WO2001009284A1

WO2001009284A1 - An antibiotic and a haloperoxidase produced by an alcaligenes microorganism

Info

Publication number: WO2001009284A1
Application number: PCT/AU2000/000901
Authority: WO
Inventors: Yung-Pin Chen
Original assignee: Enzymatics Ltd.
Priority date: 1999-07-28
Filing date: 2000-07-28
Publication date: 2001-02-08
Also published as: AU5956200A

Abstract

A new microorganism, Alcaligenes FC-88, has been discovered. This microorganism produces an antibiotic with antibiotic activity against a variety of microorganisms, including many that hitherto have been designated as antibiotic resistant. The microorganism also produces a haloperoxidase that halogenates a variety of organic compounds. A particularly advantageous feature of Alcaligenes FC-88 is that it secretes the antibiotic and haloperoxidase into the medium in which the microorganism is growing.

Description

AN ANTIBIOTIC AND A HALOPEROXIDASE PRODUCED BY AN ALCALIGENES MICROORGANISM

FIELD OF THE INVENTION The present invention relates to the production of an antibiotic and a haloperoxidase. In particular, the present invention relates to an antibiotic and a haloperoxidase isolated from an Alcaligenes spp.

BACKGROUND OF THE INVENTION

There are more than 100 microbial products in use today as antibiotics, antitumor agents and agrochemical materials. Currently, there are massive efforts underway and resources allocated in the pharmaceutical industry alone to explore the use of terrestrial bacterial fermentation products as pharmaceuticals.

Most antibiotics of microbial origin come from one group, the gram-positive soil bacteria of the order Act inomycet ales. Although these bacteria continue to be extensively studied, the rate of discovery of novel metabolites from terrestrial actinomycetes is decreasing. It is estimated that more than 90% of all bioactive cultures of these microorganisms produce previously known agents. (Fenical, W. , 1993, Chemical Studies of Marine Bacteria: Developing a New Resource, Chem. Resc. 1993,

1673-1683) .

In addition, unfortunately, infectious diseases are rapidly developing resistance to most of the important antibiotics. Many gram-negative organisms and various strains of Mycobacterium can produce β-lactases that inactivate antibiotics used in patient treatments. This is seen most dramatically in two particular strains of human pathogens, Enterococcus and Staphylococcus aureus . More than 90% of Staphylococcus aureus today are resistant to Penicillin and related antibiotics. There has been an alarming rise recently in the incidence of enterococci which are now the most common cause of infections that are acquired by patients in hospitals. Approximately 10-12% of patients in hospitals in the U.S. are reported to be infected by one of these two microbes. These microbe species can only be treated with the antibiotic vancomycin, but recently it has been reported that strains of these microorganisms have appeared that are resistant to vancomycin which was the last antibiotic weapon for defeating such pathogens (Brickner, S.J., 1997, Multidrug- resistant Bacterial Infections: Driving the Search for New Antibiotics, Chemistry and Industry, 131-135) . Thus, there is a need to find and develop new antibiotics. Since the previous microbial sources of antibiotics is becoming depleted, it is important to explore other sources, such as other microorganism groups, for new bioactive natural products.

Despite early observations (Dopazo, et al . , 1988, Inhibitory Activity of Antibiotic Producing Marine Bacteria against Fish Pathogen, J. Appl . Bact . 65, 97-101), there has been relatively little attention directed to the study of natural products from marine bacteria. Subsequent studies of marine plants and animals have shown natural product chemicals and an array of unusual secondary metabolites (Fenical, . and Jensen, P.R., 1993, Marine Microorganisms: A New Biomedical Resource, Marine Biotechnology, Vol . 1 : Pharmaceutical and Bioactive Natural

Products, David H.A. and Zaborsky, O.R., ed., Plenum Press, New York; Jensen, P.R. and Fenical ., 1996, Marine Bacteria Diversity as a Resource for Novel Microbial Products, J. Indust . Micro. 17, 346-351). Many of these chemicals have not been found in terrestrial sources

(Jensen and Fenical, 1996) . The biological functions of many of the natural product chemicals and secondary metabolites are not clear, but there are some data that support the role of some of these chemicals as antifungal, antibacterial, antitumor and anti-inflammatory compounds. Among the marine organism natural products are enzymes that halogenate hydrocarbon molecules to form halohydrocarbon molecules. The biosynthetic molecules from marine organisms that belong to this class of enzymes include haloperoxidases.

Haloperoxidases are enzymes that are capable of halogenating a variety of organic compounds using hydrogen peroxide and halide ions as substrates. The general reaction scheme is given as:

AH + H₂0₂ + H⁺ + X^"→ AX + 2H₂0 where AH is an organic substrate; X is a halide ion such as chloride, bromide, or iodide; and AX is the halogenated product. According to the enzyme capabilities in utilizing halide ions, the haloperoxidases are generally known as chloro-, bromo, or iodoperoxidases .

Haloperoxidases are unique enzymes and their reaction mechanism is not fully understood. There are two groups of haloperoxidases, depending on whether the enzymes contain a heme molecule (protoporphyrin IX) as a prosthetic group or whether the enzymes contain a non-heme molecule as the prosthetic group. The former enzymes are called heme- containing haloperoxidases and the latter enzymes are called non-heme containing haloperoxidases. The main biological sources of heme-containing haloperoxidases are algae and the main biological sources of non-heme containing haloperoxidases are bacteria. The he e- containing enzymes are mostly bromoperoxidases which produce molecular bromine and therefore are not much use to the synthetic organic chemist. However, Penicillus capitatus, which produces bromoperoxidase at neutral, and perhaps low, pH, can be used in organic chlorination reactions ( iesner et al . , 1998, J. Biol . Chem. 263,

13725), so not all heme-containing enzymes have the same halogenating abilities.

