at 4.55 mg/ml and incubated with 25 mg of USP grade trypsin for 16 hrs at 30°C in 10 mM citric acid, pH 5.80. HPLC analysis revealed an overall nisin recovery of about 95%. Moreover, subsequent removal of trypsin using a molecular weight cut off membrane yielded a recovery of 87.5% nisin and 0.38% trypsin. [0024] To determine whether insoluble trypsin treatment of nisin preparations was also effective in digestion of impurities, but not nisin itself, 1.6 liters of partially purified nisin prepared at 9.55 mg/ml was treated with 1.6 ml of TPCK treated insoluble trypsin bound to beaded agarose. After 6 hr of incubation at 30°C, and following removal of insoluble trypsin by centrifugation, the overall nisin recovery was about 90%. Trypsin-treated nisin retains antimicrobial activity [0025] Treatment of nisin preparations with trypsin, under the conditions described above, does not affect the physical structure of nisin as judged by mobility on a reverse phase HPLC column or migration by SDS-PAGE. To confirm that the biological activity of nisin is also unaltered, in vitro bactericidal activity assay was performed. Trypsin-treated nisin was analyzed for antibacterial activity against a mastitic isolate of Streptococcus agalactlae (strain #20) in a 96 well plate assay and bacterial growth was read as absorbance readings on an ELISA reader. These experiments demonstrated that the antimicrobial activity of trypsin-treated nisin is not substantially different than the activity of the control, untreated nisin. Trypsin treatment reduces the inflammatory factors present in impure nisin [0026] Bovine intra-mammary infusion of impure nisin stimulates the production of high levels of somatic cells in milk as part of an inflammatory reaction. As shown in Table 1, non- mastitic cows treated with an infusion of 30 mg partially purified nisin have somatic cell counts that are greatly elevated. In contrast, somatic cell count of milk from quarters treated with an infusion of trypsin-purified nisin is much lower and is not statistically different from quarters treated with buffer alone. WO 2004/033704 PCT/US2003/031986 -8- Other proteases exhibit selective activity towards impurities over nisin [0027] Nisin is comparatively resistant to proteolysis by trypsin under the optimized conditions described above. To determine whether nisin is also resistant to other proteolytic enzymes under similar conditions, partially purified nisin was exposed to pepsin, thrombin, V8 protease (Endo Gluc), subtilisin, papain, thermolysin, or clostripain at pH 5.80. Pepsin exposure was also performed at pH 3.50. The results showed that nisin was resistant to proteolytic digestion by the enzymes. Pepsin at pH 5.80 and thrombin appeared ineffective against the impurities as well. At pH 3.50 pepsin was effective at digesting nisin impurities, but did not digest nisin. These results demonstrate that, under the low pH conditions and nisin to protease ratios described for trypsin treatment, other proteases also exhibit selective activity towards impurities over nisin. METHODS Trypsin digestion [0028] Tryptic digestion of nisin (50 mg) was carried out in 50 ml of a buffer consisting of 25mM sodium acetate, 6 mM Tris acetate, 5 mM CaCI2, and pH 7.0. One ml of USP grade trypsin (5mg/ml) was added followed by incubation at 30°C. Thereafter, 500 µl aliqouts of trypsin were added at times 24,48,72, and 96 hours. At each time point in the experiment, 2 ml aliquots were removed for further analysis, of which 100 µl was immediately plated on blood agar plates for determination of contamination. The remaining 1.8 ml was stored at -20cC until the end of experiment for SDS-PAGE and HPLC analysis. Protease digestions [0029] For treatment of nisin with soluble trypsin, 2 liters of partially purified nisin from a fermentation product was prepared at 4.55 mg/ml in 10 mM citric acid buffer and 5 mM CaCI2. After addition of 25 mg of USP grade trypsin, the reaction mixture was incubated overnight at 30°C. The weight ratio of nisin to trypsin was approximately 400:1 (w/w). NaOH was used to adjust the pH of the digestion reaction to a range between 5.25 to 6.50. Nisin recovery and purity was determined by SDS-PAGE and HPLC. Digestion reactions were terminated by lowering the pH to 3.0 using 3N HCl. Based on the results, a pH of 5.80 was selected and used in ail subsequent reactions involving other proteases. [0030] Digestion experiments using pepsin (EC3.4.23.1), thrombin (EC3.4.21.5), V8 protease (EC3.4.21.19), subtilisin (EC3.4.21.62), papain (EC3.4.22.2), thermolysin WO 2004/033704 PCT/US2003/031986 -9- (EC3.4.24.27), and clostripain (EC3.4.22.8) were performed essentially as described for trypsin and at pH 5.80. Pepsin digestion