Okafor N. Modern Industrial Microbiology and Biotechnology

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"! Modern Industrial Microbiology and Biotechnology

method of classification difficult to sustain. In some cases they have been classified on the

basis of the producing organisms, but the same organism may produce several

antibiotics, e.g. the production of penicillin N and cephalosporin by a Streptomyces sp.

The same antibiotics may also be produced by different organisms. Antibiotics have been

classified by routes of biosynthesis; however, several different biosynthetic routes often

have large areas of similarity. The spectra of organisms attacked have also been used, e.g.

those affecting bacteria, fungi, protozoa, etc. Some antibiotics belonging to a well known

group e.g. aminoglycosides may have a different spectrum from the others. The

classification to be adopted here therefore is based on the chemical structure of the

antibiotics and classifies antibiotics into 13 groups. This enables the accommodation of

new groups as they are discovered (Table 24.1).

Table 24.1 Grouping of antibiotics based on their chemical structures

Chemical Group Example

Aminoglycosides Streptomycin

Ansamacrolides Rifamycin

Beta-lactams Penicillin

Chloramphenicol and analogues Chloramphenicol

Linocosaminides Linocomycin

Macrolides Erythromycin

Nucleosides Puromycin

Puromycin Curamycin

Peptides Neomycin

Phenazines Myxin

Polyenes Amphothericin B

Polyethers Nigericin

Tetracyclines Tetracycline

One well-known example of each group has been given to facilitate recognition of the

groups.

The nomenclature of antibiotics is also highly confusing as the same antibiotic may

have as many as 13 different trade names depending on the manufacturers. Antibiotics

are therefore identified by at least three names: the chemical name, which prove long and

is rarely used except in scientific or medical literature; the second is the group, generic, or

common name, usually a shorter from of the chemical name or the one given by the

discoverer; the third is the trade or brand name given by the manufacturer to distinguish

it from the product of other companies.

The production of antibiotics is a very wide subject and because of space limitations

only beta-lactam antibiotics will be discussed. Even among them, only penicillin and

cephalosprin will be discussed in any detail.

24.2 BETA-LACTAM ANTIBIOTICS

The Beta-lactam antibiotics are so-called because they have in their structure the four-

membered lactam ring. Figure 24.1 shows the structures of the various Beta-lactam

antibiotics.

Production of Antibiotics and Anti-Tumor Agents "!

The Beta-lactam structure is not very common in nature and besides the antibiotic

groups to be discussed it is only found in some alkaloids and some anti-metabolite toxins

including pachystermines from the higher plant, Pachystradra terminalis, wild-fire toxin

from Pseudomonas tabici and the anti-tumor antibiotics, phleomycins and bleomycins

from Streptomyces verticillus.

The Beta-lactam antibiotics include the well-established and clinically important

penicillins and cephalosphorins as well as some relatively newer members:

cephamycins, nocardicins, thienamycins, and clavulanic acid. Except in the case of

nocardicins these antibiotics are derivatives of bicyclic ring systems in which the lactam

ring is fused through a nitrogen atom and a carbon atom to ring compound. This ring

compound is five-membered in penicillins (thiazolidine), thienamycins (pyrroline) and

clavulanic acid (oxazolidine); it is six-membered (dihydrothiazolidine) in

cephalosporins and cephamycins (Fig. 24.1).

The Beta-lactam antibiotics inhibit the formation of the structure-conferring

petidoglycan of the bacterial cell wall. As this component is absent in mammalian cells,

Beta-lactam antibiotics have very low toxicity towards mammals.

Fig. 24.1 The Beta-lactam Antibiotics

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24.2.1 Penicillins

24.2.1.1 Strain of organism used in penicillin fermentation

In the early days of penicillin production, when the surface culture method was used, a

variant of the original culture of Penicillium notatum discovered by Sir Alexander Fleming

was employed. When however the production shifted to submerged cultivation, a strain

of Penicillium chrysogenum designated NRRL 1951 (after Northern Regional Research

Laboratory of the United States Department of Agriculture) discovered in 1943, was

introduced. In submerged culture it gave a penicillin yield of up to 250 Oxford Units (1

Oxford Unit = 0.5988 of sodium benzyl penicillin) which was two to three times more

than given by Penicillium notatum. A ‘super strain’ was produced from a variant of NRRL

1951 and designated X 1612. By ultraviolet irradiation of X-1612, a strain resulted and

was named WISQ 176 after the University of Wisconsin where much of the stain

development work was done. On further ultra violet irradiation of WISQ 176, BL3-D10

was produced, which produced only 75% as much penicillin as WISQ 176, but whose

product lacked the yellow pigment the removal of which had been difficult. Present-day

penicillin producing P. chrysogenum strains are far more highly productive than their

parents. They were produced through natural selection, and mutation using ultra violet

irradiation, x-irradiation or nitrogen mustard treatment. It was soon recognized that

there were several naturally occurring penicillins, viz. Penicillins G, X, F, and K (Fig.