Vanadium-containing haloperoxidases are quite abundant in the group of non-heme containing enzymes (Butler A. and Walter, J.V., 1993, Chem. Rev. 93, 1937- 1944) . These haloperoxidases are at present the major source of halo etabolites. However, it has been found that a chloroperoxidase from Pseudoinonas pyrrocinia can produce 7-chloroindole and a chlorinated pyrrolnitrin but does not contain vanadium nor a heme group (Weisner et al . , 1988). A further enzyme has been found that is capable of catalyzing the bromination of the antifungal antibiotic pyrrolnitrin. A unique flavin-containing chloroperoxidase from the marine worm Notomastus lobatus has been purified to homogeneity (Chen et al . , 1991, J. Biol . Chem. 266:23909- 23915) . This enzyme can chlorinate a wide variety of aromatic compounds. This enzyme is the first haloperoxidase reported to contain flavin. The enzyme has several unusual physical and catalytic properties, but the natural substrates of this chloroperoxidase are still unknown. Another haloperoxidase has been isolated from the microorganism Rathayibacter biopuresis (U. S. Patent No. 5,589,354). This haloperoxidase can utilize the iodide, bromide, chloride and fluoride halides. The enzyme can convert one antibiotic, cephalexin, to another antibiotic, cefaclor, by the simultaneous removal of a methyl group, and replacement of it with a chloride moiety.

It would be advantageous to find further biocatalysts which produce halogenated intermediates and novel products by new synthetic routes. These biocatalysts offer specificity of reaction and production of products that are easier to make and to isolate than when these products are produced by conventional synthetic chemical techniques .

SUMMARY OF THE INVENTION

This invention pertains to the production of an antibiotic which has antibiotic activity against a variety of microorganisms. Among the microorganisms that are susceptible to the antibiotic of this invention are methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus .

The antibiotic of this invention is produced by a hitherto unknown microorganism of the Alcaligenes genus. The particular Alcaligenes species that produces the antibiotic, identified as Alcaligenes FC-88, is a marine microorganism and is also an object of this invention. The Alcaligenes FC-88 microorganism of this invention, in addition to producing the antibiotic of this invention, also produces a haloperoxidase. The present invention also pertains to this haloperoxidase. This haloperoxidase halogenates a variety of organic compounds including phenol, substituted benzoic acids and 6-deoxy-L- galacto pyranose.

A further aspect of the present invention is that the antibiotic as well as the haloperoxidase of this invention are produced by the microorganism Alcaligenes FC- 88 and secreted into the medium that surrounds the microorganism. It is unnecessary to lyse the microorganism to recover either the antibiotic or the haloperoxidase of this invention. Thus, the microorganism can act as a miniature factory for producing the antibiotic and the haloperoxidase of this invention. The growth medium surrounding and sustaining the microorganism can be removed and replenished without the need for adding a fresh inoculum of the microorganism. The antibiotic or the haloperoxidase of the invention, or both, can then be isolated and purified from the growth medium that was removed from the microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a stained preparation of Alcaligenes FC-88 under microscopy showing the flagella.

Figure IB is a stained preparation of Alcaligenes FC-88 under electron microscopy showing the flagella.

Figure 2 is a graphical representation of the absorbance at 260nm (closed squares) and the microbial susceptibility (open squares) for 4ml fractions of eluant from a 1.8 x 10cm Roxyn 1-300 (H⁺-OH^") column.

Figure 3 is a graphical representation of the absorbance at 260nm (closed diamonds) and of the microbial susceptibility of methicillin resistant Staphylococcus aureus (closed squares) for eluant fractions from a 1.8 x 10cm Ag-lxlO column. Figure 4 is a graphical representation of the size of the microbial susceptibility inhibition zone against methicillin resistant Staphylococcus aureus for different concentrations of antibody eluants from the 1.8 x 10cm Roxyn 1-300 (H⁺-0H^~) column (open squares) and the 1.8 x 10cm Ag-lxlO column (closed squares). Insert: petrie dish showing microbial susceptibility inhibition zones for respective amounts: A and 1 are 5μg; Band 2 are 15μg; C and 3 are 25μg; and D and 4 are 35μg.

Figure 5 is a graphical representation of the absorbance at 280nm (+ symbol) and of the halogenation activity (closed triangles) for eluant fractions from a 1.8 x 12cm Ag-lx8 column.

DETAILED DESCRIPTION OF THE INVENTION The present invention pertains to a microorganism, from a marine environment, and to two substances that this microorganism produces. These substances are an antibiotic that is effective against a wide range of microorganisms, other than itself, including some microorganisms that are resistant to other antibiotics, and a haloperoxidase that halogenates a variety of aromatic and non-aromatic organic compounds. A further feature of this invention is the secretion of the antibiotic and the haloperoxidase by the microorganism into the medium in which the microorganism resides. With this property, it is possible to recover either the antibiotic or the haloperoxidase, or both, from the growth medium of the microorganism without destroying the integrity of the cells that produce these substances. The microorganism has two features, either of which render this microorganism unique. These features are: (1) the production of the antibiotic of this invention, and (2) the production of the haloperoxidase of this invention. A microorganism that is endowed with both of these features is a microbe of the Alcaligenes genus. A particular species that has both of these features has been newly discovered and has been designated as Alcaligenes FC- 88. A sample of this microorganism has been deposited under conditions of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Virginia, 20110, USA. This deposit was made on 19 April 2000 and has been designated with Accession Number PTA-1738.