was also performed at pH 3.50. For trypsin digestions using trypsin linked to a solid matrix (e.g. insoluble trypsin), 1.6 liters of partially purified nisin was prepared at 9.5 mg/ml in 10mM Tris HCI and pH 5.50. Following the addition of 1.6 ml of TPCK-treated trypsin bound to beaded agarose (Sigma Chemical Company), the reaction mixture was incubated at 30cC for 6 hours with frequent agitation. Insoluble trypsin was removed by centrifugation at approximately 4000 x g for 7 minutes. The resultant supernatant containing nisin was prepared for HPLC analysis. Mammary infusion [0031 ] Non-mastitic cows were treated with an infusion of vehicle, untreated, and trypsin- treated nisin (30 mg/10 ml) in the quarter after the 4th, 5th, and 6th milkings. The somatic cell count of the milk was recorded before, during, and after milking. High somatic cell counts were indicative of an inflammatory reaction. Statistical analysis was carried out using two sided t-Test comparisons with unequal variance. Biological activity of nisin [0032] Nisin activity was determined using an antibacterial assay against a mastitic isolate of Streptococcus agalactiae (IC 20) in a 96 well plate assay. Test samples were serially diluted into M17 10% lactose medium and then mixed with an equal volume of 104 cfu/ml. After 66 hr of incubation at room temperature, the plates were read on an ELISA reader at 450 nm. Wells containing bacterial growth gave high absorbance readings. The data was analyzed using a four-parameter curve-fitting software. WO 2004/033704 PCT/US2003/031986 -10- Table 1: Reduced inflammation in trypsin-treated nisin Somatic Cell Count of Quarter Milk (X1000) t-Test Comparison(Two sided with unequalvariance) Milking(Treatmentsgiven after 4th,5th,6th) Mean ofvehiclecontrol Mean oftrypsin/ nisingroup Mean of nisinstartingmaterialgroup Vehiclecontrol vs.trypsin/ nisingroup Vehiclecontrol vs.nisin startirtgmaterialgroup 1 25 10 23 0.196 0.775 2 33 22 37 0.372 0.845 3 72 23 30 0.376 0.696 4 21 21 24 0.395 0.644 5 58 54 2360 0.944 0.018 6 105 187 2269 0.439 0.155 7 133 300 5787 0.382 0.000 8 61 148 2842 0.360 0.008 9 88 106 2325 0.671 0.000 10 86 82 879 0.664 0.000 11 69 103 1066 0.589 0.000 12 51 81 113 0.467 0.284 13 48 60 514 0.832 0.001 14 39 36 172 0.907 0.092 15 57 53 294 0.692 0.045 16 30 37 107 0.889 0.202 17 39 40 . 128 0.825 0.123 18 28 41 80 0.691 0.210 WO 2004/033704 PCT/US2003/031986 -11- CLAIMS 1. A method for purifying a lantibiotic, the method comprising: a) providing a solution containing the lantibiotic to be purified; b) providing a proteolytic enzyme; and c) forming an incubation mixture comprising the solution containing the lantibiotic and the proteolytic enzyme, and incubating the mixture under conditions which are optimized for selective degradation of non-lantibiotic protein or polypeptide impurities, while leaving the lantibiotic substantially undigested. 2. The method of Claim 1 further comprising the step of removing or inactivating the proteolytic enzyme following digestion. 3. The method of Claim 1 wherein the provided solution of step a) is a product of fermentation. 4. The method of Claim 1 wherein the lantibiotic is selected from the group consisting of nisin, subtilin, epidermin, gallidermin, mutacin, pep5, eplcidin, epilandn, cytolysin, lacticln, staphylococcin, salvaricin, lactocin, streptococcin, sublancin, carnocin, variadn, cypemycin, connamycin, duramycin, ancovenin, mersacidin and actagurdine. 5. The method of Claim 1 wherein the lantibiotic is nisin. 6. The method of Claim 5 wherein the proteolytic enzyme is trypsin. 7. The method of Claim 6 wherein the conditions optimized for selective degradation comprise a pH of between 5.5 and 6.25. 8. The method of Claim 1 wherein the proteolytic enzyme is selected from the group consisting of trypsin, endopeptidase Arg-C, thermolysin, V8 protease, subtilisin, proteinase K, clostripain, lysyl endopeptidase, papain, endopeptidase Asp-N, enterokinase, Factor Xa, and chymotrypsin. 9. The method of Claim 8 wherein the conditions optimized for selective activity comprise a pH of between 5.5 and 6.25. WO 2004/033704 PCT/US2003/031986 -12- 10. The method of Claim 1 wherein the proteolytic enzyme is pepsin. 11. The method of Claim 10 wherein the conditions optimized for selective activity comprise a pH of greater than about 4.5. 12. The method of Claim 1 wherein the proteolytic enzyme is linked to a solid support. 13. The method of Claim 1 wherein the effective amount of the proteolytic enzyme is about 400/1 (w/w) lantibiotic/proteolytic enzyme. Disclosed is 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 dictate 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 optimized for selective proteolytic activity.

