Vous êtes sur la page 1sur 8

Mutation: Various mutagenic agents like UV irradiation Nitrogen mustard etc, have been tried, during 60s.

Recently NTG a mutagen providing extremely high mutational rate relative t its killing effect has been preferred. Various media developed by panlab program fro screening the mutant having higher yield of penicillin. One approach to improving the efficiency of this procedure is the potency index agar plate technique used in the improvement of high yielding cultures. In this case the zone size was reduced by incorporating penicillinase in the agar to reduce the sensitivity to a useful range. An important contaminant of the high potency broth turns out to be the oxidized P-hydroxy form of phenyl acetic acid, which when incorporated interferes with the semi synthetic chemistries. A clear selection techniques used by panlab involves choosing for further evaluation small colonies from plates with phenyl acetic acid as sole carbon source, except for just enough glucose to produce a small colony. Thus the small colonies were those impaired in the ability to oxidize the side chain precursor. The techniques was successful and used by several commercial users Genetics > two techniques adopted for getting recombinants with superior yield over parents Parasexual breeding: techniques involves construction of a complementation auxotrophic blocks or different resistance mutations in two strains. The parents are grown together on complete medium then selected for presence of both the markers, allowed to sporulate and stable diploid spores are isolated. Recombinants with superior yields over the parents have been reported and certain diploids have been used industrially. Protoplast fusion: Protoplast fusion followed by selection techniques had allowed it to improve penicillin productivity by 8% per year. The technique is used to introduce desirable characteristics such as stable sporulation and good growth of seed culture Recombinant DNA technique: Basic goal of any program is to increase the rate of rate limiting steps. The first approach there fore is to identify the rate limiting step. A major research effort would be needed and at the end one would identify a particular enzyme isolate it clone it. At this point one is likely face with a new rate limiting step. If one has a suitable vector and a properly sized gene bank of a high producing strain, there in principle, screening 3000 clones should allow one to obtain increased copies of each gene in the organism in turn provided that transcriptional

translational or substrate level regulation did not overcome the effect of increased gene dose, one would have created an improved strain. If one would further amplify and integrate the gene responsible into the chromosome, a more stable strain might result. In recent developments a group at Eli lilly has developed a transformation system for cephalosporium and has cloned the gene for C. acremonium cyclase. Combination of all the above techniques should continue to lead to improvements in penicillin production efficiency for several years. It should be recognized that another application of the engineering of novel B-lactams through modification of substrate specificities of key enzymes and other techniques might be quite important also. Fermentation process: Inoculum development: These stages are conducted at 250 C in shake cultures and agitated vessels. A typical vegetative or seed stage medium contains an organic nitrogen source such as corn-steep liquor and a sufficient concentration of fermentable carbohydrates such as 2% (w/v) sucrose or glucose. CaCO3 at 0.5 to 1.0 % is often included as buffer. Log phase growth is usually desirable at these stages and a mass doubling time of about six hours is achieved. Criteria for monitoring the inoculums stages include cyclic time, changes in PH residual carbon source concentration, packed cell volume and respiration rate. Fermentation stage: 1. carbon and energy source other than glucose and lactose P. crysogenum can utilize a variety of carbon and energy sources. Among these that are used for penicillin production are sucrose dextrins, and starches. A variety of crude carbohydrate sources of lower purity such as molasses has been mentioned. Animal and vegetable oils and ethanol have also been used. Use of fatty acids and lactose as the carbon and energy source for benzyl penicillin production has been compared. Under the same conditions one gram of soybean oil produced as much benzyl penicillin as 2.5 g lactose under their optimized batch conditions fed corn oil produced penicillin at a faster rate and for longer duration than lactose. Thus vegetable oils are effective carbon sources when fed. Nitrogen sources:

Untill 1975 most industrial processes included corn steep liquor as the organic nitrogen source mainly because of the presence of phenylalanine which gets converted to phenylacetic acid ( a side chain precursor). The introduction of specific side chain precursors allowed producers to substitute for corn steep liquor because of its variability and occasionally short suppy. Cotton seed meal is found to be a satisfactory substitute. Pan labs used cotton seed flour for as substitute for corn steep liquor. The importance of a continued supply of ammonium nitrogen has been demonstrated. It is essential to maintain a concentration of ammonia (by feeding ammonium sulphate) between 250 and 340 ug/ml for continued synthesis of penicillin. A lack of available ammonia nitrogen caused lysis of the mycelium and a drastic decrease in respiration relative to controls with a suitable nitrogen feed. Nitrogen is of course required for penicillin synthesis. Precursors of the penicillin side chain: The side chain attachment is nonspecific and high concentration of phenylacetic acid,or phenoxyacetic acid has to be included in the medium or in feeds to the fermentor, if the desired single component penicillin has to be obtained. The theoretical requirement for sodium phenylacetate is 0.47 g/g penicillin G and for sodium phenoxyacetateit is .50 g/g penicillin V To avoid the toxicity of sodium phenylacetate to the producing organisms and to minimize hydroxylation of these compounds by roducing organismthey are fed continuously. It has been calculated that greater than 90% conversion of phenylacetic acid into penicillin G side chain has been obtained through continuous feeding of one of its salts to maintain a low residual concentration. Sulfur source: Pan et al mentioned ammonium sulphate a common ingredient in production media as providing nitrogen and sulfur in the proportional necessary for penicillin synthesis. Phosphorus source: phytic acid in corn steep liquor has been reported as an important source of phosphorus. Phosphorus may also be added in the form of inorganic salts.