The antibiotic of this invention has antibiotic activity against a variety of microorganisms. The microorganisms that are susceptible to this antibiotic include Serratia marcescens, Micrococcus leuteus,

Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus cereus, Citrobacter freundii, Protens vulgar is, Staphylococcus aureus, and the genera Enterococcus and Pneumoniae . From this list, it can be appreciated that the antibiotic is a broad spectrum antibiotic. Further attributes of this antibiotic are that it is effective against antibiotic resistant strains of microorganisms. For example, the antibiotic has antibiotic activity against methicillin resistant Staphylococcus aureus and against vancomycin resistant Enterococcus. The antibiotic has activity against strains of microorganisms that have previously been shown to be resistant to a range of previously known antibiotics. These strains are quite pathological, particularly in clinical settings where patient mortality occurs due to infections by these microorganisms. These strains, which are susceptible to this antibiotic, include clinic isolate Enterococcus (VRE) #83, clinic isolate Staphylococcus (MRSA) #68, clinic isolate Pseudomonas aeruginosa #97, clinic isolate Escherichia coli #32, clinic isolate Pneumonia #79, and clinic isolate Citrobacter freundii that is resistant to the antibiotics Ampicillin, Cefazolin, Cefuroxine, Cephalothin, Ciprofloxacin, Gentamicin, Levofloxacin, Minocycline, Nalidixic Acid and Norfloxacin.

The antibiotic is an organic compound with the molecular formula CιoHι_{8 2}0₅ and a formula weight of approximately 246 daltons. It is apparently a glycopeptide antibiotic. The antibiotic has been given the name: Thomacin.

The haloperoxidase of this invention can halogenate a variety of organic compounds, including aromatic, saturated and unsaturated compounds. The halogenation reaction uses oxygen, typically from a peroxide such as H₂0₂, as the reaction is carried out. As a result of the halogenation reactions, the products of the reaction have one or more additional chlorine, bromine or iodine atoms than were present before the reaction. In the absence of an organic substrate, the enzyme has halide- independent catalase activity.

The haloperoxidase can halogenate phenol, a variety of benzoic acids, cinnamic acids, flavone, toluic acid and pyranoses. In particular, the haloperoxidase can halogenate phenol, 2, 5-dihydroxybenzoic acid, flavone, toluic acid, p-aminobenzoic acid, p-hydroxycinnamic acid, 3, 4-dihydroxybenzoic acid, p-hydroxybenzoic acid, 2,3- dihydroxybenzoic acid, 4-hydroxy-3, 5-dimethoxybenzoic acid, 3, 5-dihydroxybenzoic acid, 4-hydroxycinnamic acid, and 6- deoxy-L-galacto pyranose. When phenol is the substrate, the products of the halogenation reaction include 4- bromophenol, 2,4-dibromophenol, 2,4, 6-tribromophenol, 4- chlorophenol, 2,4-dichlorophenol, and 2, 6-dichlorophenol. The haloperoxidase is a monomeric enzymic; i.e. the enzyme is not composed of subunits. The molecular weight of the haloperoxidase is approximately 34,000 daltons .

Although it is possible to recover either the antibiotic or the haloperoxidase from the microorganism

Alcaligenes FC-88 by lysing or homogenizing the cells and then extracting the antibiotic or haloperoxidase from the lysate or homogenate, such a procedure is unnecessary for this microorganism. The antibiotic and the haloperoxidase are secreted by the microorganism into the medium in which the microorganism is growing. These substances are typically found in the medium extracellular to the microorganism bodies. The antibiotic or the haloperoxidase can be recovered from the growth medium without destroying the microorganisms that are producing and secreting these substances. Thus, recovery and purification of these substances is simpler and more efficient than when more traditional cytolysis techniques are necessary. The microorganisms can be grown in a suitable growth medium under suitable growth conditions of pH and temperature. As the microorganisms grow and secrete the antibiotic and/or the haloperoxidase, the growth medium can be removed from the growing microorganisms and replaced by fresh growth medium. The antibiotic and/or the haloperoxidase can be isolated and purified from the removed growth medium. Thus, a continual batch process can be used to obtain the antibiotic and/or the haloperoxidase without replenishing the microorganism stock.

The subject matter of this invention is exemplified by the following Examples. These exemplifications are not to be construed as limiting, in any way, the disclosed invention, and modifications can be made within the abilities of the skilled artisan. For example, modifications in the microorganism culture media and growth conditions can be made without seriously impairing the growth of the microorganisms and the production of the antibiotic or haloperoxidase. Likewise, other known procedures for isolating and purifying the antibiotic or haloperoxidase can be performed by the skilled artisan. In addition, the haloperoxidase reaction conditions can be altered by skilled artisans from those specifically exemplified herein. Example 1 Isolation of Microorganism and Incubation

Procedure

The marine bacterium was isolated from marine sediment obtained from a site at Perth, Australia. Approximately 2-3g of sediment was diluted with 5ml of a cold 0.1M K₂HP0₄ buffer at pH 6.5. This solution was centrifuged at 18,000 x g for 30 min. The recovered supernatant contained several bacteria as revealed by microscopy. The following media were used for subsequent procedures : Plate Medium (TBS)

(NH₄) S0₄ 1M

Basal salt* 10ml Benzoic acid 1.22g

Bacto-Agar 2g

K₂HP0₄/KH₂P0 0.1M pH 6.8

Biotin 5mg

These substances were placed in and brought to IL with distilled water.

