Biotechnological production of prostaglandin

Biotechnological production of prostaglandin

Biotechnology Advances 19 (2001) 387 – 397 Research review paper Biotechnological production of prostaglandin Emin Yilmaz* Department of Food Engine...

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Biotechnology Advances 19 (2001) 387 – 397

Research review paper

Biotechnological production of prostaglandin Emin Yilmaz* Department of Food Engineering, Faculty of Engineering and Architecture, C¸anakkale Onsekiz Mart University, 17100 C¸anakkale, Turkey

Abstract Prostaglandins (PGs) are the oxidation products of PG endoperoxide (PGH) synthase and other tissue enzymes. They occur in a tissue-specific manner and act as local hormones. Biotechnological production of PGs has been of interest, but not yet fully established. Biological tissues have been used as PG sources, but this disturbs ecological balance, and the cost of production is very high for commercial purposes. On the other hand, various microorganisms have been shown to synthesize them de novo, or biotransform precursors to active molecules, but these processes have not been further evaluated. Using mammalian enzymes in free or immobilized form is a promising new approach to synthesize PG from fatty acid substrates. Rapid enzyme inactivation during the catalysis is the main problem to be solved. Optimization of factors in the reactions and the design of special reactors that will allow removal of products continuously from the reaction medium without affecting enzyme activity need immediate attention from researchers and the pharmaceutical industry. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Prostaglandin; Biotechnology; Microorganism; Enzyme; Production

1. Introduction Eicosanoids, which include prostaglandins (PG) and oxylipins, comprise a family of structurally related lipid mediators that exhibit interesting biological activities in animals and in plants, respectively. They are synthesized from arachidonic acid (AA) that is released upon cell stimulation from membrane phospholipids (Newton and Roberts, 1982; Kuhn and

* Tel.: +90-286-213-0205; fax: +90-286-213-0155. E-mail address: [email protected] (E. Yilmaz). 0734-9750/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 4 - 9 7 5 0 ( 0 1 ) 0 0 0 7 2 - 6

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Borngraber, 1998). Almost 50 years ago, scientists in Europe and the USA observed that lipid fractions isolated from human semen induced contraction and relaxation of the human uterus. Von Euler then coined the name prostaglandin for the active component, erroneously believing that the substance was produced in the prostate gland (Curtis-Prior, 1988). In the biological systems, PGs have various tissue-dependent functions (i.e. vasodilation, platelet aggregation, pain induction). There is a great need for those molecules as pharmaceuticals in medical applications (as pyretic agents, abortives, or circulation aids). Today, they are not cheaply available in large quantities but are rather produced by complex chemical syntheses that cost thousands of dollars per gram (Holland et al., 1988). Even though the seminal fluid and vesicular gland are the two natural sources of PGs, their occurrence in other animal tissues, plant tissues, lower vertebrates, and microorganisms has been reported (Holland et al., 1988; Goetz et al., 1989; Lamacka and Sajbidor, 1997; Panossian, 1987). Using AA cascade enzymes for biotechnological production of PGs is another means of manufacturing still awaiting further research and development (Lamacka and Sajbidor, 1997). In this review, the biochemistry and biology of PGs, their synthesis through tissue usage, microbial bioproduction, biotransformation, and enzymatic production will be discussed.

2. The biology and biochemistry of PGs The presence of PGs in most tissues such as vesicular gland, kidney, and brain has been demonstrated. Most of the prostanoids are synthesized and released in response to a stimulus, while some molecular species appear to be stored as prefabricated compounds in tissues. The discovery of different PGs with three branches showing high activity and low stability made the picture even more complex. Today, stable PGs, thromboxanes, prostacyclins, and leukotrienes are well known (Curtis-Prior, 1988). The availability of substrates, the different PG-synthesizing enzymes present (and active) together with the different rates of biosynthesis and metabolism can combine to make one particular PG predominate in any particular tissue (Newton and Roberts, 1997). The main source of free AA is the cell membranes. Many different types of stimulation — physical (stretching, squeezing, vibration, electrical) and chemical (hormonal, immunological, ischaemia) — will lead to the release of free AA (Newton and Roberts, 1997). Free AA and other fatty acids are subsequently metabolized via three different pathways: (1) the cyclooxygenase (COX) pathway, forming PGs, thromboxanes, or prostacyclins; (2) the lipoxygenase (LOX) pathway, forming leukotrienes, lipoxins, hepoxilins, and hydro(pero)xy fatty acids; and (3) the cytochrome P450 (cyt-P450) pathway, forming hydroxylated fatty acids and epoxy derivatives (Kuhn and Borngraber, 1998; Smith, 1989). In the COX pathway (Fig. 1), the initial reaction is done by PG endoperoxide synthase (PGH synthase), which for simplicity is called COX. AA, which contains four double bonds, is converted via the COX pathway into the PGs of the 2-series (PGD2, PGF2, PGF2a, PGI2, TX2). The biologically active PGs are further metabolized into decomposition products that are subsequently eliminated from the body by excretion in urine. As with the biosynthesis of PGs, their metabolism and degradation occur rapidly and to different extents in different

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Fig. 1. Biosynthetic pathways of prostaglandin biosynthesis (Smith, 1989).