Full Text

WO 2004/033704 PCT/US2003/031986
METHOD OF PURIFYING LANTIBIOTICS
Background of the Invention
[0001] 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.
[0002] 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 (3-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.
[0003] 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.
[0004] 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.

WO 2004/033704 PCT/US2003/031986
-2-
Therefore, a method is needed that improves lantibiotic purity and that is practical to employ
on a commercial scale.
[0005] 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.
[0006] 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
[0007] 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.

WO 2004/033704 PCT/US2003/031986
-3-
Detailed Description of the Invention
[0008] Lantibiotics are a group of ribosomally synthesized, post-translationally modified
peptides containing unusual amino acids. Such amino acids include the thioether amino
acids lanthionine (Lan) and/or MeLan, in addition to a number of modified residues, such as
2,3-didehydroalanine (Dha) and 2,3-didehydrobutyrine (Dhb). The presence and influence of
these residues on the structure and activity of lantibiotics has been the subject of significant
research efforts.
[0009] It has been observed, for example, that the sequence-specific dehydration of
serine (to Dha) and threonine (to Dhb) results in modified amino acids with electrophilic
centers which can react with neighboring nucleophilic groups. The thioether lanthionine is
formed when the double bond in Dha is attacked by the thiol (SH) group of a neighboring
cysteine residue. As a consequence of the presence of these intramolecular bridges,
lantibiotics are polycyclic structures containing a number of lanthionine rings. The presence
- of these lanthionine rings is thought to be essential for a number of important lantibiotic
properties including, for example, maintenance of peptide rigidity and resistance to thermal
inactivation.
[0010] The present invention is based on Applicants' surprising discovery that a crude or
partially purified preparation of a lantibiotic can be subjected to protease treatment under
conditions which result in selective proteolytic activity against protein or polypeptide
impurities, without measurable proteolysis of the lantibiotic. The use of such conditions
represents an effective method for the purification of a lantibiotic.
[0011 ] The prior art teaching which relates to the sensitivity of lantibiotics to protease
digestion is mixed. Many of these reports relate specifically to the well-studied lantibiotic,
nisln. For example, Gross et al. (J. Am. Chem. Soc. 93(18) (1971) 4634-4635) reported that
nisin was trypsin-sensitive. A contradictory report was later published by Wilimowska-Pelc
(Acts Microbiol. Pol A 8(1) (1976) 71-77). More recently, Chan et al. (Int. Food Microbiol. 390
(2001) 267-281) reported that nisin is subject to tryptic digestion, but at a reduced rate. In
summary, one of skill in the art, familiar with relevant teaching of the prior art, could not have
predicted with any degree of certainty that it would be possible to identify conditions under
which non-iantibiotic protein, polypeptide and peptide impurities present in a crude or partially
purified lantibiotic containing solution could be selectively degraded by the action of a
proteolytic enzyme, without substantially degrading the lantibiotic present in the solution.