*The basal salt solution was composed of the following materials:

Deionized water 800ml

MgS0 .7H₂O 20. Og CaCl₂.2H₂0 7.5g

FeS0₄.7H₂0 - EDTA⁺ 100ml NFb trace elements (see below) 10ml

These substances were placed in and brought to IL with distilled water.

+The Fe-EDTA solution was prepared by dissolving 16g EDTA and 10.4g KOH in 180ml water. This was mixed with an iron solution prepared by dissolving 13.7g FeS0₄.7H₂0 in 364ml water. Air was buffled through the mixture overnight to oxidize the iron to the ferric form. The final solution had a pH of approximately 3 and contained 5mg Fe per ml . Solution Culture Medium (MNM) (NH₄)₂S0₄ 0.5g

MgS0₄»7H₂0 0.2g

NaCl 0. lg

CaCl₂ 0.02g FeEDTA (1.64% solution) 4.0ml

NFb trace elements 2.0ml

(MnSo₄.H₂0 1.79g, H₃B0₃ 2.8g, ZnS0₄.7H₂0 0.24g, CuS0₄.5H₂0 0.079g, and Na₂Mo0₄.2H₂0 1.26g in 100 ml distilled water) Nfb vitamin solution 1.0ml

(Biotin 0. lg and Pyridoxin 0.2g in 100 ml distilled water)

Difco yeast extract (0.1%) HEPES (3mM or lOmM) to bring pH to 6.8 Carbon source chosen from:

Malic acid 5.0g

Glucose lOmM

Benzoate 3mM

4-hydroxybenzoate 3mM These substances were placed in and brought to IL with distilled water. Bacteria Isolation lOOμL of the recovered supernatant from the initial sediment solution centrifugation was inoculated on a TBS plate and incubated at 28°C for two days. A single colony was reinoculated on a TBS plate. This process was repeated 2-3 times until a single strain was obtained. Early stationary phase cells can be grown in MNM medium using malic acid as the carbon and energy source. Growth is optimally monitored and the cells are harvested when the cell density OD reaches 0.8 at 560nm.

Example 2 Taxonomic and Identifying Characteristics of

Isolated Microorganism Morphological features were determined by electron microscopic techniques and by flagella staining.

The isolated microorganisms have the following characteristics :

1. Morphological characteristics (see Figure 1) a) Form and size: short rod with round end 1000 x 1200nm ± 31nm b) Motility: small population are motile with polar and monotrichous flageHum c) Spore: no sporulation d) Gram stain: negative e) Fluorescent pigment: negative 2. Growth characteristics on particular media a) Tryptic soy agar plate (28°C) i) Colony formation: 24 hours after inoculation ii) Shape: regular circle with an entire edge iii) Surface: smooth, low-convex, glossy iv) Color: cream v) Smell: odoriferous, stale b) Rich nutrient (28°C) i) Colony formation: 36 hours after inoculation ii) Shape: regular circle with an entire edge iii) Surface: smooth, low-convex, glossy iv) Color: cream

Physical properties a nitrate reduction + b methyl red test + c Voges-Proskauer test d Indole Formation e Gelatin hydrolysis f Starch hydrolysis g Esculin hydrolysis h Utilization of citrate + i Catalase activity + j Oxidase activity + k Urease activity 1) Arginine dihydrolase activity m) Alkaline phosphatase activity n) Aerobic or anaerobic Aerobic o) Utilization of carbohydrates and organic acids:

N-acetyl-D-galactosamine

N-acetyl-D-glucosamine adonitol

L-arabinose + D-arabitol cellobiose i-erythritol

D-fructose +

L-fructose + D-galactose + gentiobiose + oc-D-glucose + m-inositol α-lactose + α-D-lactose-lactulose maltose

D-mannitol

D-mannose

D-melibiose β-methyl D-glucoside psicose +

D-raffinose +

L-rhamnose +

D-sorbitol + sucrose +

D-trehalose + turanose + xylitol p) Growth in the presence of particular carbohydrates and organic acids: methyl pyruvate + mono-methyl succinate + acetic acid + cis-asconitic acid + citric acid + formic acid + D-galactonic acid lactone +

D-galacturonic acid +

D-gluconic acid +

D-glucosaminic acid +

D-glucouronic acid α-hydroxy butyric acid + β-hydroxy butyric acid + γ-hydroxy butyric acid p-hydroxy phenylacetic acid + itaconic acid + α-keto butyric acid + α-keto glutaric acid + α-keto valeric acid +

D,L-lactic acid + malonic acid + propionic acid + quinic acid +

D-saccharic acid + sebacic acid + succinic acid + bromo succinic acid + succinamic acid + glucuronamide + alaninamide + q) Tolerance to sodium chloride or potassium tellurite,

5% NaCl + 15% NaCl

0.03% potassium tellurite + r) Amino acid utilization as nitrogen sources: D-alanine +

L-alanine +

L-alanyl-glycine + L-asparagine +

L-aspartic acid +

L-glutamic acid + glycyl- -aspartic acid + glycyl-L-glutamic acid - histidine + hydroxy L-proline -

L-leucine +

L-ornithine +

L-phenyl alanine +

L-proline +

L-pyro glutamic acid +

D-serine -

L-serine +

L-threonine +

D, L-camitine - γ-amino butyric acid - s) Nucleoside utilization abil ■ity urocanic acid - inosine - urdine - thymidine - t) Amines utilization abi!ity: phenyl ethyl amine + putrescine

2-amino ethanol u) Phosphorylized compounds:

2,3-butanediol glycerol D,L-α-glycerol phosphate + glucose-1-phosphate glucose-6-phosphate v) Hydrolysis of compounds: α-cyclodextrin + dextrin glycogen + tween 40 + tween 80 +

4) Cellular fatty acid composition as determined by gas chromatography:

10:0 30H 0.13%

12:0 0.47%

12:0 20H 2.23%

12:0 3OH 0.18%

14:0 4.25%

15:1 W6C 0.29%

15:0 0.44%

16:0 31.18%

17:0 CYCLO 6.08%

17:0 1.07%

16:0 20H 0.17%

16:0 30H 0.38%

18:1 W9C 0.19%

18:0 2.85%

19:0 CYCLO W8C 0.28%

Based on these characteristics the isolated microorganism was classified as belonging to the genus Alcaligenes .

