DE2320696A1 - PROCESS FOR THE PREPARATION OF DEACETOXYCEPHALOSPORIN C - Google Patents

Description

Eli Lilly and Company, Indianapolis, Indiana, V.St.A. Process for the preparation of deacetoxycephalosporin C

The invention relates to a process for the production of deacetoxycephalosporin C, which is characterized in that that in an aqueous culture medium under submerged, aerobic remote orientation conditions, a microorganism which forms a penicillin N and belongs to the species Cephalosporium, Emericellopsis, Scopulariopsis, Paecilomyces or Diheterospora until a substantial amount of deacetoxycephalosporin C is formed by the microorganism in the culture medium wui'dε, breeds and then the deacetoxycephalosporin C from the Culture medium isolated.

The cephalosporin compound deacetoxycephalosporin C is represented by the following structural formula:

ON it t

HOOC - CH - (CH ₂ ) ₃ - C - N

NH ₂ 0

COOH

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- ² ", 232069Q

It differs from cephalosporin C in that the acetoxymethyl group in the 3-position of the dihydrothiazine ring of cephalosporin C has been replaced by a methyl group. This structural difference is suitably expressed by the prefix desacetoxy or deacetoxy in the name of the depicted cephalosporxn compound. Alternatively, the formal name, namely 3-methyl-7- (5'-amino-5 ^t -carboxyvaleramido) -3-cephem-4-carboxylic acid, which corresponds to the Cepham nomenclature system, is often used. For the sake of simplicity, the compound prepared according to the invention is referred to as "deacetoxycephalosporin".

Deacetoxycephalosporin C is a valuable intermediate that can be used to make cephalosporin antibiotics. For example, it can be with nitrosyl chloride under the conditions described in U.S. Patent 3,188,311 be implemented, whereby the aminoadipoyl side chain is removed and 7-aminodeacetoxycephalosporanic acid (7-ADCA) is formed. The 7-ADCA can be acylated with the desired acyl group, forming the cephalosporin antibiotic will. For example, 7-ADCA can be prepared by known methods with an active ester, with mixed anhydrides or others suitable derivatives of phenylglycine are acylated to form the known antibiotic cephalexin.

Deacetoxycephalosporin C was previously prepared by hydrogenolysis of the cephalosporin C according to the method described in US Patent 3,124,576. Because of the usefulness of this compound as an intermediate in the manufacture of cephalosporin antibiotics, there is a need for other more economical processes for the manufacture of this compound.

Fungi that form penicillin N (Cephalosporin N) of the species Cephalosporiura, Emericellopsis, Scopulariopsis > Paecilo- -nyces and Diheterospora are in an aqueous culture ^ ·

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nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts, under submerged, cultivated aerobic fermentation conditions, whereby deacetoxycephalosporin C is formed. Deacetoxycephalosporin C is separated from the other substances formed at the same time, such as cephalosporin C, penicillin N and desacetylcephalosporin C. separated by first the entire reaction solution acidified with a mineral acid such as sulfuric acid to a pH of approximately pH 2. The acidified solution will then stirred at room temperature for about 1 hour. During this time the acid-sensitive compounds become decomposed. The pH is then brought back to a pH value by adding a suitable base such as sodium hydroxide set from 6.0. The mycelium and other insolubles are filtered off and the filtered culture medium is mixed with extracted with a water-immiscible organic solvent to remove further impurities. That Deacetoxycephalosporin C is obtained from the aqueous semi-purified culture medium by chromatography in the following manner (If in the present application of culture medium or solution is spoken in the cultivation of microorganisms, it is understood to mean the medium that is used in the cultivation receives. This can also be referred to simply as "broth". The word "medium" is used in this context in the sense of English word "broth" used.) The aqueous medium is first chromatographed over activated carbon. the Carbon column is washed with water, to remove impurities further to remove, and then the activity is eluted with 50% aqueous acetone. The eluate is concentrated and passed through an anionic exchange haz. The deacetoxycephalosporin C is thereof, preferably with a 0.15N sodium acetate solution, eluted. The eluates are then evaporated to dryness or lyophilized, using a crude deacetoxycephalosporin C preparation received. The raw material is further chromatographed on a cellulose column or cleaned over silica gel. The active factions, the Deacet-

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oxycephalosporin C are either concentrated to a small volume or evaporated to dryness. That Concentrate or the dry solid residue is dissolved in a minimal amount of isopropanol and the solution becomes poured into diethyl ether to precipitate deacetoxycephalosporin C as the sodium salt.

In the method according to the invention, fungi of the species Cephalosporium, Emericellopsis, Scopulariopsis, Paecilomyces and Diheterospora, which produce penicillin N, in a nutrient medium cultured containing assimilable sources of carbon, nitrogen and inorganic salts, wherein Deacetoxycephalosporin C is formed.

The microorganisms useful in the method of the invention have been found with the exception of Emericellopsis as members of the Fungi imperfect class! classified because they do not form sexual spores. You will continue into the class Moniliales, the family Moniliaceae and the species Cephalosporium, Scopulariopsis, Paecilomyces and Diheterospora classified. The microorganisms of the present invention that produce ascospores have been incorporated into the Species Emericellopsis, the consummate sexual state of Cephalosporium. The organisms are accordingly classified as Ascomycetes of the Eurotiales class in the Eurotiaceae family.

The above classification of fungi useful in the present invention is based on those observed morphological and cultural characteristics. A taxanomic description of a number is given in the following sections of fungi, which are considered examples of the respective species.

The reference to "Maerz and Paul color plates" relates refer to the color plates in Maerz and Paul, Dictionary of

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Color, McGraw-Hill Book Co., Inc. New York (1950).

General morphology - Cephalosporium

The spore-bearing cells are phialides that. straight from the vegetative hyphae or from germ duct fibers of the hyphae or from coremia. The phialides are hyaline, conical, i.e. tapering to a point, and form phialospores (= vial spores) at the tip, which are either in the form formed by beads or as fragile chains. The spores are generally non-septate, spherical to short cylindrical. The colonies vary from smooth to moist without aerial mycelium to granular and flaky with aerial raycel. The mycelium generally contains funicular hyphae. The color the colony is diverse and ranges from colorless, i.e. white, to yellow to pink and dark gray or brown.

Morphology and Cultural Characteristics - Cephalosporium Strains

In the following taxanomic descriptions of Cephalosporium sp., The cultures form an incomplete stage or a "step imperfecti", which is characteristic of the species Cephalosporium.

Cephalosporium sp. NRRL 54-4-5

Sporogenic cells are formed on unbranched phialides, which are arranged alternately, and arise directly from the synnematous hyphae. Phialids have essentially uniform diameters, with the tips are slightly conical. "The phialides are unicellular and have an average diameter the base of 2.8 / u, their length ranges from 14 to 25 / u and averages 21 / u. The phialospores generally occur in chains, but usually terminate in clusters of spores in older crops. The vial spores are hyaline, but appear slightly ocher to medium yellow-pink in the mass (Maerz and Paul color plate 2-9A).

I ,, I, _#ii ^

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The vial pores range in size from 1.4 to. 4.2 / U χ 2.8 to 4.9 / U, an average of 2.4yU χ 3.5 / U. The spurs are polysymmetrical and oval to oboval in overall shape.

The growth of the fungus is different on different media » Cultures grow rapidly on rich media such as V-8 Juice Agar and Lactose-Glycerin-Peptan-Agar and reach diameters larger than 50 mm in 10 days. The air growth is flaky, white at first and then turns slightly ocher in older crops. the Aerial hyphae generally contain erect to semi-erect Synnemata, with some prostrate or prostrate funiculare. Hyphae are present. On synthetic Media, growth is slow, aerial mycelium is scarce or absent, and sporulation is scarce. The vegetative Color is medium orange (Maerz and Paul plate 10-7F). *

Cephalosporium chrysogenum ATCC 14615 A taxanomic description of this fungus is given by R.S. Sukapure and M.J. Thirmalachar, Studies on Cephalosporium Species from India I, Mycologia LV, 563-569 (1963).

Cephalosporium sp. NRRL 5712

Colorless aerial hyphae appear on all subsequent agar colonies in 3 days and yellowish-pink tones in 5 days.

Colonies grown on Czapek ¹ s solution agar show excessive flaky through. funicular hyphae with both erect and prostrate synnemata. They are radially folded, round, and have a complete, unbroken margin, and reach 38 mm from a "16 mm" zone. After 14 days, no fruiting or seeding occurs in these colonies They are moderately yellowish pink, their reverse color is an intense orange.

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Potato dextrose agar colonies are slightly grayish yellow. The colonies expand from an inoculated zone with a diameter of 18 mm to a zone with a Diameter of 37 mm. P.D.A. Colonies (Potato Dextrose Agar) show poor aerial hyphae formation, they appear velvety and are funicular and are erect and prostrate synnemas. The phialides are excessive and arise from the synnemata, funiculi and the proliferating ones Hyphae and form conidia in chains and in moist ones Tangles. This medium is used to further study the conidial stage.