Factors limiting duration of the fed batch Fermentation The duration of penicillin Fermentation can be limited by the oxygen transfer capacity of the fermentor. Since a certain growth u is required to maintain high q pen (penicillin productivity) values. At sometime in the Fermentation the cell concentration will exceed the oxygen supply necessary to maintain high qpen values and synthesis will decay. Morphology and penicillin production While studying morphology and penicillin production it was observed that pellet type growth very high dissolved oxygen loads were necessary to obtained reasonable penicillin productivity The highest penicillin yield could be obtained when mycelium was in short fragments and agitation was not sufficient to cause excessive mechanical damage. The observations from other groups also confirmed this that filamentous mycelium produced a higher penicillin titer than mycelium in pellet form. It was also found that a strain that grew in pellet form had a higher oxygen uptake rate and produced double the penicillin titer compared to the strain that exhibited the filamentous form. A conclusion which can be drawn is that the genesis of larger, dense pellet must be prevented and mycelium in the filamentous form or small fluffy loose (hairy pellet) forms seems preferable for penicillin production. Good penicillin production has been observed with both filamentous and pelleted mycelium

Broth rheology Mycelial broth exhibits a sheer thinning behavior. The apparent viscosity defined as the ratio of the shear stress to the shear rate, decreases with increasing shear rate. This leads to relatively low viscosities in regions of higher shear rates. (near the impeller in a stirred vessel) and a very high viscosities in the region of markedly diminished shear rate (near the wall or cooling coils in a fermenter where stationary zones occurs. The highly viscous mycelial broth cause serious problems in the transfer of momentum heat and mass.

Transport phenomenon in fermenter influences the fermentation conditions. These conditions influence growth morphogenesis and product formation. Use of pellet growth form has the advantage of eliminating the broth viscosity problem to a great extent; however the transport of nutrient into the pellets can be diffusion limited. The volumetric oxygen transfer coefficient KLa falls with increasing viscosity and filamentous mycelial broth can quickly become oxygen limited. The poor oxygen transfer is probably a result of low bubble formation rates, increased bubble coalescence rates and decreased broth flow rates around the fermenters or diffusion limitation into mycelial flocs. Mycelial hyphae reside per variable period in the well aerated primary flow region and the more stationary secondary flow region which can be deficient in oxygen. To overcome this mixing problem high external power input results in smaller flocks and a decrease of secondary flow region size and consequently in an increase of oxygen uptake rate, oxygen exchange and heat removed but also in increased heat generation. Microscopically a pelleted morphology seems energetically more efficient than a filamentous morphology. Nutrients for mycelial growth and penicillin production: Nutrient media and feeds The nutrient media used by the penicillin G and v producer are closely guarded trade secrete. Production of submerged fermentation started with lactose corn steep liquor medium. It mainly contains glucose, lactose, salts phenyl acetic acid, antifoam and corn steep liquor. The use of additional salts depends upon the quantity of corn steep liquor used. PH during fermentation is controlled by addition of alkali or ammonia and acid. Lactose was initially partially and finally completely replaced by a continuous feed during fermentation.

Batch fermentation: Submerged penicillin fermentation has a long history of development since first trials during world war. For this process a lactose corn steep liquor medium was developed. Lactose was found to be a

penicillin production improving carbon source. Lactose is a slow enzymatically hydrolyzed carbohydrate. The slow rate of enzymic hydrolysis prevents accumulation of hexose, which can cause inhibition of the penicillin synthesis. Corn steep liquor helps in improving the yield of penicillin since it contains materials like amino aids, polypeptides, lactic acid minerals etc. which are required for penicillin Production. A disadvantage of substrate with a higher degree of reduction than glucose such as ethanol, fatty oils and 1-phenyl-n-alkanes is a higher heat production. Oxygen demand and a considerable higher heat production. Oxygen demand and heat generation by the organism increase with the degree of reduction on the substrate. As a consequence of use of such substrates; aeration and cooling capacity limits of the fermenter are reached at as lower total carbon dosage than when glucose is the only carbon and energy source. The use of fatty oils as consumable antifoam was found to affect oxygen transfer and to cause a decrease in dissolved oxygen tension. The metabolism of consumable antifoam resulted in an increased growth rate and a decreased penicillin production rate. Penicillin strains have been selected which give a lower viscosity broth. As results the amount of biomass which is normally limited by the oxygen transfer capacity could be increased resulting in high yields. Strains with reduced level of phenyl acetic acid oxidase. Biochemically deficient mutants and strains resistant to a wide variety of mitotic inhibitors, heavy metals and sulfur and amino acid analogs yielded high percentage of superior strains. Tolerance to phenyl acetic acid, sensitivity to iron penicillin acylase activity etc. The presence of phenylalanine present in corn steep liquor acts as a side chain precursor. Phenylalanine can be broken down to phenylacetic acid which in turn is precursor for penicillin side chain. After the glucose and corn steep liquor carbon compounds are metabolized and rapid mycelium development has occurred, lactose utilization begins and slow mycelial growth results. During the rapid growth phase the rate of antibiotic production was comparatively low. The maximum penicillin synthesis occurred during the lactose utilization phase, the slow growth phase. After the lactose was exhausted autolysis begins and the onset of a decrease in penicillin concentration was observed. The fermentation were harvested before this point. N- source: the corn steep liquor nitrogen compounds are deaminated and ammonia nitrogen is formed. The nitrate is utilized when organic nitrogen is no longer available. Owing to nutrient utilization the