The characteristics of the isolated microorganism were compared to the characteristics of another species from the Alcaligenes genus . The results of this comparison are shown in Table 1.

Table 1

Comparison of the morphological and physiological characteristics of the Alcaligenes species

The characteristics of the isolated murine bacterium differ from those of other species of the Alcaligenes genus by the following:

1. The fatty acid composition profile of the isolated bacterium is different. In particular, the percent of 16:0 cellular fatty acid is higher than the percent of this fatty acid in other microorganisms of this genus .

2. The carbohydrate utilization profile of the isolated microorganism is unique. In particular, the isolated bacteria utilize aromatic compounds such as 4- hydroxybenzoate and benzoic acid. Other Alcaligenes species are not able to utilize these compounds. The isolated microorganism is also able to utilize L-arabinose, D- fructose, L-fructose, α-lactose, sucrose, glucose, D- galactose, gentiobiose, carboxylic acids, and many other carbon sources. The other Alcaligenes species, with the exception of A. xylosoxidans, only utilize D-glucose and D- gluconate. 3. The amino acid utilization profile of the present isolate is unique. It has been shown to utilize most amino acids as a nitrogen source.

The isolated bacteria's flagella are polar (Figure 1), whereas the flagella of A. faecalis, A. odorans, and A. denitrificans are peritrichous with the exception of an unnamed Alcaligenes-like bacterium, which has been designated a Group I'VE bacteria by the CDC, that has flagella that are polar and lateral.

Based on this comparison as well as the physical and physiological characteristics of the isolated microorganism, this bacterial species is unique and has been named Alcaligenes FC- 88.

Example 3 Antibiotic Preparation and Properties A sample of the bacterium isolated in Example 1 was grown in a 120ml flask containing MNM medium containing lOmM malic acid as the carbon source. Incubation took place with shaking at 2,000rpm at 28°C for 3-4 days to allow maximum growth of the bacterial inoculum to occur. The incubation solution was then centrifuged at 11,950 x g for 15min. The pellet was discarded and the supernatant was passed through a Vac4 CAP 0.2μm gel membrane. A total of 150ml of fluid was collected.

Aliquots of 150μL of this filtered supernatant were placed on antibiotic susceptibility disks to assess the subsequent zone diameter for various microorganisms. The size of the zone diameter is a measure of the various antibiotic susceptibilities of microorganisms to the supernatant from Alcaligenes FC-88. The test microorganisms were obtained from 0.85% saline suspensions of the microorganisms adjusted to an OD of 0.1 at 600nm (See Cappuccino and Sherman, 1998, Microbiology & Laboratory Manual, pp. 253-254, Addison Wesley Longman, Inc.).

The susceptibility of the test microorganisms was interpreted according to the following criteria:

For comparison purposes, a 30μg cephalothin disk (9mm) containing cefadroxil, an antibiotic of the cephalosporin class, gives the following zone diameters using standard procedures for laboratory control microorganisms: The antibiotic susceptibilities of various microorganisms to the Alcaligenes FC-88 supernatant were:

These results demonstrate that the supernatant of the medium containing Alcaligenes FC-88 microorganisms has antibiotic activity against Serratia marcescens, Micrococcus leuteus, Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa, and Bacillus cereus.

Example 4 Purification of Antibiotic from Alcaligenes

FC-88.

A sample of the bacterium isolated in Example 1 was grown in MNM medium containing lOmM malic acid as the carbon source. Incubation took place with shaking at 2, 000 rpm at 28°C and pH 3.5 for 3 days. The incubation solution was then centrifuged at 11, 950 x g for 15min. The supernatant was collected and passed through a 0.2μm filter. The filtrate (3ml) was loaded onto a 1 x 6cm DEAE- cellulose column that had been equilibrated with 25mM Tris- HCl pH 7. 0. The load solution OD₂₆o was 1.377. Fractions of 1.2ml were collected from the column when it was washed with 0.1M NaCl in 25mM Tris-HCl pH 7.0. The collected fractions had the following ODs: Fraction Number OD₂₆₀ 1 0.055 2 0.394 3 0.509 4 0.543 5 6.000 6 3.046 7 0.145 8 0.034

Peak fraction Number 5 was assayed for antibiotic activity against representative microorganisms with the following results:

Organism Zone Diameter (mm) and interpretation

S. pyogenes 27 (S)

S. aureus 18 (R)

B . cereus 35 (S)

These results demonstrate that the antibiotic material from the supernatant of the medium containing Alcaligenes FC-88 microorganism can be isolated by DEAE- cellulose column chromatography. The material from peak fraction Number 5 has antibiotic activity against S. pyogenes and B. cereus, but not against S. aureus.