Malt extract agar gives colonies with a diameter of 37 mm from an inoculated zone 16 mm in diameter. The colonies are flaky to funicular or funicular and yielded most of the synnemata, which is conidic stage however rare. The colonies are moderately yellowish-pink and the Reverse color is faint orange "

Modified Y-8 juice agar gives a colony that contains one 37 mm in diameter based on an inoculated k-one with a diameter of 17. The aerial hyphae are somewhat longer than those of the colonies of Czapelsisdiuras The colony is moderately yellowish pink and the reverse color is brownish-yellowish-orange. It is flaky to funicular " with mainly underlying synnemata. The phialides with conidia occur only in moderate numbers.

The phialides are hyaline, pointed and have lengths in the range of 55.8 to 92.1 / u, the average length is 66.2 / rev. They are tapered from 2.3 / u in "to the base to a width of 0.5 / U at the top.

The unicellular, hyaline, elongated to elliptical Conidia have lengths in the range from 5.6 to 7.7 / u and Widths in the range from 2.5 to 3.5 / u; their average Size is 6.8 to 3.1 / U. . ■, ■

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Cephalosporin sp. NRRL 5716

Form colonies on the agar media described above no macroscopically visible, free aerial mycelium, but rather an almost smooth, flat, moist, pasty one Mycelium, which is marked by slightly radial folds.

The solution agar from Czapek results in a colony consisting of an inoculated area with a diameter of 20 mm can reach a diameter of 32 mm. The phialides arise from tightly packed, prostrate synnemata and spread randomly from the mycelial surface. This colony is pale yellowish-white and has a smooth overall periphery. The reverse color is -white. It educates a soluble, pale yellowish green pigment.

The potato dextrose agar colonies resemble "the Czapek colonies in size, color, phialid formation, consistency and pigment. They differ in that the potato dextrose colonies are delimited by a narrow, radially folded, fringed edge. After 14 days the soluble pigment becomes more intense.

Malt extract agar colonies are similar to those on the solution agar by Czapek, but the soluble pigment formation does not occur. This colony is on both of them for up to 7 days Surfaces white and after 14 days it turns pale greenish yellow on both surfaces. . _.

Modified V-8 juice agar yields a colony that grows from distinguishes the above colonies in various properties. It is light brown and has relatively deep, narrow radial folds. The reverse color is pale brown and that Mycelium penetrates the agar well. Loose, colorless aerial hyphae tufts dot the surface.

Microscopically, this colony consists of a mat of closely

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woven, irregular hyphae, the cells of which are swollen and distorted. The growth rate is similar to that of colonies on Czapek agar solution. The formation of phialides is greatest on this medium. They form wastefully from the colony surface and from the hyphae, which are present as tufts. These phialideh are hyaline, unicellular and pointed and are material outgrowths of the hyphae filaments. Their size is in the range from 20 to 46.5 / U length and from 2.3 to 4.7 / U width at their base, on average they are 39.37 / U χ 4.65 / U. At the tips they are 0.5 / U wide.

The conidia are connected in chains or moist tufts. They are hyaline, unicellular, smooth, and in general spindle-shaped. A few are sausage-shaped with almost pointed ends, some are boat-shaped. They are from 3.5 to 7.0 / U long and from 1.4 to 2.1> u wide, their average size is 5.0 / U χ 1.5 / U.

Cephalosporin sp. NRRL 5718

Colony formation in aerial mycelium on solution agar from Czapek is moderate and the colonies are colorless. The hyphae are irregular and have swollen, distorted cells.

Potato dextrose agar gives a flat, ringed or Stripe-drawn colony with alternating bands that differ in the amount of air growth. The growth is funicular and moderately synnematic, with phialides arising from the funiculi, synnemata and proliferating hyphae, the conidia form. The moist conidia form chains or tufts. This colony is weak on both surfaces yellowish-white. It grows from an inoculated area with a diameter of 17 mm to a colony that has a Has a diameter of 40 mm, in 10 days at 26 ° C.

The conidia, the drawing with rings or stripes,

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- ΊΟ -

hypha formation and colony size are on malt extract agar same as on potato dextrose agar. · One gets 4 mm colorless border. The malt extract colony is weak greyish-white and the reverse color is pale yellowish-white.

Although the growth rate can be compared to that of the above media, colonies show modified V-8 juice agar the strongest overgrowth of hyphae and conidia. This colony is flaky to funicular and contains both erect and prostrate synnemas. It is supported by a 3 mm wide, flat, · almost surrounded by a velvety edge that is somewhat wavy. The mycelium is pale yellowish-pink and slightly greyish-white on the Edge. The reverse color is light grayish-dark yellow. The colony border is in sharp contrast with the main part the colony.

The phialides are hyaline, unicellular and pointed structures. They are from 18.9 to 29.4 / u long and 2.8 / u at their base wide, the average size is 24.6 χ 2.8 / u.

The conidia are hyaline, unicellular and elliptical. You are = from 3.5 to 4.2 / U long and 2.1 / U wide, the average Size is 4.1 αϊ χ 2.1 / u.

Cephalosporium sp. NRRL 5719

This unfinished state of the mushroom is called the genus Cephalosporium corda classified. It does not become a completed stage educated.

The colonies are · rar, colorless to Lösungsagar of Czapek and non-spore forming up to 10 days at 26 ⁰ C. The presence of numerous spores on the Agaroberflache indicates that no germination occurs on this agar. The colonies on corn extract agar tend to grow relatively flat and have an inoculated area with a diameter of 17 mm

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gives colonies with a diameter of 29 mm. The periphery is sharply defined and slightly curved. Occasional tufts of flaky to funicular hyphae extend over the lower, proliferating hyphae surface. These colonies are very pale pinkish-white and the reverse color is white. Some phialides with terminal chains and tufts of conidia arise from the proliferating funiculi and individual hyphae filaments. Potato dextrose agar gives a flat, notched, or serrated colony that is oyster white on both faces and that grows from an inoculated area 18 mm in diameter to a colony 26 mm in diameter. Interwoven The aerial mycelium contains slightly flaky hyphae loosely with funiculösen Hyphensträn- gen from which numerous phialids arise, some of which are branched laterial. The conidia form at the tip of the phialides in moist tufts and rarely in chains. With age, the spores collect in the moisture on the hyphae filaments and funiculi. Modified V-8 juice agar gives the best air growth. The white aerial hyphae turn beige with numerous spores. The surface is irregularly flaky and contains both fun. ⁴ ouli as well as synnemata and shows a slightly notched edge. The phialides form laterally on both the funiculi and the synnemata. The sporulation occurs like on potato dextrose agar.

The phialides are unicellular, hyaline and from 17 to 26.4 / u long and 3 / u wide at their base. The average . ■ Size is 11 / u χ 3 / U, tapering to 1.5 ai.

The conidia are hyaline, thick-walled, somewhat rough and spherical \ shape to elliptical. They are from 4.2 to 5> 25vU long and from 2.1 to 3.5 / U wide, their average size is 4.64 / U χ 2.85 / U.

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Cephalosporium sp. NRRL 5720

Growth on solution agar from Czapek at 26 ° C during 10 days is moderate and aerial mycelium is not formed. The colony is mostly below the surface or in the water, only widens 3 to 5 mm and has an indefinite edge.

After 10 days at 26 ^° C., potato dextrose agar results in a colony which enlarges from an inoculated zone with a diameter of 12 mm to a zone with a diameter of 37 mm. This colony is drawn with rings and stripes and contains alternating bands with and without aerial mycelium. It is flaky to funicular and synnematic. The colony is yellowish white and the reverse color is pale yellow. The phialides arise from the funiculi, the synnemata and the proliferating hyphae.

Malt extract agar colonies reach a diameter of ' 31 mm when growing on an inoculated area with a diameter of 16 mm. The colony is marked with rings and stripes and has a narrow, unbroken border Air hyphae limited. The drawing with rings and stripes, the conidial steps and color are as described for the colonies on potato dextrose agar.

Modified. V-8 juice agar gives colonies that make up the most Contains aerial mycelium. These colonies grow into one from an inoculated area with a diameter of 13 mm Colony with a diameter of 37 mm. The aerial mycelium is dense and woven together, strongly funicular, flaky and almost wool-like. The colony is pale yellowish pink. The reverse color shows alternating rings that are faint yellowish-tan in the center than a darker ring, etc.

The phialides are long, pointed, tubular, with hyaline structures, which are bulbous in their base. she

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are from 15.4 to 24.5 / u long, from 2.1 to 3.5 / u at their Base wide and have average sizes of 19.9 x 2.5 / U.

The conidia are generally smooth, some are warty, hyaline and 'spherical to sub-spherical.' They have a diameter ranging from 2.8 to 4.9 / u and average diameters of 3.65 / rev.

Cephalosporium sp. NRRL 5721

On Czapek agar · growth is economical, non-pigmented and non-spore forming. The colonies on malt extract agar reach a diameter of 28 mm ^{in 7 days at 26 °} C. from an inoculated zone with a diameter of 15 mm. The aerial mycelium is flaky to velvety. Microscopically it is funicular and provided with synnemata that are either erect or lying. The colony is drawn with rings or stripes, shows a border and an inner border with different tints. It is moderately yellowish pink and is surrounded by a border that is pale yellowish pink. The reverse color is pale yellowish green.