24.2).

Penicillin G (benzyl penicillin) was selected because it was markedly more effective

against pyogenic cocci. Furthermore, higher yields were achieved by supplementing the

medium with phenylacetic acid, analogues (phenylalanine and phenethylaninie) of

which are present in corn steep liquor used to grow penicillin in the United States.

Present day penicillin-producing strains are highly unstable, as with most industrial

organisms, and tend to revert to low-yielding strains especially on repeated agar

cultivation. They are therefore commonly stored in liquid nitrogen at – 196° or the spores

may be lyophilized.

Penicillin has since been shown to be produced by a wide range of organisms includ-

ing the fungi Aspergillus, Malbranchea, Cephalosporium, Emericellopsis, Paecilomyces,

Trichophyton, Anixiopsis, Epidermophyton, Scopulariopsis, Spiroidium and the

actionomycete, Streptomyces. The only type of penicillin produced by actinomycetes how-

ever is Penicillin N (with the chemical structure D-a (d- aminoadipyl) penicillin usually

accompanied by cephamycins and/or deacetyl – 3 – 0-carbamoylcephalosporin C.

24.2.1.2 Fermentation for penicillin production

The inoculum is usually built up from lyophilized spores or a frozen culture and

developed through vessels of increasing size to a final 5-10% of the fermentation tank. As

the antibiotic concentration in the fermentation beer is usually dilute the tanks are

generally large for penicillin and most other antibiotic production. The fermentors vary

from 38,000 to 380,000 liters in capacity and in modern establishments are worked by

computerized automation, which monitor various parameters including oxygen content,

Beta-lactam content, pH, etc.

The medium for penicillin production now usually has as carbohydrate source

glucose, beet molasses or lactose. The nitrogen is supplied by corn steep liquor. Cotton

Production of Antibiotics and Anti-Tumor Agents "!!

seed, peanut, linseed or soybean meals have been used as alternate nitrogen sources. The

nitrogen source is sometimes exhausted towards the end of the fermentation and it must

then therefore be replenished. Calcium carbonate or phosphates may be added as a

buffer. Sulfur compounds are sometimes added for additional yields since penicillin

contains sulfur. The practice nowadays is to add the carbohydrate source intermittently,

i.e. using fed-batch fermentation. Lactose is more slowly utilized and need not be added

intermittently. Glucose suppresses secondary metabolism and excess of it therefore limits

penicillin production. The pH is maintained at between 6.8 and 7.4 by the automatic

addition of H

or NaOH as necessary.

Precursors of the appropriate side-chain are added to the fermentation. Thus if benzyl

penicillin is desired, phenylacetic acid is added. Phenyl acetic acid is nowadays added

continuously as too high an amount inhibits the development of the fungus. High

yielding strains of P. chrysogenum resistant to the precursors have therefore been

developed.

Fig. 24.2 Natural and Biosynthetic Penicillins

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Penicillin production is stimulated by the addition of surfactants in a yet unexplained

mechanism. The temperature is maintained at about 25°C, but in recent times it has been

found that yields were higher if adjusted according to the growth phase. Thus, 30-32°C

was found suitable for the trophophase and 24°C for the idiophase. Aeration and

agitation are vigorous in order to keep the components of the medium in suspension and

to maintain yield in the highly aerobic fungus.

Penicillin fermentation can be divided into three phases. The first phase (trophophase)

during which rapid growth occurs, lasts for about 30 hours during which mycelia are

produced. The second phase (idiophase) lasts for five to seven days; growth is reduced

and penicillin is produced. In the third phase, carbon and nitrogen sources are depleted,

antibiotic production ceases, the mycelia lyse releasing ammonia and the pH rises.

24.2.1.3 Extraction of penicillin after fermentation

At the end of the fermentation the broth is transferred to a settling tank. Penicillin is

highly reactive and is easily destroyed by alkali conditions (pH 7.5-8.0) or by enzymes. It

is therefore cooled rapidly to 5-10°C. A reduction of the pH to 6 with mineral acids

sometimes accompanied by cooling helps also to preserve the antibiotic. The

fermentation broth contains a large number of other materials and the method used for

the separation of penicillin from them is based on the solubility, adsorption and ionic

properties of penicillin. Since penicillins are monobasic carboxylic acids they are easily

separated by solvent extraction as described below.