WO 2004/033704 PCT/US2003/031986
-4-
This discovery is particularly important given the long-felt need for an improved, cost-
effective, high purity method for lantibiotic purification which yields a lantibiotic preparation
suitable for pharmaceutical use.
[0012] The conditions for protease digestion determined to be effective in connection
with the present invention can be characterized as selective. That is, the conditions
established for the incubation that includes the impure lantibiotic and a proteoiytic enzyme
are conditions under which the proteoiytic enzyme operates selectively to degrade non-
lantibiotic impurities. Parameters which can be varied to modulate these selective conditions
include altered pH ranges, reduced protease to lantibiotic ratios, reduced temperatures, etc.
This listing of parameters which can be altered to optimize the selective operation of the
proteoiytic enzyme is not intended to be comprehensive as those skilled in the art will
recognize other parameters that can be altered to effect the desired goal.
[0013] One of skill in the. art can readily determine optimal conditions for a particular
proteolytic enzyme. If produced commercially, the manufacturer typically provides such
Information in packaging materials shipped with the enzyme. Alternatively, it is a matter of
routine experimentation to empirically determine such conditions.
[0014] Optimal conditions, however, are not necessarily selective conditions. Under
incubation conditions which are optimal for a given proteoiytic enzyme, both the lantibiotic, as
well as protein or polypeptide impurities, may be degraded. Generally speaking, selective
conditions are empirically determined. A starting point for such a determination is the
manufacturer's recommended optimal condition. If, under these conditions, both lantibiotic
and impurities are degraded, the conditions may be "de-tuned" arrive at the selective
conditions. For example, as discussed below, the reported optimal pH for the enzyme trypsin
has been reported to be about 8.0. However, the optimized selective conditions described in
the Specification include, under otherwise identical incubation conditions, a pH within the
range of about 5.50 to about 6.25.
[0015] In the Exemplification section which follows, the extensively studied lantibiotic
nisin was employed. However, as indicated above, representatives of the lantibiotic genus
share a number of structural features, some of which have been identified as contributing to
their relative resistance to proteolysis. Lanthionine rings, for example, have been specifically
cited in this regard. Thus, while the Exemplification section focuses primarily on the

WO 2004/033704 PCT/US2003/031986
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lantibiotic nisin, the principles established herein would be expected to apply to all lantibiotics
due to the presence of their defining structural characteristics. Other lantibiotics include, for
example, subtilin, epidermin, gallidermin, mutacin, pep5, epicidin, epilancin, lacticin,
cytolysin, staphylococcin, salvaricin, lactocin, streptococcin, sublancin, carnocin, variacin,
cypemycin, connamycin, duramycin, ancovenin, mersacidin and actagurdine.
[0016] The methods of the present invention are particularly well-suited to the proteolytic
purification of a lantibiotic present in crude or partially purified fermentation broth. In the.
fermentation of nisin, for example, peptide analysis by GC/MS/MS has identified over 40
peptides that co-purify with nisin. The Exemplification section which follows clearly
demonstrates the effectiveness of trypsin in the degradation of the co-purifying impurities.
[0017] With respect to the proteolytic activity of trypsin, the optimal pH has been reported
to be about 8.0. Under otherwise identical incubation conditions, the optimized selective
conditions described in the Specification include a pH within the range of about 5.50 to about
6.25. Similar conditions fall within the selective range for other proteases including, for
example, endopeptidase Arg-C, thermolysin, V8 protease, subtilisin, proteinase K, pepsin,
papain, clostripain, lysyl endopeptidase, endopeptidase Asp-N, enterokinase, or Factor Xa.
Preferably, conditions for selective digestion of nisin impurities by these other proteases
comprise pH in the range of about 5.50 to 6.25. In the preferred embodiment, the pH is 5.80.
With respect to pepsin, a pH of 3.5 is within the selective limits.
[0018] The selective protease digestion conditions, as defined by the selective digestion
of non-lantibiotic impurities, can be achieved in a variety of ways. This can include, for
example, low protease to lantibiotic ratio, decreased temperature, altered pH, etc. The
various relevant parameters can be altered independently or in combination.
[0019] Following completion of proteolytic digestion, it may be desirable to remove the
protease. A particularly convenient method for facilitating removal of the protease following
digestion is to provide the protease attached to a solid support (e.g., an agarose or a
magnetic bead). In this format, the solid support is easily separated from the incubation
mixture following digestion. Alternatively, simple sizing column steps may be employed to
effect removal of the protease. Other techniques 1OT removing proteases iottowing proteotyfc
digestions are known in the art, including chromatography techniques such as those based
on affinity, ion exchange, and hydrophobicity. *