Potato dextrose colonies growing from a zone of inoculum with a diameter of 15 mm are notched by a ... colorless, veil-like border, which is 8 mm wide. The main surface of the colony has one Diameter of 30 mm and contains a flaky to funicular, white, short air felt. After 14 days the colony turns pale yellow-brown, the reverse color is moderately orange-yellow, with the periphery having a weaker tone.

Modified V-8 juice agar colonies grow from an inoculated one Zone with a diameter of 15 mm to zones with a diameter of 38 mm including a 3 to 4 mm wide, veil-like, colorless, somewhat notched edge. The main body of the colony contains funicular hyphae filaments that

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are woven together in a ribbon-like pattern. The colony is somewhat flaky and shows slight radial folds. she is pale brown with a yellowish-brown reverse color.

All agars show synnematic - 'hyphae, but this condition is best observed on potato dextrose and modified V-8 juice agar. · Smooth, unicellular, hyaline Phialides arise from the synnemata and the proliferating funiculi and are found in the potato-dextrose agar colonies described.

These phialides are in the range from 9.1 to 38.5 / U long and from 2.1 to 2.8 / U broad at their base. You own average Sizes from 22.26 / u χ 2.5 / u and are pointed to 0.7 / U. The conidia are unicellular, smooth, hyaline and spherical to sub-spherical. They are 2.8 to 4.9 / U long and 2.8 to 4.2 / U wide, on average they have Sizes from 3.6 / u χ 3.1 / u.

Cephalosporium sp. NRRL 5722

On solution agar from Czapek they reach a diameter of 32 mm from inoculated areas of 14 mm. The mycelium is flat, colorless on both surfaces, radially furrowed and free from aerial mycelium. Malt extract agar colonies grow into colonies with a diameter of 36 mm from an inoculated area with a diameter of 20 mm. The aerial hyphae are funicular and have both upright and lying synnemata, they are radially furrowed and have a flat, surrounded by a smooth, 2 mm wide margin. The phialides arise from both the funiculi and the synnemata with good conidial development. Malt extract colonies are pink-yellow-brown and their reverse colors are medium-yellowish-white. the Colonies growing on potato dextrose agar are funicular, appear to be velvety and contain short, erect hyphae filaments. The conidia are rare. The culture results in a grayish white colony with a medium gray

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brown, 2 mm wide border and the reverse color is medium yellow. It reaches a diameter of 30 mm from an inoculated area of 16 mm diameter. On modified V-8 juice agar, the colonies have pale grayish-white aerial hyphae that turn beige to buff-colored when sporulated. The reverse color is light brown with a yellowish-white, 3 mm border or hem. The inoculated area with a diameter of 15 mm grows to a colony having a diameter of 34 mm in 10 days at 26 ⁰ C. This colony contains a relatively deep, flake to funiculären airfelt and is strongly synnematon.

The conidial stage was observed in all three media. The phialides are hyaline, unicellular, conical and pointed. In their base they have widths in the range from 1.4 to 3.5 / u and taper to 1.4 / u; they are from 10.5 up to 25.9 / U long and the average size is 20.2 / U χ 3.75 / U. The conidia are unicellular, hyaline, elliptical to oval to oboval and form either chains or slimy (mucoid) tufts. They are from 2.1 to 2.8 / u wide and from 3.5 to 4.9 / u long, the average size is 4.0 / U χ 2.45 / U.

Cephalosporium sp. NRRL 5723

This culture is characterized by its imperfect level and belongs to the species Cephalosporium corda. No perfect level is evident. The growth is on all agars limited and a series of irregular lobes are observed extending radially from the inoculated site.

A colony is formed on solution agar from Czapek, which is irregularly notched to lobular (= small-lobed) and which The rags are fringed. The irregular flap formation results in differences in diameter that range from 25 to 30 mm and expand from a 12 mm inoculated zone.

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The free air components are rare and become microscopic observed They consist of scattered phialides, synnemata and aerial hyphae fibers. This colony is on both surfaces colorless.

Colonies on potato dextrose agar are deeply and irregularly lobed and form only limited white aerial hyphae and are surrounded by a smooth, 3 mm wide border. They have diameters ranging from 30 to 35 mm. As well as the erect as well as the lying synnemata result in phialides, which form spores in chains or moist clusters. The vegetative mycelium is flat, light grayish-brown, the reverse color is pale yellowish-brown.

The malt extract colonies are strong and irregularly small-lobed and they have a narrow, radially folded edge. They are medium-brownish-pink, the reverse color is weak yellowish pink. The formation of phialides and conidia is similar as with the potato dextrose colonies, but stronger.

Modified V-8 juice agar yields a colony that varies in diameter up to 40 mm, starting from an inoculated area with a diameter of 15 mm. An uneven, The colony is surrounded by a small-lobed, colorless, 3 mm wide margin located below the surface. This colony is flaky to funicular, with the synnemata and aerial hyphae appear as a flat surface and a denser and deeper growth is observed at the edge. It is weak brownish-pink and the reverse color is schwaqh brownish-yellow. The phialides arise from the synnemata, funiculi and proliferating hyphae. /

On potato dextrose agar and on modified V-8 juice agar Phialides are observed in the colonies and from these Colonies described. They are hyaline, ..unicellular, with bulbous base and they slowly taper to one

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Tip with a width of 1 / u. They are average from 12.5 Ms 23.0 / U long and at their base from 2.1 to 2.8 / U wide. The average size is 19.0 χ 2.3 / u.

The conidia are unicellular, hyaline, smooth, and elliptical to oval. They are from 2.8 to 5.6> u long and from -2.1 to i 3.5 / u wide. The average size is 4.48 ^ u χ 2.8 / rev.

Cephalosporium sp. NRRL 5724

Air-free hyphae are rare on all media described below and appear either as flaky tufts or as scattered individual strands on a moist-looking " Mycelium on.

Inoculum which was applied to an area with a diameter of 17 mm on solution agar according to Czapek and which was ^{incubated at 26 °} C. for 10 days, grows into a colony with a diameter of 39 mm. Jagged spots on an unevenly jagged periphery seem to connect in the center of the colony by radial rows with less development. Scattered phialides arise on the flat mycelium surface and support moist tufts of conidia. The mycelium is pale yellowish-white. The reverse color is white.

Malt extract agar colonies grow from an inoculated area with a diameter of 18 mm to colonies with a diameter of 37 mm. This type of colony "has a 4 mm wide Edge that is veil-like and the outer edge of which is unclear. The main part of the colony is flat and smooth and possesses a circular pattern of irregularly arranged tufts separating the colony from the edge. Scattered Phialides, bearing moist conidia balls, occur randomly over the pale pinkish-yellow-brown surface. The reverse color of the colony is pale yellowish white.

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Potato Dextrose Agar yields when used with a zone 14 mm in diameter, a colony 35 mm in diameter. The edge is 4 mm wide, veil-like and mainly submerged. This colony is flat and smooth and contains scattered, flaky up funicular tufts of hyphae. The phialides form conidia in short chains or moist clumps, but are relatively rare and well-dispersed on the surface. The conony is pale orange-yellow on both sides, but slightly less colored on the opposite side. The colored part is irregularly lobed. .

Modified V-8 juice agar colonies grow from an inoculated one Area with a diameter of 15 mm to colonies with a diameter of 34 mm. A egg-like, 3 bis A 7 mm wide margin surrounds a denser central surface, the The edge is irregularly lobed or frayed. Free air components are sparse. The almost doughy, flat central Part is pale yellowish-white on both surfaces. The mycelial cells are swollen and distorted. It won't soluble pigment formed. The culture on this agar resembles C. chrysogenum.

The vial spores are smooth, unicellular, hyaline, and elliptical. They are from 4.2 to 5.6 λι long and from 2.1 to 3.5 / u wide, the average size is 5.27 / U χ 3.58 λι.

The phialides are hyaline, tubular and are formed laterally on proliferating hyphae. They are from 10.5 to 19.6 / rev long and from 2.1 to 3.5 / u wide at their base. The average Size is 15.89 / U χ 2.5 / U. They run conical and are 1.0 / u wide at their tips.

Cephalosporium sp. NRRL 5725

This culture hardly grows on solution agar according to Czapek and forms a non-pigmented myelium with an indefinite border.

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The culture grows moderately well on potato dextrose agar and on malt extract agar. Overgrowth occurs on modified V-8 juice agar. The colonies are after 10 days of cultivation at 26 ° C.

An inoculated area develops on Malζextrakt agar with a diameter of 15 mm to a colony with a diameter of 30 mm · It has a pale brownish white color. The reverse color is colorless. The central part the colony is characteristically synnematic in a network of funicular to flaky hyphae. It occurs Slightly curved edge from short aerial hyphae, intertwined with both erect and lying synnemata.

Modified V-8 juice agar results in a colony that is uniformly dense and has radial grooves is marked. The periphery is slightly curved. The colony is brownish-gray and is at the furrows and a little darker on the periphery. The reverse color is pale yellowish brown. It's funicular up, flaky and contains both erect and recumbent synnemas. The result is a colony with a diameter of 32 mm an inoculation area with a diameter of 15 mm.