In another experiment, a bacterial inoculum was grown as previously described. The incubation solution was centrifuged, the supernatant was filtered, and the filtrate was loaded onto a 1 x 10cm DEAE-cellulose column as previously described. Two DEAE-cellulose columns were loaded. Elution of loaded material from these columns was done using 0.15M KCl in 25mM Tris-HCl pH 7.0 buffer. Fractions of the eluate were assayed for antibiotic activity against representative microorganisms with the following results :

These results again demonstrate that the antibiotic material can be concentrated by DEAE-cellulose chromatography. In this instance, antibiotic activity by the supernatant of the medium of Alcaligenes FC-88 against S. aureus was achieved.

The antibiotic material was also purified by Roxyn 1-300 (H⁺-0H^") column chromatography. A bacterial inoculum was grown in 2 liter flasks containing 800ml of MNM medium with lOmM malic acid as the carbon source. Incubation took place on a platform shaker operated at 200 cpm at 30°C for 2 days. The incubation solution was centrifuged at 11,950 x g for 30min. The supernatant was collected and passed through a 0.2μm filter. A 150ml portion of the filtrate was applied to a 1.8 x 10cm Roxyn 1-300 (H⁺-OH^~) column that had previously been equilibrated with distilled water. The column was washed with 30ml of distilled water, followed by 60ml each of 0.15M, 0.3M, and 0.5M NaCl solutions. Fractions (4ml) were collected at a rate of 10ml/hr. The absorbency at 260nm was monitored for each fraction and microbial susceptibility was determined for the fractions associated with the absorbency peaks. The OD₂₆₀ and microbial susceptibility for the eluant factions are shown in Figure 2.

The Roxyn 1-300 (H⁺-OH^") chromatographic procedure yielded several absorbency peaks within the eluted fractions. Two of these absorbency peaks also contained material with antibiotic potency. The microorganisms that were susceptible to the antibiotic material in these absorbency peaks included Staphylococcus aureus and Streptococcus pyogenes.

A further column chromatographic procedure was performed to purify, concentrate and assay the antibiotic activity of the material in the supernatant of growth media containing Alcaligenes FC-88. In this procedure, Alcaligenes FC-88 cells were grown in MNM medium containing lOmM malic acid in 800ml cultures in 2L flasks. Incubation took place on a platform shaker operated at 200 cpm at 30°C for 2 days. The incubation solution was centrifuged at

11,950 x g for 30min. The supernatant was collected and passed through a 0.2μm filter. A 200ml portion of the filtrate was loaded onto a 1.8 x 10cm Ag-1 x 10 column that had previously been equilibrated with distilled water. The column was then washed with 50ml distilled H₂0 followed by

50-80ml each of 13%, 30% and 60% formic acid. Fractions were collected and monitored at OD₂₆o as well as by susceptibility of methicillin resistant Staphylococcus aureus using the antibiotic disk assay as previously described. The antibiotic was eluted from the column with 60% formic acid. The results of the monitoring is shown in Figure 3. The singular peak in the antibiotic activity of the collected fractions coincided with only one OD₂₆o absorbency peaks. The material in these fractions (52-63) was combined and used in subsequent antibiotic susceptibility assays. It is noteworthy that the formic acid purified antibiotic from the supernatant of growth media containing Alcaligenes FC-88 displayed activity against methicillin resistant Staphylococcus aureus (Figure 4).

The formic acid column chromatographically purified antibiotic was assayed for antibiotic susceptibility against a variety of microorganisms using the procedures previously described. The results of these assays are shown in Table 2.

Table 2

Antibacterial Activity by Antibiotic Produced by

Alicaligenes FC-88

These results show that this antibiotic is effective against a wide range of microorganisms including several microbes that are resistant to many other antibiotics.

The fractions from any one of the chromatographic procedures that contain the peak antibiotic activity can be pooled and the contents concentrated by air or vacuum dessication. Alternatively, the contents can be concentrated by lyophilization. The antibiotic material can also be concentrated by passing the solution containing the material through a desalting Sephadex G10 column with the collection of fractions that contain the peak activity (which corresponds to the peak OD₂₆o) •

When chloroethane, ethanol or methanol are added ca 1:1 to the chromatographic fractions that contain the antibiotic material, the material does not precipitate.

The antibiotic material is stable at pH as low as 2-3. The material will retain antibiotic activity when stored at 4°C for over 6 months.

The antibiotic has a molecular formula of CιoHι₈N0₅ with a formula weight of 246 as determined from

NMR, mass spectrometry and FTIR of the material purified by the chromatographic techniques. The antibiotic has been given the name: Thomacin.

Example 5 Isolation and Characterization of Haloperoxidase from Alcaligenes FC-88

A sample of the bacterium isolated in Example 1 was grown in a 2L flask containing MNM medium with L-malic acid as the carbon source. Incubation took place with shaking at 2,000rpm at 28°C for 72h. The incubation solution was then centrifuged at 11,950 x g for 30min. The pellet was discarded and the resultant liter of supernatant was passed through a 0.2μm filter. A 200ml portion of the filtrate was placed on a 1.8 x 12cm Ag-lx8 column that had been pre-equilibrated with distilled H₂0. The column was then washed with 50ml of distilled H₂0. The haloperoxidase enzyme was eluted from the column with 100ml of a 0-13% formic acid gradient pH 4.5. Fractions of 4ml were collected. The fractions containing peak haloperoxidase activity were pooled and concentrated by centrifugal ultrafiltration. The molecular weight of the native haloperoxidase obtained from the Alcaligenes FC-88 growth medium supernatant was determined by gel permeation chromatography on a calibrated 1.8 x 45cm Bio-Gel A-0.5M column (Bio-Rad, Richmond, Calif.). The column was equilibrated at 4°C with 50mM Tris-HCl buffer, pH 6.0. The molecular weight standards and the haloperoxidase were detected spectrophotometrically at 280nm. The molecular weight of the haloperoxidase was independently determined by polyacrylamide and SDS polyacrylamide gel electrophoresis carried out by the methods of Laemmli (1970) . Samples for the denaturing (SDS) gel were boiled in Laemmli sample buffer for 5min before loading. The gels were run for 2-3h at 120V in a Bio-Rad mini slab gel apparatus. Molecular weight standards for the SDS gels were phosphorylase B (97,400), bovine serum albumin (66,200), ovalbumin

(42,700), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400). The proteins in the gels were visualized with Coomassie Brilliant Blue R. The metal content of pure enzyme solutions was also determined.