WO 2004/033704 PCT/US2003/031986
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[0020] It should be noted that although it may be desirable to remove the proteases after
the impurities have been digested, it is not necessary to do so for all applications. For
example, if the purified lantibiotic is intended to be consumed with a food product, the
protease may not have to be removed in order to satisfy relevant regulations. For example,
trypsin has been granted Generally Recognized As Safe (GRAS) status for certain food
applications.
EXEMPLIFICATION
Nisin is sensitive to proteolvsis by trvpsin at neutral to slightly alkaline pH and high trypsin to
nisin ratio
[0021] Nisin has two internal lysine residues that are potential targets of the proteolytic
enzyme trypsin. To determine whether nisin could be digested by trypsin, a digestion
reaction mixture was prepared where a nisin preparation, partially purified nisin from a
fermentor run, was exposed to trypsin under conditions optimal for trypsin activity. These
conditions included a reaction pH of 7.0 and a nisin to trypsin ratio of about 10:1 (w/w).
Samples were incubated at 30°C and allqoutes were removed for analysis every 24 hr,
beginning at time zero. Coomasie blue staining of SDS-PAGE demonstrated that, under the
conditions employed, nisin was degraded by trypsin. Moreover, quantitative HPLC analysis
of nisin at the various time points revealed that nisin recovery began to diminish by 24 hours
following exposure to trypsin. After 96 hr of treatment, more than 90% of nisin was
degraded.
Nisin is resistant to trypsin digestion at low pH and low trvpsin to nisin ratio
[0022] Peptide analysis has identified over 40 peptides that co-purify with nisin. All of
these peptides have been identified as being products of Lactococcus fermentation. Since it
was determined that nisin, at neutral to slightly alkaline pH and high trypsin to nisin ratios, is
subject to proteolysis by trypsin, conditions were altered in an attempt to identify conditions
under which trypsin would not degrade nisin but would degrade the peptide impurities in the
nisin sample. Reaction mixtures containing nisin preparations, at a concentration of 11
mg/ml, and USP grade trypsin at a concentration of 27.5 µg/ml, were incubated overnight at
room temperature (400:1 w/w). The pH of the mixtures ranged from 5.25 to 6.5. While
minimal hydrolysis of both nisin and impurities occurred below pH 5.50, quantitative HPLC
analysis indicated that recovery of nisin was best at or below pH 6.25. These results
indicated that conditions involving dilute trypsin and pH between 5.50 and 6.25 are optimal

WO 2004/033704 PCT/US2003/031986
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for removal of peptide impurities from nisin preparations with minimum loss of nisin to tryptic
digestion.
[0023] To further optimize the conditions for removal of peptide impurities from nisin
preparations by tryptic digestion, two liters of partially purified nisin was prepared at 4.55
mg/ml and incubated with 25 mg of USP grade trypsin for 16 hrs at 30°C in 10 mM citric acid,
pH 5.80. HPLC analysis revealed an overall nisin recovery of about 95%. Moreover,
subsequent removal of trypsin using a molecular weight cut off membrane yielded a recovery
of 87.5% nisin and 0.38% trypsin.
[0024] To determine whether insoluble trypsin treatment of nisin preparations was also
effective in digestion of impurities, but not nisin itself, 1.6 liters of partially purified nisin
prepared at 9.55 mg/ml was treated with 1.6 ml of TPCK treated insoluble trypsin bound to
beaded agarose. After 6 hr of incubation at 30°C, and following removal of insoluble trypsin
by centrifugation, the overall nisin recovery was about 90%.
Trypsin-treated nisin retains antimicrobial activity
[0025] Treatment of nisin preparations with trypsin, under the conditions described
above, does not affect the physical structure of nisin as judged by mobility on a reverse
phase HPLC column or migration by SDS-PAGE. To confirm that the biological activity of
nisin is also unaltered, in vitro bactericidal activity assay was performed. Trypsin-treated
nisin was analyzed for antibacterial activity against a mastitic isolate of Streptococcus
agalactlae (strain #20) in a 96 well plate assay and bacterial growth was read as absorbance
readings on an ELISA reader. These experiments demonstrated that the antimicrobial
activity of trypsin-treated nisin is not substantially different than the activity of the control,
untreated nisin.
Trypsin treatment reduces the inflammatory factors present in impure nisin
[0026] Bovine intra-mammary infusion of impure nisin stimulates the production of high
levels of somatic cells in milk as part of an inflammatory reaction. As shown in Table 1, non-
mastitic cows treated with an infusion of 30 mg partially purified nisin have somatic cell
counts that are greatly elevated. In contrast, somatic cell count of milk from quarters treated
with an infusion of trypsin-purified nisin is much lower and is not statistically different from
quarters treated with buffer alone.