Potato dextrose agar gives a colony with a diameter on an inoculated area with a diameter of 15 mm of 28 mm. The colony is thin, veil-like, and funicular. The periphery is slightly curved. There are Faintly radial folds are present, which run to the curved notches. The colony is colorless on both surfaces.

Diverging phialides arise from the funiculi, Synnemas and proliferating hyphae. These phialides are running out slightly conical, they are hyaline, smooth and unbranched. They are 3 / u wide at their base and 0.5yu at their tips

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wide. They have lengths in the range from 9.5 to 35 »O / ^u » the average size is 22 / U χ 3 / U ·

The conidia are in moist clusters or chains on the Tips of the phialides formed. These conidia are hyaline, smooth, unicellular and subspherical 'to elliptical. they are from 2.1 to 3.5 / u wide and from 2.1 to 4.2 / u long. The average Size is 3.43 / u χ 2.63 / u.

Emericellopsis - General Morphology

Cleistothecia are spherical, round (globrous) and have a diameter of 25 to 120 / u with a sub-hyaline peridia. The ascospores are unicellular, ellipsoidal, "dark-walled with 3 to 6 characteristic longitudinal wings. The ascocarps. __'_ ' * contain from some (less than 5) to many 8-spore asci, which contain vanishing walls (= small walls).

Morphology and cultural characteristics of Emericellopsis strains

Emericellopsis sp. NRRL 5713

This fungus is a member of the Eurotiaceae family and he has both a conidial and an ascogenic stage Step. A16OO5 is classified as an Emercellopsis by Beyma.

Dense air growth is obtained on solution agar according to Czapek, but no type of spores. The colony extends from an inoculated zone with a diameter of 18 mm at 26 ^° C. for 10 days to an area with a diameter of 37 mm. The aerial hyphae are first white and then become moderately yellowish-pink. The reverse color is vivid orange Synnemata and funicular hyphae are formed.

Malt extract agar results in a synnematic, flaky to funicular colony with a diameter of 32 mm from an inoculation zone with a diameter of 18 mm. The colony

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is pale orange-yellow and the reverse color is pale yellowish-pink. No fruiting occurs within 14 days on.

Potato dextrose agar yields a colony that is one diameter of 32 mm, starting from an inoculated zone with a diameter of 18 mm. The colony is with Rings and stripes drawn, contains little short, white aerial mycelium on a pale orange-yellow, cloudy mat, whose reverse color is pale yellowish pink. Somewhat scattered perithecium can be seen, but the conidia are in Abundance available.

Modified V-8 juice agar yields a colony consisting of an inoculated zone 18 mm to grows to a zone with a diameter of 31 mm. It is folded radially, funicular to flaky, slightly yellowish-pink with an inverse color of medium orange. The Phialids arise from synnemata, funiculi and proliferating hyphae «However, the ascogenic stage reduces the conidial stage.

Cleistothecia are black, spherical and spherical to sub-, spherical "with thick walls of pseudoparenchymatic cells. They have a diameter of 57.4 to 131.7 / U, the average diameter is 86.57 / U. -Two or more Asci are included in the cleistothecium. ' The asci are hyaline, thin-walled and fragile. The average diameter is 28 / u. They contain 8 brown, oval ascospores, those with 3 to 5 weakly serrated wings, the 1.5 / U wide are decorated longitudinally. These spores are from 6.3 to 8.4 / u long and 4.2 / u wide. The average size is 7.4 / u χ 4.2 / u.

The phialides are hyaline, smooth, and occasionally branched. They are in the range 35 to 70 ai long and an average eat three ο «i G /! U © ί

49 / U long. They are 2.5 u wide at their base and slowly taper to 0.5 u at their tips. The conidia collect in moist clusters at the tips of the phialides. They are smooth, hyaline and elliptical and from 2.5 to 3.0 / u wide and from 3.5 to 8.4 "/ u long, the average size is 8.0 χ 2.8 αι. Phialidenbranches can be up to 54 / u. Be long and also form conidia.

Emericellopsis sp. NRRL 5714

This fungus is a member of the Eurotiaceae family and forms both a conidial and an ascogenic stage and is characterized as a species of Emercellopsis by Beyma. Excessive Cleistrothecia appear on a modified V-8 juice agar that gives a colony, its center practically black-is. The remaining area of the colony is dark reddish gray. The reverse color is brownish-orange. Colonies in this agar are drawn with rings or stripes what is best observed on the opposite surface, Radially folded, notched and flaky to funicular. Within 3 weeks it will turn into a black, soluble pigment educated. This colony grows from one at 26 ° C in 10 days inoculated zone with a diameter of 20 mm to a colony with a diameter of 37 mm. On this agar there is also a moderately dense conidial step.

Potato Dextrose Agar yields one colony by one inoculated zone with a diameter of 20 mm. a colony with a diameter of 38 mm grows in 10 days at 26 ° C. Numerous undeveloped cleistothecia are formed. The conidia are relatively heavy. The mycelium is funicular and developed both erect and recumbent synnemata. This colony is pale yellowish pink and is sized by a 3 mm Edge of colorless, air-free hyphae surrounded «The reverse color of the medium is orange-yellow.

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Malt extract agar colonies can be transformed from an inoculated zone with a diameter of 20 mm to colonies with a diameter grow from 37 mm. Good conidial formation is observed, but the ascogenic stage does not develop. The colony is pale orange-yellow on both surfaces. It is funicular and develops both erect and recumbent synnemata.

The air components develop poorly on solution agar according to Czapek. The colony is relatively flat and shimmering. It reaches 44 mm in diameter from an inoculated zone with a diameter of 20 mm. This colony is pale yellowish green on both surfaces with a 4 mm colorless border of hyphae below the surface. The hyphae are irregularly shaped, distorted and provided with vacuoles.

Cleistothecia on modified V-8 juice agar are dark brown to black, glabrous, spherical to subspherical and have a diameter of 28 to 85 Ai. The average diameter is 56.6 ai. They contain subspherical asci that are colorless and fragile. These asci have a diameter of 18.6 ai and each contain 8 brown ascospores. From 3 to 5 wings are arranged longitudinally on the spores and 2 / U wide. The ascospores have sizes in the range from 8.4 to 11.2 ai in length and from 4.9 to 7.0 / u in width, the average sizes are 10.0 5.6 ai.

A typical Cephalosporium conidium step is observed on potato dextrose agar. The phialides arise from funiculi, synnemata and proliferating hyphae. They are unicellular, hyaline, and tapered slightly from base to tip. Their base is 2 ai wide, the tips are 0.5 ai wide. They are 20 to 60 ai long and an average of 40 χ 2 ai in size. Conidia, which are epically formed by the phialides and which form moist clumps, are hyaline, smooth, uni-

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~ 24

cellular and elliptical. They are from 2.1. up to 4.2 / U wide and from 3> 5 to 7.0 / U long. The average size is 3 / u χ 5 / u.

Emericellopsis sp. NRRL 5717

This fungus forms both an ascogenic and a conidial stage. It is classified as a member of the Eurotiaceae family. This fungus is classified as an Emercellopsis by Beyma. Both of these fruiting stages are described on modified V-8 juice agar. However, they also kick with potato dextrose agar. In solution agar after Czapek or in malt extract agar, no fruit formation is noticeable at 26 ° C in 14 days.

In solution agar according to Czapek, a colony with a diameter of 35 mm is obtained, the periphery of which is undamaged and in the is essentially circular. This colony expands from an inoculated zone with a diameter of 18 mm. on The densest aerial hyphae appear in this medium and the colony appears velvety. The microscopic examination shows synnemas and proliferating funicular hyphae. It is moderately yellowish-pink and the other side is up to yellowish-orange vivid orange. .

Malt extract agar results in a colony which is essentially free of air components, is flat, dull and has a faint yellowish orange color. The other side is pale yellowish white. The rate of growth is similar to that of the colony in the agar to Czapek.

Potato dextrose agar colonies grow from an inoculated Zone 16 mm in diameter to colonies 35 mm in diameter. The colony surface is flaky up wool-like. There are both erect and recumbent synnemas that make phialides. This colony is pale yellowish-pink and the reverse side is pale orange-yellow.

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, 2320698

Modified V-8 juice agar results in a colony emerging from an inoculated zone with a diameter of 20 mm a colony with a diameter of 39 mm grows. Synnematic, flaky to funicular mycelium supports a strong conidium level. Numerous hyaline cleistothecia will be Formed in 6 days, become black, glabrous, and spherical to sub-spherical in 10 days. They contain numerous hyaline, fragile asci, which in turn contain 8 ascospores. These ascospores are medium brown and oval and adorned longitudinally with 2 to 6 serrated or serrated wings that are 0.5 / U wide.

Cleistothecia occur in aerial hyphae or they can be partially or fully immersed in the agar. They range in diameter from 16 to 116 / u, average diameters of 50 Ai, and can contain as few as 2 asci. The asci have a diameter of 29 / U. The ascospores contain relatively thick walls, they have lengths in the range from 5.6 to 7.0 / u and widths in the range from 2.8 to 4.9 Ai, the average size is I6yu χ 3 »8vu.