The enzyme characteristics of the haloperoxidase were determined using the following reaction system when phenol was the substrate: enzyme 100-200μl, H₂0₂3% lOμl, substrate (e.g. phenol) 0.1M, lOOμl; KBr 0.1M, lOOμl or KCl 0.1M, lOOμl; KH₂P0₄/K₂HP0₄ 0.1M buffered at pH 7.0 to final amount of 1ml. The products of the enzyme reaction were determined by gas chromatography (GC) , HPLC or by TLC.

For the GC determinations (Chen, et al . , 1991, J. Biol . Chem. 266: 23,909-23,915), the enzyme reaction mixture was extracted with 2ml of pentane. The extract was placed in a Varian model 3,300GC equipped with a fused silica capillary column (15m x 0.53mm ID) coated with a 1.5μm film of cross-linked SE-54 phase (DB-5, J&W Scientific, Folsom, CA) . The analyte separation was monitored by an electron capture detector with integrator. The sample size of 0.1ml was chromatographed with the aid of a temperature program (100°C to 200°C at 10°C/min) with N₂ flow of 30ml/min. For the HPLC determinations, the column was Bond Pak 39 x 300mm (Waters Part No. 27324) with a running buffer of 0.2M ammonium acetate pH 5.6. Acetonitrile (15ml) was added to 100ml of this buffer and the resulting solution was taken to 1 liter with distilled H₂0. The enzyme was eluted with this solution. For the TLC determinations the samples were run on silica gel, TLC (Merck) with distilled H₂0: MEOH of 1:1. The substrate and hydrogenated product were distinguished by their characteristic Rf values.

The Ag-lx8 column chromatographic separation of the filtrate obtained from Alcaligenes FC-88 growth media yielded two symmetrical peaks of which one coincided with peak halogenation activity (Figure 5) . The enzyme elution occurred at a lower concentration of formic acid.

Based on the molecular weight determinations, the molecular weight of the haloperoxidase enzyme is 34,000. The enzyme is a monomer and is not composed of subunits.

The enzyme halogenates a wide variety of organic compounds. For example, phenol can be halogenated to produce mono-, di- and tri-halogenated phenol compounds. Thus, 4-bromophenol, 2, 4-dibromophenol, 2,4,6- tribromophenol, 4 chlorophenol, 2,4-dichlorophenol and 2,6- dichlorophenol can be produced from phenol when the haloperoxidase and either KBr or KCl are present. The enzyme oxidizes Cl^~, Br^" and I^" with optimum pH values of 4.5, 5.0 and 6.0, respectively, in the presence of 400μM H₂0₂. The enzyme has halide-independent catalase activity in the absence of an organic substrate. The results of further halogenation studies are shown in Tables 3 and 4. Table 3

Chlorination of Organic Compounds by Alcaligenes . FC-88

Haloperoxidase

Reaction: organic compound l-2mg/ml; KCl IM, 20μl; H₂0₂ 3%, lOμl; and buffer NaAc 0.1M pH 3.5, brought to final volume of 1ml. Incubation was at 28°C overnight.

Table 4 Bromination of Organic Compounds by Alcaligenes . FC-88

Reaction: organic compound l-2mg/ml; KBr IM, 20μl H₂0 3%, lOμl; and buffer KH₂P0₄ 0.1M pH 6.0, brought to final volume of 1ml. Incubation was at 28°C overnight.

These results demonstrate the range of organic substrates that can be halogenated by the enzyme.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS :

1. An isolated Alcaligenes spp. which produces an antibiotic that is active against Serratia marcescens, Micrococcus leuteus, Escherichia coli. Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, Enterococcus and Pneumoniae .

2 . An isolated Alcaligenes spp. according to, claim

I, wherein said Staphylococcus aureus is methicillin resistant Staphylococcus aureus .

3. An isolated microorganism according to claim 1 or Claim 2, wherein said Alcaligenes is Alcaligenes FC-88.

4. An isolated Alcaligenes spp. which produces a haloperoxidase that halogenates phenol; 2,5- dihydroxybenzoic acid; flavone; toluic acid; . p- aminobenzoic acid; p-hydroxycinnamic acid; 3,4- dihydroxybenzoic acid; p-hydroxybenzoic acid; 2,3- dihydroxybenzoic acid; 4-hydroxy-3, 5-dimethoxybenzoic acid; 3, 5-dihydroxybenzoic acid; and 6-deoxy-L-galacto pyranose.

5. An isolated Alcaligenes spp according to claim 4 wherein said Alcaligenes is Alcaligenes FC-88.

6. An antibiotic produced by an Alcaligenes spp. that has antibiotic activity against Serratia marcescens, Micrococcus leuteus, Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus cereus,

Staphylococcus aureus, Enterococcus and Pneumoniae .

7. An antibiotic according to claim 6, wherein said antibiotic is produced by Alcaligenes FC-88.

8. An antibiotic according to claim 7, wherein said antibiotic is present in Alcaligenes FC-88 growth media after incubation of said Alcaligenes FC-88 in said growth media.