The fermentation beer or broth is filtered with a rotary vacuum filter to remove mycelia

and other solids and the resulting broth is adjusted to about pH 2 using a mineral acid. It

is then extracted with a smaller volume of an organic solvent such as amyl acetate or

butyl acetate, keeping it at this very low pH for as short a time as possible. The aqueous

phase is separated from the organic solvent usually by centrifugation using Podbielniak

centrifugal countercurrent separator (Chapter 9).

The organic solvent containing the penicillin is then typically passed through

charcoal to remove impurities, after which it is back extracted with a 2% phosphate buffer

at pH 7.5. The buffer solution containing the penicillin is then acidified once again with

mineral acid (phosphoric acid) and the penicillin is again extracted into an organic

solvent (e.g. amyl acetate). The product is transferred into smaller and smaller volumes of

the organic solvent with each successive extraction process and in this way, the

penicillin becomes concentrated several times over, up to 80-100 times. When it is

sufficiently concentrated the penicillin may be converted to a stable salt form in one of

several ways which employ the fact that penicillin is an acid: (a) it can be reacted with a

calcium carbonate slurry to give the calcium salt which may be filtered, lyophilized or

spray dried. (b) it may be reacted with sodium or potassium buffers to give the salts of

these metals which can also be freeze or spray dried; (c) it may be precipitated with an

organic base such as triethylamine.

When benzyl penicillin is administered intramuscularly it is given either as the

sodium (or potassium) salt or as procaine penicillin. The former gives high blood levels

but it quickly excreted. Procaine penicillin gives lower blood levels, but it lasts longer in

the body because it is only slowly removed from the blood. It is produced by dissolving

sodium or penicillin in procaine hydrochloride.

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24.2.1.4 Production of semi-synthetic penicillins

In the late 1940s it was shown by labeling experiments that penylacetamide derivatives

were directly incorporated into the benzyl penicillin molecule. The possibility was

recognized of inducing the mold to produce new antibiotics antibiotic by the

introduction of various precursors. Phenoxymethyl penicillin (penicillin V) which had

greater acid stability than penicillin G, allythiomethyl penicillin (Penicillin O) which

was less likely to induce allergic reactions and butylthiomethyl penicillin (Penicillin S)

were thus produced. The natural penicillins (formed in unsupplemented media) and the

biosynthetic (produced by the addition of specific side-chain precursors) are indicated in

Fig 24.2 The high expectations of making new penicillins by the introduction of side-

chains during fermentation, did not however, result in many new pencillins.

In 1959 6-amino penicillanic acid (6-APA) was isolated from precursor-starved

P. chrysogenum fermentations and this ushered in the era of semi-synthetic penicillins

and indeed other semi-synthetic antibiotics. Today the only ‘natural’ penicillins used are

benzyl penicillin (Penicillin G) and phenoxymethyl penicillin (Penicillin V). All others

are semi-synthetic.

In preparing semi-synthetic penicillins, 6-APA is not produced by starving P.

chrysogenum of precursors, because yields are low. It is prepared by cleaving from

penicillin G or penicillin V, the 6-acyl group by chemical means or with enzymes

(acylases) produced by a wide range of microorganisms including bacteria, yeasts, and

molds and even mammals (hog kidney acylase). The various acylases have different

substrates. Actinomycete and mold acylases usually attack penicillins with aliphatic

side-chains, e.g. penicillin V or phonoxymetyl penicillin (Penicillin); penicillin G is

attacked more slowly. On the other hand, bacterial acylases attack penicillin G rapidly.

Immobilized enzymes and cells are being used in these processes.

The introduction of the acyl side chain is done by reacting 6-APA with a suitable

derivative of a carboxylic acid, usually a chloride, in organic solvents under anhydrous

or aqueous conditions. In the latter system it is done in acetone-water mixtures in the

presence of sodium bicarbonate. The resulting penicillins can be extracted by solvent

extraction as already described, followed by charcoal treatment.

Semi-synthetic penicillins were developed to meet some of the short-comings of benzyl

penicillin. Some of the desired properties were greater intrinsic activity against Gram-

positive bacteria, increased antibacterial spectrum including Gram-negative organisms,

gastric acid stability and oral absorbability and resistance to beta-lactamases, (i.e.

enzymes which open the Beta-lactam ring). All penicillins to some extent bind to the

serum albumin and therefore reduce the quantity of the antibiotic available to attack

microorganisms. The less binding therefore, for pencillins of otherwise equal activity, the

more bactericidal. The final desirable property is reduced ability to induce

hypersensitivity, a phenomenon which occurs in 9-10% of the population.