The phialides are branched or unbranched, hyaline, smooth and pointed. Phialide branches can be 30 Ai long. In general, the phialides are 30 to 70 / u long and 1.5 / u wide at their base. They are on average 51.3 / u long. .Conidia occur at the ends of the phialides and form moist clusters. The conidia are hyaline, smooth, spindle-shaped, approximate elliptical shapes and from 8.4 to 14.0 / u long. Their width is in the range from 3.5 to 6.3 / u. The average conidia size is 4.7 x 11.5 / U.

Emericellopsis sp "- NRRL 5446

Cleistothecia are excessively formed on V-8 juice agar and moderately on potato dextrose agar. The growth is sparse on Czapek ¹ s agar. Cleistothecia are dark brown to brownish-black, glabrous, spherical to subspherical and have

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Diameter in the range from 3i> to 68 M ₉ the average diameter is 50 / u. The peridium "is about 5 / u thick, weakly" brown and contains closely intertwined hyphae. The asci are subspherical and in the range of 21 / U in diameter. The asci are & -sporous and form one to many asco per ascocarpia. The ascospores are ellipsoidal to oval, 5.6 to 7 _f 0 / U χ 8.4 to 10.5 / U, on average 6.2 to 10.1 / U, dark brown, smooth with prominent, ie developed, wings, in general four extending longitudinally, about 2 / U, and equidistant from the ascospore wall. Irregular nicks or cuts appear on the edges of the wings. The surfaces of the ascospores are otherwise smooth. ^:

A cephalosporium conidial step is also formed, but it is economical in all media. On potato dextrose agar The synnematic aerial or lying hyphae give rise to unbranched phialids, which are hyaiin, average sizes of 38 / U χ 1.6 / U and point towards the tips are conical. The spores generally appear in short Chains from 5 to 6 and ending in a damp ball. The conidia are hyaiine, unicellular, subspherical to oval, 5.5 to 6.5 / u 10 to 12 yu, generally 5.8 / u χ 10.6 / u.

The culture grows moderately on V-8 juice agar and potato dextrose agar V-8 juice agar, the colonies are flaky up funicular, the aerial mycelium is white to pale brown, the substrate mycelium appears blue-gray. The color on the other hand is bluish-orange in young cultures.

Emericellopsis sp. NRRL 544? .

This organism is a Cleistothecia fungus that belongs to the family Eurotiaceae. The gleistothecia are formed in excess within 5 days on V-8 juice agar, sparingly on potato dextrose agar. Very poor growth occurs on the Czapek agar.

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The ascocarpies are spherical, glabrous _s öiraisi big black, have a diameter of 25 bia ·. 120 / U, im. Generally, about 70 / α * The PerMiuja is transparent ₉ hyaline to light brown, and 5 to 6 / U thick "The AsGocarpien _s are random-like arranged in the SUBSTR atmycel ₉ siithalten one to many Asci * The asci are spherical Ms subkugslförmig, irregularly in their contour, shrinking ₉ have a diameter of 14 to 1SyU and contain 8 spores each. The ascospores are ellipsoidal to oval, dark brown, unicellular, with four pale brown, long! Tudinal wings, which have moderately serrated edges and which can extend up to 2.1 ai .

A conidial step is also formed in excess on potato dextrose agar and moderately on V-8 juice agar. The conidiophores (= conidia carriers) are phialides which arise from the funicular, lying aerial hyphae. The conidiophores are hyaline, unbranched, smooth, mainly 3 / U χ 28 ai, with basal septations. The conidia are unicellular, spherical, subspugelförraig to oboval, polysymme metric and have sizes in the range from 2.8 to 8.4 / U χ 3 »5 to 11 / u, on average 7 x 6.5 / u · The conidia are in spherical heads that merge with neighboring conidial heads

The cultures grow moderately, the reverse color is brownish-orange, the surface growth is colorless to bluish-gray, flaky to funicular.

The imperfect state of this organism is a form of Cephalosporium.

Morphology and cultural characteristics of Scopulariopsis Scopulariopsis sp. NRRL 5715 The characteristics of this culture correspond to those of Raper and Thorn are described in Scopulariopsis Bainier (1),

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The formation of conidia with "either smooth or whispered Walls formed from both the lateral and terminal phialides, but not entirely, as well as the color samples and the colony description show that this culture is best. between group III of Raper and Thorn, caused by Scopulariopsis brevicaulis var. glabra thorn is represented, and group IX, represented by S. diversispora, fits.

The growth of this fungus on solution agar according to Czapek is economical and colorless. There is no fruiting.

A colony is formed on potato dextrose agar which ^{grows within 10 days at 26 °} C. from an inoculated zone with a diameter of 22 mm to a colony with a diameter of 34 mm. The sporulation is moderate and limited to the inoculated zone, which contains nutrient medium that was carried over into the inoculum * The mycelium is colorless on both surfaces and has a smooth, even border. .

Malt extract agar yields a colony from an inoculated Zone with a diameter of 15 mm has increased its diameter to 33 mm. This colony is pale yellowish pink on both surfaces with a colorless, 4 min wide, radially folded edge. Both upright and lying synnemata and funicular hyphae form phialides, the conidia develop, generally in chains, but occasionally in moist clusters.

Modified V-8 juice agar gives the best conolia development of air components. This colony reaches from an inoculated zone with ^a: diameter of 15 mm a diameter of 33 mm. A comparatively flat, 3 mm wide edge zone has not grown well and is marked by sporulation pens or fingers that run radially. Of the

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larger central part of this colony is pale dark yellow, the result of relatively strong sporulation. This area is strongly synnematic. The reverse color is medium brown, after 10 days this color darkens to a light chocolate brown.

To describe the conidial stage of this culture, 'became the Used potato dextrose agar. Phialides arise from synnemata and funiculi, some are terminal extensions of the conidiophores. They are uniformly shaped on hers Base enlarged and become pointed. They are 2.1 / u wide at their base and 0.5 / u wide at their tips. The Phialids are from 17 to 34 / U long and on average 19.4 / U χ 2.1 / U great.

The conidia are either coarse-walled or smooth, hyaline, non-septate, spherical to sub-spherical and typically have truncated round parts, which are common to the genus Scopulariopsis are characteristic. The smooth spores are often elliptical to oval, generally smaller and they are obviously undeveloped. Both types of spores are observed on modified V-8 juice agar colonies. the smooth conidia are from 3 »5 to 4.9 / U long and from 1.4 to 2.1 / U wide, on average their size is 4.1 / U χ 1.9yU The coarse or rough spores are from 3.5 to 5.6 / U long and from 2.8 to 4.2 / U wide, their size is average 4.3 / u χ 3.6 / U, These rough conidial surfaces vary from verrucous (= provided with small warts) to echinulate (»sea urchin-like) and resemble Penicillium spores. Up to 14 days no perfect stage occurs.

References: K.B. Raper and C. Thorn, 1949. A manual of the penicillin. The Williams and Wilkins Company, Baltimore, 697-701.

Morphology and cultural characteristics of Paecilomyces Paecilomyces carneus NRRL 5711

A13215 is classified as a strain of Paecilomyces carneus (Duche ¹ and. Heim) in the order Moniliales.

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The colonies are generally velvety to flaky and; the depth "of the air felt varies according to the substrate Growth on solution agar according to Czapek is relatively thin and something funicular. The colonies are generally colorless and become somewhat discolored as the conidia develop. The peripheries are sometimes slightly notched or sawed, and on agar according to Czapek they are covered by a band of deep submersem Stressed mycelium. The reverse colors are bright greenish Tints to yellow tints, and brown tints appear in some media.

The branched conidiophores, which are from 100 to 300 / u long, arise from the agar or from proliferating aerial hyphae. Bifurcating branches can reach lengths of 40 / U. Secondary branches or sub-branches that are from 10 to 15 / u long occur frequently. Sterigmata are formed in numerous kinds on the conidiophores and their branches. They can occur pointedly as a single sterigma or in hosts up to 5. They can be used as coils lengthways the main axis with internodes of 5 to 20 / u occur. in the in general, the sterigmata in Wirtein are very divergent. Metulae are less common, but sometimes they occur in Wirtein, with only a single sterigma attached to it. The individual sterigmata, which are strongly divergent, occur often longer along the conidiophores and their branches. . . '.

The form of the Sterigmata typifies the genre. They are always lanceolate and are practically cylindrical from their base to about 75% of their length, where they taper more sharply and form a long thin tube with about 5.0 λχ χ 0.5.

The conidia are unicellular, subspherical to elliptical, dry and finely roughened and sea urchin-like to small prickly. They form in long chains and the spores shine through

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a connecting bridge together zn be a part of it can at the free conidia as little _$ droopy tip that is less than 1 / u long IMD 1 / u wide vferden recognized. These surface properties are best observed on an oil immersion lens using contrast phase lighting. In addition to these conidia, there are thick-walled, terminal macrospores (4.9 / U χ 3 * 5 / u) on some hyphae that are 100yu χ 3 / u long _? recognizable and which arise freely from the mycelium mat.