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Other proteases exhibit selective activity towards impurities over nisin
[0027] Nisin is comparatively resistant to proteolysis by trypsin under the optimized
conditions described above. To determine whether nisin is also resistant to other proteolytic
enzymes under similar conditions, partially purified nisin was exposed to pepsin, thrombin,
V8 protease (Endo Gluc), subtilisin, papain, thermolysin, or clostripain at pH 5.80. Pepsin
exposure was also performed at pH 3.50. The results showed that nisin was resistant to
proteolytic digestion by the enzymes. Pepsin at pH 5.80 and thrombin appeared ineffective
against the impurities as well. At pH 3.50 pepsin was effective at digesting nisin impurities,
but did not digest nisin. These results demonstrate that, under the low pH conditions and
nisin to protease ratios described for trypsin treatment, other proteases also exhibit selective
activity towards impurities over nisin.
METHODS
Trypsin digestion
[0028] Tryptic digestion of nisin (50 mg) was carried out in 50 ml of a buffer consisting of
25mM sodium acetate, 6 mM Tris acetate, 5 mM CaCI2, and pH 7.0. One ml of USP grade
trypsin (5mg/ml) was added followed by incubation at 30°C. Thereafter, 500 µl aliqouts of
trypsin were added at times 24,48,72, and 96 hours. At each time point in the experiment, 2
ml aliquots were removed for further analysis, of which 100 µl was immediately plated on
blood agar plates for determination of contamination. The remaining 1.8 ml was stored at
-20cC until the end of experiment for SDS-PAGE and HPLC analysis.
Protease digestions
[0029] For treatment of nisin with soluble trypsin, 2 liters of partially purified nisin from a
fermentation product was prepared at 4.55 mg/ml in 10 mM citric acid buffer and 5 mM
CaCI2. After addition of 25 mg of USP grade trypsin, the reaction mixture was incubated
overnight at 30°C. The weight ratio of nisin to trypsin was approximately 400:1 (w/w). NaOH
was used to adjust the pH of the digestion reaction to a range between 5.25 to 6.50. Nisin
recovery and purity was determined by SDS-PAGE and HPLC. Digestion reactions were
terminated by lowering the pH to 3.0 using 3N HCl. Based on the results, a pH of 5.80 was
selected and used in ail subsequent reactions involving other proteases.
[0030] Digestion experiments using pepsin (EC3.4.23.1), thrombin (EC3.4.21.5), V8
protease (EC3.4.21.19), subtilisin (EC3.4.21.62), papain (EC3.4.22.2), thermolysin

WO 2004/033704 PCT/US2003/031986
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(EC3.4.24.27), and clostripain (EC3.4.22.8) were performed essentially as described for
trypsin and at pH 5.80. Pepsin digestion was also performed at pH 3.50. For trypsin
digestions using trypsin linked to a solid matrix (e.g. insoluble trypsin), 1.6 liters of partially
purified nisin was prepared at 9.5 mg/ml in 10mM Tris HCI and pH 5.50. Following the
addition of 1.6 ml of TPCK-treated trypsin bound to beaded agarose (Sigma Chemical
Company), the reaction mixture was incubated at 30cC for 6 hours with frequent agitation.
Insoluble trypsin was removed by centrifugation at approximately 4000 x g for 7 minutes.
The resultant supernatant containing nisin was prepared for HPLC analysis.
Mammary infusion
[0031 ] Non-mastitic cows were treated with an infusion of vehicle, untreated, and trypsin-
treated nisin (30 mg/10 ml) in the quarter after the 4th, 5th, and 6th milkings. The somatic cell
count of the milk was recorded before, during, and after milking. High somatic cell counts
were indicative of an inflammatory reaction. Statistical analysis was carried out using two
sided t-Test comparisons with unequal variance.
Biological activity of nisin
[0032] Nisin activity was determined using an antibacterial assay against a mastitic
isolate of Streptococcus agalactiae (IC 20) in a 96 well plate assay. Test samples were
serially diluted into M17 10% lactose medium and then mixed with an equal volume of 104
cfu/ml. After 66 hr of incubation at room temperature, the plates were read on an ELISA
reader at 450 nm. Wells containing bacterial growth gave high absorbance readings. The
data was analyzed using a four-parameter curve-fitting software.