ph decreased first, dropped slightly when lactose was utilized. For good penicillin yield the PH should stay between 6.8 & 7.4. Fed batch fermentation: Improve penicillin yields were initially obtained by daily or more frequent addition of carbon compounds. Hexose monomers (glucose) or carbohydrate compounds readily hydrolyzed to hexose monomers were found to be the best carbon sources. Easily fermentable carbon sources such as glucose or sucrose however have to be added continuously in a controlled amount during the fermentations to establish optimal condition for formation of both biomass and penicillin and to prevent accumulation of hexose in the broth. Glucose feeding initially is not practiced. The amount of lactose present in the medium once it gets exhausted, a glucose or sucrose solution was continuously fed to the broth. Lactose present in the medium helps to control the growth rate , optimum for the penicillin production in the later stages of fermentation lactose is replaced by glucose feed, the rate of which was decided by lactose consumption data. The lower limit of sugar feed rate is determined by the amount needed to prevent autolysis and upper limit by oxygen limitation. With glucose or sucrose feed the ffermentation time could be extended considerably. Fatty oils containing a high contnt of oleic acid and linoleic acid such as lard, soybean and corn oil were very efficiently used as crbon and energy sources when fed to the ffermentation as an antifoam. Ammonia has been fed both as a nitrogen source and to control the ph. It was found essential to maintain the residual concentration of ammonia at about 0.3 to 0.4 kg/m3 to obtain a high specific penicillin production rate and a continued synthesis of penicillin. The residual concentration of the penicillin side chain precursor as phenylacetic acid in the broth must be controlled with 0.1 to 1.0 kgPAA/m3. Low concentration results in production other than penicillin G and high concentration can cause toxicity and hydroxylation by producing organism. The penicillin production improvement due to development of a fed batch process has been regarded as result of both fermentation process and strain improvement. The improvement of the specific production rate of the strain was estimated to be 20fold. The present maximum specific penicillin G production rate is said to be about 8-9 units/mg (mycelial dry weight). Efforts have been made to device chemically defined medium. The penicillin production was lower than when complex media was used, but product purification was easier. Chemically defined media lack buffering capacity. CaCO3 was added to the medium toprevent low PH values. To increase buffering capacity KH2PO4 was also used.

Alternative raw materials: Carbon sources: the carbon source represents a comparatively large part of the overall manufacturing costs for the penicillin production. It is estimated to be about 12% or more. Pentoses, hexoses, mixture of hexoses disaccharides polyols dextrines starch hydrolysate starches molasses can be metabolized comparably or less efficiently than glucose. Addition of fatty oils in addition to lactose to a glucose fed was found to increase penicillin titers. Ethanol was employed as a carbon feed source and therefore as the major carbon and energy source. Acetic acid was used as carbon feed source together with glucose in fermentation with a low production strain. Acetic acid is quickly metabolized by P. crysogenum at ph 6-7 via TCA cycle when 20% of the carbon source in the feed was acetic acid penicillin V titers were about 25% higher than in fermentation where glucose was the only carbon source in the feed. This might be explained by an increased availability of precursor s of cysteine and valine for penicillin biosynthesis which might be related to the effect of fatty oils. Nitrogen source: because of the variability of composition of corn steep liquor, substitutes were sought. Cotton seed meal, different kinds of brands with considerable amount of phytic acids phosphorus, penicillin mycelium can be regarded as usable substituents. Inorganic or defined organic nitrogen source in chemically defined media are also usable substituents. Precursor sources Some 1-phenyl-n-alkanes are suitable carbon, energy and precursor sources for penicillin G ffermentations. Using 1-phenyln-decane and 1-phenyl-ndodecane 70-80% was converted to phenylacetic acid and produced more than 90% benzl penicillin. The penicillin potency being comparable to the fermentation with phenyl acetic acid as precursor.

Vous aimerez peut-être aussi