24.2.2 Cephalosporins

Cephalosporins in general have a broader spectrum than penicillins and are less likely to

induce allergic reactions among those who react to penicillins. The first cephalosporin

was discovered in 1948 and was produced by Cephalosporium acremonium. It was later

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shown that the organism in fact produced five antibiotics. Five of these were steroids (and

therefore hydrophobic) and were active only against Gram-positive bacteria. A

hydrophilic component active against both Gram-positive and Gram-negative bacterial

was also found and named cephalosporin N. While purifying cephalosporin N, another

compound, cephalosporin C was discovered. It was latter found that cephalosporin C

was stable to acid and to pencillianse; most importantly it was active against Gram-

negative bacteria although it had about one-tenth the activity of cephalosporin N against

Gram-positives. With further study Cephalosporin N was found to be a penicillin and

renamed Penicillin N. Cephalosporin is now known to be produced by as wide a range of

micro-organisms as produce penicillins and these include fungi and actinomycetes. The

natural cephalosporins and cephamycins are given in Fig 24.3.

24.2.2.1 Production of cephalosporin

While two of the clinically important penicillins (Penicillin G & V) are produced entirely

by fermentation the rest being semi-synthetic, all of the cephalosporins in use on the other

hand are semi-synthetic. They however have as their starting point Cephalosporin C

produced by fermentation, otherwise the production of both antibiotics is similar.

24.2.2.2 Strain of organism used

The original strain of Cephalosporium acremonium (C Ml 49, 137 – Common Wealth

Mycological Institute, Kew Gardens, London) produced by violet mutagenesis, C.

acremonium 8650. This latter organism is the parent of most of the various commercially

used C. acremonium. In passing, Cephamycins, (Fig. 24.3) are produced by Streptomyces

lipmanni and S. clavuligenis.

24.2.2.3 Fermentation

The medium used for cephalosporin fermentation is same as used for penicillin N.

Paraffins have however been used to produce several cephalosporins. Methionine,

arginine, ornithine, spermine, cadaverine, and lysine have been shown to increase

cephalosporin production.

24.2.2.4 Extraction of cephalosporin after fermentation

While penicillins are carboxylic acids, cephalosporin C is amphotheric having both

alkaline and acidic properties. For this reason it cannot be extracted directly into organic

solvents. Cephalosporin C is more commonly isolated by ion exchange and precipitation.

The broth is acidified to a low pH after filter-aid filtration in a rotary vacuum filter. The

broth usually contains penicillin N and deactylacephalosporin. The low pH destroys

penicillin N and converts the deactylcelphalosporin to cephalosporin G.

While 6-APA can be made during fermentation by starving P. chrysogenum of the side-

chain precursor, 7-amino cephalosoporanic acid (7-ACA) cannot be produced by

fermentation even if precursors are not added; neither can it be produced by the

enzymatic side-chain cleavage as with 6-APA. The production of 6-amino

cephalosporanic acid is therefore by chemical means. 7-ACA is obtained by the chemical

removal of the Alpha-amino adipic side chain of cephalosporin when preparing

Production of Antibiotics and Anti-Tumor Agents "!%

Fig. 24.3 Natural Cephalosporins and Cephamycins

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cephalosporins which have Alpha-3 acetoxymethyl group or a derivative from it.

However, cephalosporins with a 3-methyl substituent (deacetoxy-cephalosporanic

acids) are derived from penicillins. The more complex nature of cephalosporins in

comparison with penicillins offers greater opportunities for the production of semi

synthetics and besides 6-ACA, 6-ADCA (6 amino decephalosporanic acid) is also used

as a basis for the production of semi synthetic cephalosporins. The methods of their

preparation are similar to those described for the semi-synthetic penicillins.

24.2.2.5 Use of cephalosporins

Among cephalosporins, cephalothin occupies the same position as benzyl-penicillin,

which continues to be widely used despite some of its deficiencies and the presence of

newer products. Cephalothin is broad-spectrum although ineffective against some

Gram-negative organisms such as Proteus and Pseudomonas. It is administered preferably

intravenously because it is poorly absorbed and because intra-muscular pain is high in

some individuals. New cephalosporins have been produced which are effective against

Gram-negative organisms e.g. cefazolin and cefenandole. Oral cephalosporins include

cefatrizine and cefachlor.

