La prescripción en el derecho comparado

The most significant commercial product from mycelial fermentations is the production o f antibiotics. Products such as penicillin, tetracycline, erythrom ycin, cephalosporins, cephalomycins and clavulanic acid are made at large scale from 50 to 200 m^ (Buckland and Lilly 1993). The widespread use of penicillin since the 1950s has meant that a large amount of literature can be found regarding penicillin production and its process developm ent. However, only a small number of literature examples exist concerning the production of antibiotics such as erythromycin and clavulinic acid. The lack of published papers is presumably due to the commercial sensitivity of the research.

Erythromycin is produced from the filamentous actinomycete Saccharopolyspora erythraea. This organism is similar to Streptomyces sp. except that the cell wall contains large am ounts o f m esodiam inopim elic acid and no m ycolic acid. This led to reclassification of the filamentous, spore producing organism from S trep to m yces erythreus to S. erythraea in 1987 (Holt, 1989). The wild type strain NRRL 2338 was first obtained from a soil sample obtained in the Philippines by the Lilly Research Laboratories, Indianapolis in 1952 (McGuire et at., 1952). The therapeutic action of erythromycin is mainly bacteristatic action at low concentrations 0.01-0.5 |xg mL'^ by binding covalently to the 50 S sub unit of the intact prokaryotic ribosomes. Bactericidal action has been reported at high concentrations above 2.5 pg mL'^ (against S. aureus).

Erythromycin is a macrolide antibiotic which consists of a 14 membered macrocyclic lactone ring to w hich sugars are attached (Garrod et at., 1981). All the types of erythromycin shown in figure 1.1 have a desosamine ring but vary from four R groups. Erythromycin A and B have cladinose sugar, whereas erythromycin C has a mycarose sugar. Erythrom ycin A is usually the main product with B, C, D, E and F as minor components. The precise route and influences on the biosynthetic pathway is still under investigation however, five primary metabolism components are known to be involved in erythrom ycin production, propianate, 2-methylmalonate and oxygen, glucose and S- adenosyl- L-methionine (Corcoran and Hahn, 1975). A proposed pathway to produce the macrolactone ring attached to two sugar residues (figure 1.2), involves the sequential condensation of 6 methylmalonates to a propionate primer, followed by ring closure and sugar attachment (Higashide, 1984). Erythromycin production has been observed to take place during the transition from logarithmic growth phase to stationary phase when a specific nutrient becomes growth rate limiting (Stark and Smith, 1961) or after the growth

CH OH HO- CH CH O R OH Erythromycin _Ri R2 R3 _R4 A OH CHg CH3 H B H C H3 C H3 H C OH H C H3 H D H H C H3 H E OH C H3 CH2 -— 0 F OH C H3 CH2OH H

Figure 1.1 Chemical structure of erythromycin (Corcoran and Hahn, 1975, Higashide, 1984)

propionyl - C oA + methylmalonyl - CoA

polyketide synthase

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T D P - desosam ine, glycosyltran sf era se

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phase (Queener and Day, 1986). The carbon and nitrogen source decrease during growth phase and some erythromycin production occurs. Stark and Smith, 1961 argued that once the nitrogen source is depleted, carbohydrate is then available for antibiotic production. Hence, the majority of erythromycin at a fast linear production rate occurs after the growth phase. Trilli et a i (1987) found evidence to suggest that erythromycin production was strongly linked to the growth phase from the close relationship between the specific growth rate and specific erythromycin production rate from a phosphate limited chemostat culture. Nitrogen limiting media have been found to produce greater yields of antibiotic than carbon limited due to the repressive effect of ammonium salts on erythrom ycin production (Flores and Sanchez, 1985, Potvin and Peringer, 1994). Glucose, fructose or sucrose are all useful carbon sources for erythromycin production (C orum et al., 1954) although D-glucose may have a repressive effect at high concentration (100 gL*0 (W allace et al., 1992, Escalante et a i , 1992). Glycine, glutam ine, alanine, serine, valine, leucine, threonine, betaine and aspargine are appropriate nitrogen sources for growth and erythromycin production (Stark and Smith, 1961). Typical com plex industrial media for erythromycin production include corn starch, soybean meal, corn steep, and soybean oil as used by Abbott Laboratories (Martin and Rosenbrook, 1967). The fed batch control of erythromycin with carbon source feeding is the usual industrial procedure for optimising erythromycin production (Warren, 1994). The mineral requirements for erythromycin production include magnesium and phosphate which are the most critical, followed by iron, zinc, cobalt, and calcium (Stark and Smith, 1961). O f the cited literature which use pH control, the optimum for erythromycin production is between pH 7-7.2 (Osman et al., 1968, W arren, 1994) and the optimum temperature is between 30 - 35®C (Kuznetsov, 1985). Only a few studies have been published on the impact of the engineering environment on erythromycin production which is necessary for process optimisation. Brinberg (1959) showed that erythromycin production was unaffected by the aeration rate when using a nutritionally weak medium containing 2% glucose and oxygen transfer rates o f 18 mg0 2.L*kmin‘^ were satisfactory. However, in a richer media erythromycin production only occurred with more vigorous aeration (29.6 mg0 2.L‘kmin-i). It was suggested that it was due to an increase in cell mass or a specific action in supplying a greater amount of a specific intermediate.

Nash (1974) studied the effect of carbon dioxide on the growth and erythromycin production o f S. erythraea in a 6 8 L stirred tank. Carbon dioxide was added to the incoming air at 0.1 vvm or 11% of the inlet air from 15 h into the 138 h fermentation. The exposure o f the cells to increased partial pressure of carbon dioxide inhibited synthesis o f erythrom ycin by 40% but had no effect on growth. The inhibition of erythromycin production was not due to a reduction in pH as it remained between 6 .8 and 7.0 in both carbon dioxide sparged and unsparged systems.

Paca et al. (1978) found that erythromycin production was proportional to the power dissipation which was studied up to 4000 Wm'^ in a 300 L stirred tank. This may have been due to limiting effect of the DOT which was below 30% saturation during the production phase for the range of power inputs studied. Klein (1994) found that the specific erythrom ycin production rate (qery) was reduced by 3 fold when the DOT remained below 10% for the duration of 10 h during a 100 h 5. erythraea fermentation in a 7 L stirred tank, when compared to the production at DOTs above 60%. However, the different DOTs regimes occurred from increased stirred speed, 250 to 500 rpm, which may also effect erythromycin production from the influence of agitation on morphology. Klein (1994) also provided evidence to suggest that erythrom ycin production was reduced and cell breakage (protein release) increased by a 2 fold increase of energy dissipation from 3000 to 14400 Wm'^ which correlated well with the breakup theory of Smith et al. (1990). Both Carrington et al. (1992) using a 20 m^ bubble column and Roman and Gavrilescu (1994) with a 20 m^ stirred tank showed that k^a decreased during the ferm entation o f S. rimosus and Carrington showed that k^a decreased exponentially with increasing broth viscosity.