WO 2004/033704 PCT/US2003/031986
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Table 1: Reduced inflammation in trypsin-treated nisin
Somatic Cell Count of Quarter Milk (X1000) t-Test Comparison(Two sided with unequalvariance)
Milking(Treatmentsgiven after 4th,5th,6th) Mean ofvehiclecontrol Mean oftrypsin/ nisingroup Mean of nisinstartingmaterialgroup Vehiclecontrol vs.trypsin/ nisingroup Vehiclecontrol vs.nisin startirtgmaterialgroup
1 25 10 23 0.196 0.775
2 33 22 37 0.372 0.845
3 72 23 30 0.376 0.696
4 21 21 24 0.395 0.644
5 58 54 2360 0.944 0.018
6 105 187 2269 0.439 0.155
7 133 300 5787 0.382 0.000
8 61 148 2842 0.360 0.008
9 88 106 2325 0.671 0.000
10 86 82 879 0.664 0.000
11 69 103 1066 0.589 0.000
12 51 81 113 0.467 0.284
13 48 60 514 0.832 0.001
14 39 36 172 0.907 0.092
15 57 53 294 0.692 0.045
16 30 37 107 0.889 0.202
17 39 40 . 128 0.825 0.123
18 28 41 80 0.691 0.210

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CLAIMS
1. A method for purifying a lantibiotic, the method comprising:
a) providing a solution containing the lantibiotic to be purified;
b) providing a proteolytic enzyme; and
c) forming an incubation mixture comprising the solution containing the lantibiotic
and the proteolytic enzyme, and incubating the mixture under conditions which
are optimized for selective degradation of non-lantibiotic protein or polypeptide
impurities, while leaving the lantibiotic substantially undigested.
2. The method of Claim 1 further comprising the step of removing or inactivating the
proteolytic enzyme following digestion.
3. The method of Claim 1 wherein the provided solution of step a) is a product of
fermentation.
4. The method of Claim 1 wherein the lantibiotic is selected from the group consisting of
nisin, subtilin, epidermin, gallidermin, mutacin, pep5, eplcidin, epilandn, cytolysin,
lacticln, staphylococcin, salvaricin, lactocin, streptococcin, sublancin, carnocin,
variadn, cypemycin, connamycin, duramycin, ancovenin, mersacidin and actagurdine.
5. The method of Claim 1 wherein the lantibiotic is nisin.
6. The method of Claim 5 wherein the proteolytic enzyme is trypsin.
7. The method of Claim 6 wherein the conditions optimized for selective degradation
comprise a pH of between 5.5 and 6.25.
8. The method of Claim 1 wherein the proteolytic enzyme is selected from the group
consisting of trypsin, endopeptidase Arg-C, thermolysin, V8 protease, subtilisin,
proteinase K, clostripain, lysyl endopeptidase, papain, endopeptidase Asp-N,
enterokinase, Factor Xa, and chymotrypsin.
9. The method of Claim 8 wherein the conditions optimized for selective activity
comprise a pH of between 5.5 and 6.25.

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10. The method of Claim 1 wherein the proteolytic enzyme is pepsin.
11. The method of Claim 10 wherein the conditions optimized for selective activity
comprise a pH of greater than about 4.5.
12. The method of Claim 1 wherein the proteolytic enzyme is linked to a solid support.
13. The method of Claim 1 wherein the effective amount of the proteolytic enzyme is
about 400/1 (w/w) lantibiotic/proteolytic enzyme.

Disclosed is 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 dictate 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
optimized for selective proteolytic activity.

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Patent Number

214235

Indian Patent Application Number

00688/KOLNP/2005

PG Journal Number

06/2008

Publication Date

08-Feb-2008

Grant Date

07-Feb-2008

Date of Filing

20-Apr-2005

Name of Patentee

IMMUCELL CORPORATION

Applicant Address

56 EVERGREEN DRIVE, PORTLAND, ME 04103, UNITED STATES OF AMERICA.

Inventors:

#	Inventor's Name	Inventor's Address
1	COUGHLIN RICHARD T.	92 WOODLANDS DRIVE, FALMOUTH, ME 041054, UNITED STATES OF AMERICA.
2	CRABB JOSEPH H.	68 PLEASANT HILL ROAD, FALMOUTH, ME 04105, UNITED STATES OF AMERICA.

PCT International Classification Number

C12P 21/06

PCT International Application Number

PCT/US2003/031986

PCT International Filing date

2003-10-07

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10/268,037	2002-10-09	U.S.A.