On solution agar according to Czapek, the colonies can reach a diameter of 32 mm in 10 days at 26 ° C., after which hardly any or limited further growth outwards is observed. They are relatively flat, with a velvety to flaky surface, and moderately funicular. The surface and sub-surfaces grow leather-like. The sub-surfaces form compartments which sharply delimit the colony. After 5 days the colonies are white and. become yellowish-white (ISCC-NBS, 92) and after 10 days weary polar bodies (Maerz and Paul, 9-B-2) .. At zimehinsadsr eonidia number one recognizes a pale pink tint * The tMcsli ^ color is dark - yellowish green (ISCC-NBS, 137), civett green (Maerz and Paul, 22-F-8). After 3 weeks the reverse color is light yellow-green (ISCC-NBS, 119), red-green (Maerz and Paul, "19-D-1). The conidial structures vary from individual sterigmata, which are either pointed or strongly divergent, related on the main axis of the conidiophores or their branches, up to hosts with 2 to 5 sterigmata arranged at the apex. Occasionally there are one or two coils of sterigmata which make the conidiophores under the whorl at the apex with internodes of 5 up to 20 / U. The Sterigmata are smooth-walled and lanceolate, have lengths in the range from 10.5 to 16.5 / U and are on average 14.35 Ai x 2.1 / u, with the width measured along the widest surface The individual divergent sterigmata can reach 20 / U.

i. i ι ...

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The conidiophores ■ form bifurcating branches and these branches can form sub-branches. The -Conidiophores are smooth-walled and 2 to 4 / u wide. Long chains of hyaline, sea urchin-like to small prickly Conidia, which are connected by a connecting bridge, arise from the sterigmata. In the crowd are these conidia pale pink. They are subspherical to elliptical and are from 2.1 to 3.5 / U long and from 1.75 to 2.8 / U wide. The average size is 2.3 x 3.3 / U

Colonies grow up to 30 mm in size on malt extract agar 10 days with little further change. The center can be speckled and the colony may extend outward in radial folds. An almost white, 3 mm, velvety edge - pale yellow-green (ISCC-NBS, 121), sea foam green (Maerz and Paul, 18-A-1) - results a flaky to velvety center that is light greenish-gray (ISCC-NBS, 154), lichen-like green (Maerz and Paul, 26-A-2). The reverse side is provided with rings or stripes, in the center greenish-yellow and of a concentric one Surrounding ring pattern, which is light yellowish green (ISCC-NBS, 115), over green (Maerz and Paul, 18-E-6). The forms and patterns of conidia, sterigmata and conidiophores are as previously described. The conidia have widths in the range from 2.1 to 3> 5 / U and lengths in the range from 2.8 up to 4.9 / u, the average size is 3.3 x 4.5 / u. The sterigmata are from 12.6 to 14.0yU long and from 2.8 to 4.9 / U wide, the average size is 13 »2 χ 3.6 Ai. The conidiophores and branches are just like on Agar to Czapek. In addition, individual macrospores, which can be referred to as aleurospores, at the top the hyphae filaments formed from the agar and the proliferating Aerial hyphae mats appear to arise.

A similar rate of growth occurs on potato dextrose agar. The colony is slightly notched. That

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The center is somewhat flaky, almost a velvety felt, which is also somewhat funicular and it is made up of a circle periphery surrounded, which consists of short aerial hyphae. The center is yellowish-gray (ISCC-NBS, 93), colored as Wood ash (Maerz and Paul, 27-A-1); the periphery is medium gray (ISCC-NBC, 265), platinum colored (Maerz and Paul, 45-A-3). The reverse color is greyish-olive green (ISCC-NBS, 127), bronze green (Maerz and Paul, 16-J-7).

The relationship of A13215 to P.carneus, Duche and Heim, is unmistakably as suggested by A.H.S. Brown and J. Smith in "The Genus Paecilomyces Banier ...", Trans.Brit.Mycol.Soc. 40 (1), 17-89 (1957). A13215 sterigmata appear possibly to be longer. The aleurospores observed on potato dextrose agar were also observed in other Paecilomyces species as described by Raper and Thorn in the Manual of the Penicilli.

Further penicillin N-forming cultures that are used in the inventive Procedures conditioned by their taxonomic view Similarities with Paecilomyces carneus NERL 5711 were used as members of the genus Paecilomyces classified. Two such cultures are identified as Paecilomyces carneus A.T.C.C. 16329 and P. carneus NRRL 2622 ".

Morphological characteristics of Diheterospora chlamvdosporia

The phialids occur singly or as threads or as single or branched conidiophores. The phialides are somewhat swollen at their base and tapering to a slender point. The vial spores are egg-shaped to short-cylindrical, smooth, hyaline, generally 1.5 to 2 / u χ 2 to 5yu. The aleuriospores common to the species are characteristic, are large (15 to 30 χ 10 to 20 / u), wall-shaped, thick-walled, somewhat brown-pigmented and multicelluloar and arise on short stems.

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The colonies are white to £ is white, grow very quickly, dense-flaky with an even, abrupt edge. No soluble pigment is formed.

As previously mentioned, the fungi which are useful in the method of the invention are further characterized by that they produce penicillin N. In addition, the fungi described above have the same morphological properties in the formation of vial spores from phialides, which are formed either individually from hyphae or from branched or unbranched conidiophores. The invention Emericellopsis organisms are the perfect sexual stage of Cephalosporium and have this morphological level Properties not. They show a conidium (Cephalosporium) that has these properties.

The major number of the above-described strains of Cephalosporium, Emericellopsis, Scopulariopsis, Paecilomyces and Diheterospora was made available by the permanent culture collection without any restriction in terms of availability Northern Regional Research Laboratory, Agricultural Research Serive, United States Department of Agriculture, Peoria, Illinois 61604, deposited where the cultures were deposited at the previously given taxanomic description after the name

of the corresponding culture as input

numbers have been assigned. ,. The remaining cultures that have the latter designation ATCC own, namely Cephalosporium chrysogenum ATCC 14615 and Paecilomyces carneus ATCC 16329, were used without limitation for availability at the American Type Culture Collection's permanent culture collection, 12301 Parklawn Drive, Rockville, Maryland 20852. .

As previously mentioned, an organism used in the present invention becomes aerobic after a submerged Cultivated fermentation process. The culture medium that

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different media gsin ^ for example kaas sas -different carbon sources Yer-wendsn such as sucrose, glucose _s starch and similar carbon sources. Usually the nitrogen sources can be selected from a large number of compounds such as soybean meal, soybean meal or dust, cottonseed oil, peanut flour, amino acids, amino acid mixtures, peptones, etc.For reasons of economy in production, because of the maximum yield and ease of isolation, certain media are preferred, for example are Molasses is a preferred source of carbohydrates, and preferred sources of nitrogen are soybean meal and amino acids.

Inorganic nutrient salts that can be incorporated into the culture medium include the common salts capable of are, sodium, potassium, ammonium, calcium, phosphate, sulfate, chloride, bromide, nitrate, carbonate, iron (II), Ferrous, magnesium, manganese ions and similar ions result. The fungi of the invention require similar to others Microorganisms that produce antibiotics, certain indispensable trace elements for growth, their development ^ and their metabolism. Such trace elements can be added to the fermentation medium, but they are in the "Generally present in sufficient quantity as impurities in the other ingredients that make up the culture medium be added. -.

The Cephalosporium, Emericellopsis, Scopulariopsis, Paecilomyces and Diheterospora organisms according to the invention can be used in devices of small size such as in 1 l ^ shake flasks are cultivated, small amounts of deacetoxycephalosporin C being obtained. For a manufacture of the cephalosporin compounds on a larger scale, the organisms are submerged in fermentation tanks on a larger scale, cultivated aerobic fermentation conditions.

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When producing deacetoxycephalosporin C on a larger scale, the spores of the organisms are placed on an agar inclined surface held. The spores from the inclined surface are used to form a vegetative medium with little Inoculate volume. The vegetative medium is incubated, producing a strong fresh, deeply growing culture of the Microorganism. This vegetative growth is then used as an inoculum for the fermentation medium on a larger scale used. In some cases it may be desirable to use additional vegetative medium as an inoculum for the fermentation medium to use. Such second stage vegetative media or organ media are generally used when the volume of the fermentation medium is significantly or substantially greater than the volume of the first vegetative Medium. In this way the spores of the organisms are first placed in a small volume of vegetative medium cultured, obtaining the inoculum for a vegetative medium with a larger volume. The vegetative medium with The larger volume then forms a sufficient concentration of organism to allow the fermentation to begin quickly initiate in a fermentation tank on a large scale. The vegetative medium can have the same composition as the fermentation medium, or it can have additional Contain ingredients to support the growth and formation of organisms on a small scale.

It has been observed that the microorganisms of the invention deacetoxycephalosporin C within a pH range grow and form from about pH 6.2 to about pH 8.0. During submerged, aerobic fermentation in tanks in on a large scale, the pH of the medium decreases from an initial pH from about 6.5 to a final pH of about 7.5.

The organisms according to the invention can be grown at temperatures between approximately 20 and 35 ^{° C.} The Deacetoxy

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cephalosporin C appears at a temperature of about 26 ° C to be formed in optimal yields.