9. An antibiotic produced by Alcaligenes FC-88.

10. An antibiotic according to claim 9, wherein said antibiotic has a molecular formula of Cι₀Hι₈N₂0₅.

II. An antibiotic according to claim 9 or claim 10, wherein said antibiotic has antibiotic activity against methicillin resistant Staphylococcus aureus.

12. An antibiotic according to any one of claims 9 to

11, wherein said antibiotic has antibiotic activity against vancomycin resistant Enterococcus .

13. An antibiotic according to any one of claims 9 to

12, wherein said antibiotic has antibiotic activity against clinic isolate Enterococcus (VRE) #83.

14. An antibiotic according to any one of claims 9 to

13, wherein said antibiotic has antibiotic activity against clinic isolate Staphylococcus (MRSA) #68.

15. An antibiotic according to any one of claims 9 to

14, wherein said antibiotic has antibiotic activity against clinic isolate Pseudomonas aeruginosa #97.

16. An antibiotic according to any one of claims 9 to 15, wherein said antibiotic has antibiotic activity against clinic isolate Escherichia coli #32.

17. An antibiotic according to any one of claims 9 to

16, wherein said antibiotic has antibiotic activity against clinic isolate Pneumoniae #79.

18. An antibiotic according to any one of claims 9 to

17, wherein said antibiotic has antibiotic activity against the clinic isolate CitroJbacter freundii that is resistant to Ampicillin, Cefazolin, Cefuroxine, Cephalothin, Ciprofloxacin, Gentamicin, Levofloxacin, Minocycline, Nalidixic Acid and Norfloxacin.

19. A haloperoxidase produced by an Alcaligenes spp. that halogenates phenol; 2, 5-dihydroxybenzoic acid; flavone; toluic acid; p-aminobenzoic acid; p- hydroxycinnamic acid; 3, 4-dihydroxybenzoic acid; p- hydroxybenzoic acid; 2, 3-dihydroxybenzoic acid; 4-hydroxy- 3, 5-dimethoxybenzoic acid; 3, 5-dihydroxybenzoic acid; and 6-deoxy-L-galacto pyranose.

20. A haloperoxidase according to claim 19, wherein said Alcaligenes is Alcaligenes FC-88.

21. A haloperoxidase according to claim 19 or claim

20, wherein said haloperoxidase is present in the growth media after incubation of said Alcaligenes spp. in said growth media.

22. A haloperoxidase according to claim 21, wherein said haloperoxidase is recoverable from said growth media without cytolysis of said Alcaligenes spp.

23. A haloperoxidase according to any one of claims 19 to 22, wherein said haloperoxidase is secreted by said Alcaligenes spp. into the medium surrounding the Alcaligenes spp microorganisms.

24. A haloperoxidase according to any one of claims 19 to 23, wherein said haloperoxidase has a molecular weight of about 34,000 daltons.

25. A method for producing an antibiotic with antibiotic activity against Serratia marcescens, Micrococcus leuteus, Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, Enterococcus and Pneumoniae comprising the step of incubating a Alcaligenes spp. in a suitable growth medium under appropriate conditions whereby said antibiotic is produced.

26. A method according to claim 25 wherein said antibiotic is recoverable from said growth medium without cytolysis of said Alcaligenes spp.

27. A method according to claim 25 or claim 26, wherein said antibiotic has a molecular formula of Ci₀H₁₈N₂0₅.

28. A method for producing an antibiotic with a molecular formula of Cι₀Hι₈N₂0₅ comprising the step of incubating Alcaligenes FC-88 in a suitable growth medium under appropriate conditions whereby said antibiotic is produced.

29. A method according to claim 28, wherein said antibiotic is recoverable from said growth medium without cytolysis of said Alcaligenes FC-88.

30. A method for producing a haloperoxidase that halogenates phenol; 2, 5-dihydroxybenzoic acid; flavone; toluic acid; p-aminobenzoic acid; p-hydroxycinnamic acid; 3, 4-dihydroxybenzoic acid; p-hydroxybenzoic acid; 2,3- dihydroxybenzoic acid; 4-hydroxy-3, 5-dimethoxybenzoic acid; 3, 5-dihydroxybenzoic acid; and 6-deoxy-L-galacto pyranose comprising the step of incubating a Alcaligenes FC-88 in a suitable growth medium under appropriate conditions whereby said haloperoxidase is produced.

31. A method according to claim 30, wherein said haloperoxidase is recoverable from said growth medium without cytolysis of Alcaligenes FC-88.

32. A method according to claim 31, wherein said haloperoxidase is secreted by said Alcaligenes FC-88 into said growth medium.

33. A method according to claim 32, wherein said haloperoxidase has a molecular weight of about 34,000 daltons .

34. A method for producing a haloperoxidase comprising the step of incubating Alcaligenes FC-88 in a suitable growth medium under appropriate conditions whereby said haloperoxidase is produced.

35. A method according to claim 34, wherein said haloperoxidase is recoverable from said growth medium without cytolysis of said Alcaligenes FC-88.

36. A method according to claim 35, wherein said haloperoxidase is secreted by said Alcaligenes FC-88 into said growth medium.

37. A method of treating a bacterial infection comprising the step of administering to a mammal in need thereof an effective amount of an antibiotic according to any one of claims 1 to 18.

38. A method of treating a bacterial infection comprising the step of administering to a mammal in need thereof an effective amount of a haloperoxidase according to any one of claims 19 to 24.

39. A method of sterilisation comprising the step of treating an article with an effective amount of haloperoxidase according to any one of claims 19 to 24.