24.2.3 Other Beta-Lactam Antibiotics

24.2.3.1 Cephamycins, (7-Methoxycephalosporins)

Three cepham antibiotics produced by Streptomyces were discovered in 1971. A cepham

with a methoxy group at position C-7 was produced by Streptomyces lipmanni while Strep-

tomyces clavuligenis produced cephalosporin (A-16886-A) and 7-methoxycephalosporin

(A-16886-B) with carbamoxy-loxymethyl function at C-3 position. Penicillin N was pro-

duced simultaneously in both strains. Soon afterwards several species of Streptomyces

able to produce 7-methoxycephalosporins were found (Fig. 24.1).

As with other cephams and penicillin N, the production of cephamycins is stimulated

by methionine. They are generally broad-spectrum though the extent to which they affect

Gram-positive and Gram-negative organisms vary among different compounds in the

group. Cephamycin C is for instance specially active against Proteus and E. coli. They are

resistant to hydrolysis by cephalosporinases produced by cephalosporin-resistant

bacteria. They appear most important for the present as starting points for the synthesis

of new semi-synthetic cephams.

24.2.3.2 Nocardicin

The nocardicins were first isolated in 1976 using a super sensitive mutant strain of E. coli,

strain ES 11, whose minimum inhibitory concentrations on penicillin G (100)

Cephalosporin (400) and Nocardium A were 0.8, 0.4, and 0.4 respectively. They are

produced by Nocardia uniformis subspecies suyamanensis. This antibiotic is novel among

the Beta-lactams (Fig. 24.1) in that it is monocyclic (i.e., no ring is fused to the Beta-lactam

ring). In comparison with the cepham, cephazolin, it is highly effective against the Gram-

negative Proteus and Shigella but has limited activity against Pseudomonas. An enhanced

activity occurs however when Pseudomonas is treated in vivo. Against Gram-positive

bacteria and yeasts and molds it is completely ineffective. Norcadicin A and B differ

slightly in their structures and are very non-toxic to mammals.

Production of Antibiotics and Anti-Tumor Agents "!'

24.2.2.3 Clavulanic acid

This antibiotic was first described in 1976 using another novel method of isolation. In

this method, agar plates containing 10 mcg/ml of benzylpenicillin are seeded with Beta-

lactamase – producing Klebsiella aerogenes. Test samples which did not inhibit the

organisms without the introduced penicillin give a zone of inhibition on the test plate

when a diffusible Beta-lactamase inhibitor was present. Clavulanic acid, cephalosporins

and penicillin N are produced by strains of Streptomyces clavuligerus using the above

method. Clavulanic acid was not however discovered with the classical method. It is a

weak antibiotic but has broad-spectrum activity against bacteria. However, it has an

obvious potential value if it can be used along with penicillinase-susceptible antibiotics

of greater potency than itself. Structurally it resembles cephalosporins.

24.2.3.4 Thienamycins

Thienamycin was first described in 1976. Like the cephalosporins and the cephamicins it

was discovered as a fermentation product with broad spectrum activities and was

produced by Streptomyces cattleya. Thienamycins are reported to be broad spectrum even

at lower concentrations while being as non-toxic as the known natural and semi-

synthetic Beta-lactams.

24.3 THE SEARCH FOR NEW ANTIBIOTICS

In the 1970s the view was that the fight against communicable diseases was about to be

won. The rates of bacterial disease were falling through vaccination and the effectiveness

of the available antibiotics. Many pharmaceutical companies decided to focus attention

away from anti-microbial drugs production as there seemed to be little need for new

compounds. The situation has changed and there now appears an urgent need for new

antimicrobial drugs. This section will discuss the need for new antibiotics, the classical

methods for searching new antibiotics, and some of the newer methods for prospecting

them.

24.3.1 The Need for New Antibiotics

24.3.1.1 The problem of multiple resistance to existing antibiotics

Microorganisms have developed multiple resistance to many of the antibiotics currently

in common use. This is due to several factors some inherent in the nature of

microorganisms, others relating to use (or misuse) of antibiotics by humans. Some of the

factors pertaining to the human use of antibiotics include the wide spread and sometimes

unnecessary use of antibiotics, the prophylactic use of antibiotics, and the use of low

doses of antibiotics for encouraging the growth of farm animals. In the case of bacteria,

their sheer numbers means that there is a potentially large number of genotypes waiting

to be selected and their short generation time fuels the rapidity of this selection. Finally

their ability to horizontally transfer genetic materials through plasmids, transposons,

and by conjugation and transformation set the stage for the (almost) inevitability of the

development of resistance among microorganisms, especially bacteria.