The maximum formation of deacetoxycephalösporiri C occurs, when the organism is grown in tanks on a large scale for a period between about 4 and 7 days. if however, if one grows in devices on a smaller scale, such as in 1 l shake flasks, the growth of the organisms proceeds faster and deacetoxycephalosporin C becomes in one shorter time, for example during 2 or 3 days.

Since the final pH in large scale fermentation medium appears to reach pH 8.0 or higher, can the yield of Deacetoxycepahlosporin C adversely affected will. It is therefore desirable to adjust the pH of the fermentation medium on a large scale from time to time to be checked during fermentation. It appears that the pH values reach such values before the time at which a maximum formation of antibiotics occurs. The pH can be suitably adjusted by adding a suitable Acid or a suitable buffering agent to the fermentation medium can be adjusted lower.

The formation of deacetoxycephalosporin C during fermentation can be followed by chromatographically analyzing test samples of the fermentation broth. Alternatively can the presence of deacetoxycephalosporin .C by bioautographs being checked. The organism Pseudomonas solanacearcum can be used in bioautography determine the organism.

As with most submerged, aerobic fermentations, sterile air is bubbled through the culture medium to make it more effective To achieve growth of organisms and to increase the formation of fermentation products. The volume of air, that is forced through the culture medium is generally

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- mean at least about G, 2 vol. air / min / vol »culture medium, However, there may be an increased speed in the passage air often has a beneficial effect on the formation of deacetoxycephalosporin C own.

Most of the fungi useful in the process, in addition to deacetoxycephalosporin, form C and the characteristic Penicillin N also desacetylcephalosporin C, cephalosporin C and occasionally the lactone that is produced with desacetylcephalosporin C is formed, as well as other unidentifiable metabolites. Some of the aforementioned compounds are acid labile. It is therefore in the extraction of Deacetoxycephalo sporin C desirable from the fermentation medium, to treat the entire fermentation medium for a short time at an acidic pH value in order to remove some of the Destroying impurities. In the case of fermentations on a small scale, the penicillin N can be reduced by adding a penicillinase to the medium.

The deacetoxycephalosporin C fermentation product is made from the filtered fermentation broth so treated and from the other constituents of the fermentation medium separated by chromatography on an ion exchange resin. It is further purified by chromatography over cellulose or silica gel. . and then failed and recrystallizedο

At the beginning, the filtered fermentation medium becomes a preliminary Subjected to purification procedures, with a first extraction with a water-immiscible organic Solvents like n-butanol, amyl acetate is made to remove impurities to remove. The extracted medium can then be further prepared by chromatography over activated Carbon to be purified. The extracted medium is chromatographed on a column with activated carbon, e.g. Pittsburgh 12 χ 40

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Carbon, and the column is then washed with water to remove water-soluble ₉ colored impurities and water-soluble inorganics. The fermentation products are then eluted from the carbon with 50% acetone-water. The eluate is concentrated to an aqueous phase, which is then chromatographed over a basic / anionic exchange resin. Among the basic anionic exchange resins that can be used are those of the polystyrene quaternary airanonium type, for example those sold under the names Dowex 1, Dowex 2 (Dow Chemical Cc, Midland, Mich.) And those sold under the names IRA-400, IRA-45, IRA-68 (Amberlite, Rohm and Haas, Philadelphia, Pa.) Are available. The resins can be in the hydroxyl cycle, the acetate cycle, the pormate cycle, or other blessed cycles. The concentrated eluate from the carbon acid is added to the anionic exchange resin and then the resin is washed with barrels. The deacetoxy cephalosporin C is eluted from the exchange resin in salt form with a suitable weak base, for example the eluting solvent is, if the column is used in the acetate cycle, an aqueous solution of sodium acetate at a concentration of approximately 1N or less. The concentration of the sodium acetate solution is not critical. However, in order to avoid the presence of excess inorganic salt (sodium acetate) in the eluate from the exchange column, the desirable concentration of sodium acetate is between approximately 0.1n and 0.5n. A particularly preferred concentration for the elution, which avoids excess salt in the eluate, is 0.15n. If the exchange resin is used in the formate cycle or in other suitable cycles, the corresponding salt in aqueous solution can be used as the eluting solvent. For example, ammonium formate can be used when the resin is in the formate cycle. Many fractions are collected and the fractions containing deacetoxy-

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contain cephalosporin C, determined by bioautogram, are united. The combined eluates are then chromatographed over activated carbon, over excess to remove inorganic salts, e.g. sodium acetate, which was used as the salt solution for elution. the Carbon column is washed with water to remove these inorganic water-soluble salts and the deacetoxycephalo sporin C is then removed from the column with a 50% acetone-water mixture. The eluate becomes dry evaporated or, alternatively, the acetone is distilled off and the concentrated aqueous solution is then lyophilized, Deacetoxycephalosporin C is obtained as a solid crude product in both processes.

The crude preparation of Deacetoxycephalosporin C is chromatographed over cellulose or silica gel or over other suitable non-ionic adsorbents further purified. The deacetoxycephalosporin C is eluted from the column with acetone: water, 80:20, and many fractions are collected * The fractions containing deacetoxycephalosporin C, determined by paper chromatography or bioautograph, using of a test organism of Pseudomonas solanacearcum are pooled and concentrated to a small volume or evaporated to dryness. The highly concentrated aqueous residue or the dry residue will then dissolved in a minimal amount of isopropyl alcohol and die.alcoholic solution is poured into a large volume of diethyl ether poured. The purified Deacetoxycephalosphorin C is in the form of the salt, which the aqueous eluent, that had been used in the elution of the anionic exchange resin, is obtained. For example, if Sodium acetate was used as the eluent, that will Deacetoxycephalosporin C obtained as the monosodium salt. The salt form of Deacetoxycephalosporins C is filtered and dried.

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In the process according to the invention, the following conditions are preferably used for the isolation. The entire fermentation broth is acidified to pH 2 by adding sulfuric acid and then the medium is stirred for 1 hour at room temperature. The pH of the medium is then adjusted to 6.0 with sodium hydroxide and the basified medium is then filtered using filter aid to remove mycelium and other insolubles. The aqueous medium is then extracted with n-butanol to remove further impurities and then the extracted medium is chromatographed over activated carbon. The carbon column is washed with water and the fermentation product is eluted therefrom with 50% acetone-water. The eluate is concentrated to an aqueous phase. The aqueous phase is chromatographed over an anionic quaternary ammonium exchange resin in the acetate cycle, preferably the quaternary ammonium polystyrene resin which is commercially available and is referred to as Amberlite IRA-68 (Rohm and Haas, Philadelphia, Pa.). The exchange resin is first washed with water and then the deacetoxycephalosporin C and other compounds which are also formed are eluted with 0.15N aqueous sodium acetate. The active eluates are absorbed on activated carbon and the column is washed with water to remove water-soluble salts such as excess sodium acetate carried over from the resin eluates., The deacetoxycephalosporin C is removed from the carbon with 50% acetone: water eluted. The eluate is then evaporated to dryness, giving crude deacetoxycephalosporin C as the sodium salt. The crude material is preferably purified by chromatography over a cellulosic acid (Avicel pH 101). The cellulose column is eluted with acetonitrile / water (80:20) and many fractions are collected. The fractions containing deacetoxycephalosporin C, determined by bioautograms, are combined and then evaporated to dryness in vacuo. The dried solid

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Deacetoxycephalosporin C residue is dissolved in a minimal amount of isopropyl alcohol. The alcohol solution is then poured into diethyl ether to precipitate the deacetoxycephalosporin C monosodium salt. ^:

Another method of isolating deacetoxy, cephalosporin C, the eluate from the anionic exchange resin is concentrated in the acetate cycle by evaporation and a 20% aqueous solution of sodium acetate becomes the concentrate added ·. The concentrate is stirred with cooling to precipitate the deacetoxycephalosporin C monosodium salt.

The salt form of deacetoxycephalosporin C can be converted to the free acid by methods commonly used used to convert salts into acids. For example the salt can be passed over a cationic exchange resin, the acid being obtained in free form.

The chromatography system that is used to detect deacetoxycephalosporin Identifying C in crude fermentation media or in resin eluate fractions is made using performed by Whatman No. 1 chromatography paper in descending order. The solvent system that you use for Development used is acetonitrile: water, 80:20 vol.:VoI*, the chamber with the vapors of the solvent mixture, that of n-propanol: pyridineacetic acid: acetonitrile: water in ratios of 45: 30: 9: 40: 36, expressed by volume per volume, consists, is saturated.

The developed chromatograms are then used as bioautograms, using Pseudomonas solanacearcum used as a microorganism for the identification.

To simplify chromatography, it is desirable to to treat the test sample with a penicillinase,

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before the chromatogram is performed.

The following examples illustrate the invention without representing it restrict.

example 1

Separate spores of Cephalosporium sp. Strain NRRL 5445 Cephalosporin chrysogenum ATCC 14615, Emericellopsis sp. Strain NRRL 5446 and Emericellopsis sp. Strain NRRL 5447 separately introduced on a slant plate made of nutrient agar with the following composition:

Ingredients % (w / v) lactose 1

Glycerin 1

Soy peptone 0.25

soluble substances, obtained by ₂

Drying of grain and malt extracts 0.25

Magnesium sulfate heptahydrate 0.05

Iron (II) sulfate heptahydrate Ö, 001

Calcium carbonate 0.2

Traces of mineral solution ^ 0.625

Agar. . - 2

Water to the desired volume

1. Enzymatic extract of soybean meal

2. "Produlac", National Distillers Products Co.

3. 'The solution contains the following salts per

100 ml deionized water: Weight · salt

* 0.102 g CuSO ₄ .5H ₂ O

0.018 g FeSO ^ .7H ₂ O

0.126 g ■. MnCl ₂ .4H ₂ 0

0.024 g of ZnSO ₄ .7H ₂ O

The slants were incubated at a temperature of 26 ⁰ C for 7 days. The mature sloping cultures were

I ι. ■

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¹ ; .Ί · ;. : it

rilem distilled water and covered with a sterile The stick was scratched slightly, giving the spore suspensions.

' 1

The spore suspensions thus obtained were each used to inoculate two separate sterile growth media of the following composition:

Components % (W / v) Peanut flour 2 malt extract 2 Corn water 0.5 Magnesium sulfate heptahydrate 0.025 Potassium dihydrogen phosphate., 0.1 Dipotassium hydrogenphosphate 0.05 Calcium chloride dihydrate 0.01 Trace mineral solution 0.625.

Water to the desired volume.

1. Footnote 3 as above _t . _t . , ,,

The pH of the vegetative medium was checked before treatment adjusted to pH 6.5 in the autoclave. The inoculated vegetative media were poured on a rotary chute at a temperature of 26 ° C! device with 250, rpm with a Incubated for 48 hours.

The incubated vegetative media were then used as inoculum for the fermentation medium in a ratio of Λ% vegetative medium per volume of fermentation medium. The sterile production media that were used had the following composition:

¹ ■ · * ■■■ "I ■■» ■■. ·. · ■ · < ^! -) Ι ·. .- ,, j - .. j j-

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- 45 - Components % (Wt. 2320696 Sucrose 4th / VoI) Glycerin 1 Sodium glutamate O. »5 Peanut flour 2 Iron (II) ammonium sulfate hexahydrate 0.3 Potassium nitrate 2.0 • Calcium carbonate 0.3 Water on volume

The use of surfactants or defoaming agents Occasionally occurs when conducting a large scale fermentation. The pH of the Fermentation media was adjusted to pH 6.5 before sterilization. The inoculated fermentation media were then incubated with stirring at a temperature of 26 ° C for 5 days; during fermentation became sterile Air through the fermentation media at a rate corresponding to approximately 1/2 volume air / volume culture medium / min passed through. The final pH of the fermentation media was pH 7.5.

In each case 15 l of the complete media obtained as described above were brought to a pH value with sulfuric acid acidified by 2. The acidified complete media was stirred at room temperature for 1 hour and then was stirred the pH value is adjusted to pH 6.0 with sodium hydroxide solution. The media was filtered and the aqueous filtrates were with n-butanol to remove insoluble substances in suspension extracted. The aqueous phases were then passed through columns loaded with activated carbon and the columns were washed with distilled water. The eluate from the water wash was discarded. The columns were then eluted with 50% aqueous acetone. The eluates were evaporated in vacuo to the aqueous phase, which then on columns chromatographed with Amberlite IRA

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a quaternary ammonium polystyrene resin in the acetate cycle were packed. The columns were washed 'first' with water and then the water was discarded. The active fermentation products were eluted from the columns with 0.15N aqueous sodium acetate solution. Many factions were collected and the fractions from each individual experiment containing deacetoxycephalosporin C were combined.

The presence of deacetoxycephalosporin C in the eluate fractions was determined in each case by bioautograms. The following system was used in each experiment: The first chromatography was performed in descending order using untreated Whatman No. 1 chromatographic paper. Acetonitrile: water (80%: 20%) was used as the eluting solvent. The bottom of the chromatography chamber was coated with a solvent mixture which contained 300 ml of a solution of 31.5% n-propanol, 25 "pyridine, 7.5% acetic acid and 30% water and which was diluted to 100 ml with acetonitrile Localization of deacetoxycephalosporin C on the chromatogram was carried out by performing a bioautogram with the developed chromatogram and using Pseudomonas solanacearcum as the detection organism. .- ■ '.,'

The fractions obtained by the chromatographic procedure described above and containing deacetoxycephalosporin C were united. The combined fractions were then absorbed onto a column that was filled with activated carbon, and then the column was first using distilled water'washed to soluble inorganic To remove substances such as sodium acetate. After that ^ the column was eluted with 50% aqueous acetome. The eluate was evaporated to dryness in vacuo, leaving a crude solid preparation of deacetoxycephalosporin C sodium salt received.

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The crude material was further passed over by chromatography purified on a column packed with cellulose (Avicel pH 101). The antibiotic was from the cellulose column with a solvent mixture containing acetonitrile: water (80:20) contained, eluted. Many fractions were collected, and then the fractions containing deacetoxycephalosporin C contained, combined using the chromatography system described above for determination. The United Fractions were then evaporated to dryness and ΓΙ the dry residue containing deacetoxycephalosporin C was dissolved in a minimal amount of ilsopropanol. the The alcohol solution was poured into an amount of ether about 20 times the volume of the isopropanol solution. Under With stirring, the sodium salt of deacetoxycephalospron C formed as a precipitate. The precipitate was filtered off and air dried to give 126 mg of the antibiotic as the sodium salt.

Example 2

Following the procedure described in Example 1, spores became of Emericellopsis sp. NRRL 5714 in a vegetative medium stage cultured using essentially the same vegetative medium. The cultured medium then became used to inoculate a production medium of the following composition:

Ingredients '" % (wt./VaI)

Soluble starch 1.0

Dextrose ■ ·. . * 4.0

Soybean meal 1.0 calcium carbonate, ... , /,. , 0.5

Antifoam agent, 0.1,. Water on volume

The pH of the production medium was adjusted to 6.5 before the inoculation and before the treatment in the autoclave.

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The fermentation is carried out for 5 days at about 26 ⁰ C and deacetoxycephalosporin C is produced as described in Example 1 "and isolated ^1..:

Example 3 '.....

According to the culture method described in Example 1, spores of Scopulariopsis sp. NRRL 5715 germinated and grown in the vegetative medium described in Example 1. The vegetative medium is used to inoculate a fermentation medium which has the composition as described for the fermentation medium in Example 1. ^The fermentation is carried out for 5 days at about 26 ⁰ C and deacetoxycephalosporin C is isolated from the fermentation medium by the procedures described in Example 1.

Example 4 I; . ::

The media and culture methods described in Example 1 are used and method of recovery and cultivated and fermented Paecilomyces carneus NRRL 5711, being deacetoxycephalosporin C is obtained.

Example 5

The culture and isolation methods and media described in Example 1 are used, and Diheterospora chlamydosporia NRRL 372.8 is cultured and fermented, with deacetoxycephalosporin C being formed. "-

■ • ■ ι · ι '■ - ■ · ". ■ .-' ■; .... Ii:;.. Ι-1. T.. ..I |. ,, ι:.

3098467 1067 ^! '

Claims (3)

Claims., 1. Process for the production of deacetoxycephalosporin C, characterized in that in an aqueous culture medium under submerged, aerobic fermentation conditions a penicillin N-producing microorganism belonging to the genera Cephalosporium, Emericellopsis, Scopular riopsis, Paecilomyces or Diheterospora, breeds, until a substantial amount of deacetoxycephalosporin C is formed by the microorganism in the culture medium, and then the deacetoxycephalosporin C is isolated from the culture medium. 2. The method according to claim 1, characterized in that the microorganism Cephalosporium corda NRRL 5445 used. 3. The method according to claim 1, characterized in that that Cephalosporium chrysogenum ATCC 14615 is used as the microorganism. 4. The method according to claim 1, characterized in that that Emericellopsis sp. NRRL 5446 used. 5. The method according to claim 1, characterized in that that Emericellopsis sp. NRRL 5447 used. 6. The method according to claim 1, characterized in that that the microorganisms Cephalosporium sp. NRRL 5711, Cephalosporium sp. NRRL 5716, Cephalosporium sp. NRRL 5718, Cephalosporium sp. NRRL 5719, Cephalosporium sp. NRRL 5720, Cephalosporium sp. NRRL 5721, Cephalosporium sp. NRRL 5722, Cephalosporium sp. NRRL 5723, Cephalosporium NRRL 5724 or Cephalosporium sp. NRRL 5725 used. 30 9 * 8 46/1067 -: = ■. '■ ■ ■■■ ⁽ ■ - ■ - .. ,, V'<"V- ^.: 7. The method according to claim 1, characterized in that the microorganisms Emericellopsis sp. URRL 5713, Emericellopsis sp. NRRL 5714 or Emericellopsis sp. MRRL ₁ 5717 used. 8. The method according to claim 1 ', characterized in that that Scopulariopsis sp. NRRL 5715, Paecilomyces carneus NRRL 5711 or Diheterospora chlamydosporia NRRL 5728 used. 3 0 9 8 48/106 7 > ^, '/