tissues. The decomposition pathways for various PGs are different but there are common principles in the metabolism: (1) oxidation of the OH-group at C-15, forming corresponding keto derivatives; (2) b-oxidation of the carboxyl terminus, forming di-nor PGs; and (3) woxidation of the methyl terminus, forming dicarboxylic compounds (Kuhn and Borngraber, 1998; Newton and Roberts, 1997). PGs show very diverse functions depending on the kind and the tissue in which they are found. At the functional level, it appears that many PGs are rapidly biosynthesized, exert their biological effect, and are rapidly metabolized, thereby functioning as autocoids or local hormones (Newton and Roberts, 1997). PGE and prostacyclin act as vasodilators and prevent platelet aggregation, and PGI2 strongly dilutes coronary blood vessels, while TXA2 induces platelet aggregation. PGE can induce pain when combined with bradykinin, or itching when given together with histamine. PGE is also a powerful pyretic agent when injected intracerebroventricularly. PGs have actions on sperm migration, dysmenorrhea, labor and parturition, luteolysis, and circulatory functions in the uterus and placenta, in part associated with foetal growth. Prostaglandins PGE2 and PGF2a have found extensive use as abortives. PGs are diarrheic in some cases, and also inhibit the gastric secretion to protect epithelium of the gastrointestinal tract. PGEs inhibit the response to adrenergic nerve stimulation and release of noradrenalin from the nerve endings (Curtis-Prior, 1988; Swinney et al., 1998). In

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addition, PGs have roles in control of body temperature, onset of rheumatoid arthritis, and some bone cancers (Newton and Roberts, 1982). The observation of their very low concentrations (10 9 M) and synthesis in most tissues support the concept to state that PGs are local hormones. They act via the receptors analogous to adrenergic receptors coupled to G proteins. Once induced, concentrations of second messengers (cAMP, IP3, IP4, DAG, Ca2 + , etc.) change by stimulating adenylate cyclase, phospholipase C, opening/closing Ca2 + and K + channels, or promoting Na + /H + exchange (Smith, 1989).

3. The ways of PG production PGs are used in medicine, scientific research, and pharmaceuticals. They are used as parturifacients in obstetrics and gynaecology, intestinal movement promoters after abdominal operations, and medicine for the treatment of chronic arterial obstruction and erectile dysfunctions. Obviously, there is a great demand for them, so that several approaches have been considered to produce them. Semisynthesis by using tissues, microbial biosynthesis and biotransformations, and enzymatic production are the main ways of production (Lamacka and Sajbidor, 1997). 3.1. Semisynthesis by using tissues Bundy et al. (1972) describe semisynthesis of PGs from the coral Plexaura homomalla, which is found in the Caribbean area. Ester derivatives of both 15S- and 15R-PGA2 are epoxidized, then reduced and separated by silica gel chromatography. The procedure is tedious and expensive. Synthesis of PGs from P. homomalla and human seminal fluid has also been described by Srivastava (1980). The coral contains about 1.3% R-enantiomers of PGs, and they are converted to the S-enantiomers by epoxidation and reduction. Human seminal fluid contains large amounts of 19-hydroxy derivatives. PGs are extracted and purified by chromatography, and purity is controlled by bioassay. There is an ecological danger of using coral as PG biosource, hence, other sources must be found. The synthesis of PGs by follicle walls of brook trout (Salvelinus fontinalis) mature ovary was investigated by using [14C]AA by Goetz et al. (1989). Synthesis was significantly greater in follicles taken postovulation as compared to those sampled prior to germinal vesicle breakdown. The highest conversion of PGs to PGE2 (19.4%), and a small amount to PGF2a (1.6%) was observed. Occurrence of PGs and related compounds in plants was shown by Panossian (1987). The seeds of Bryonia alba contain trienic dioxyacids, and germinating beans contain traumatine. PGF2a has been discovered in the extract of germinating seeds of Pharbitis nil, and it showed that the content increases during flowering and in darkness. Yellow onion (Allium sepa) and other onions were shown to contain PGA1 (0.0001%) and other PGs. The red algae, Gracilaria lichenoides includes 0.07% and 0.1% PGE2 and PGF2, respectively.

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PGF2a (1–1.5 ng/g) was found in Kalanchoe blossfeldiana v. Poeln. There are some indications of the presence of PG-like compounds in sugar cane, bananas, and coconut, but this is not yet confirmed. There is no study reporting large-scale extraction of PGs from the plant sources. Mammalian gland tissues have long been used as PG biosources. These tissues include ram, hog, and beef adrenal and vesicular glands. The serious drawbacks of semisynthetic ways are the requirements for internal organs of animals, low stability of the tissue enzymes and the products, and expense of the process (Lamacka and Sajbidor, 1997). 3.2. Microbial biosynthesis and biotransformations A series of 15-deoxy-PG1 analogs were synthesized chemically in the laboratory, and their biotransformations by Rhizopus arrhizus, Rhizopus stolonifer, Aspergillus ochraceus, and Curvularia lunata fungi were studied by Holland et al. (1988). All products were isolated by extraction, purified by chromatography to homogeneity, and fully characterized by IR, H-NMR, and C-NMR analyses. They concluded that all those fungi are able to synthesize prostanoids carrying hydroxy groups at C-18 or C-19 position. Sadney and Willetts (1989) described the biotransformation of norbornanone to bridgehead lactone, which is used in chemical synthesis of prostanoid analogues by cyclopentane-grown Pseudomonas sp. NCIB 9872. The biotransformation was best at pH 7.1–7.3 with a substrate concentration less than 15 mM, and with added nonionic detergent and sonication of medium before incubation. This method can be combined with chemical synthesis to Table 1 De novo eicosanoid-synthesizing bacteria and fungi (Lamacka and Sajbidor, 1995) Microorganism Bacteria belonging to Mycobacterium, Pseudomonas, Micrococcus, Streptomyces, Nocardia Pseudomonas aeruginosa, Propionibacterium acnes Acinetobacter calcoaceticus Actinomyces spheroides Fungi belonging to Ascomycetes, Phycomycetes, Basidiomycetes, Deuteromyces, Saccharomyces cerevisiae, Dipodascopsis uninucleata, Dipodascopsis tothii, 33 Lipomycetaceae strains Aspergillus sp., Mortierella sp., Fusarium sambucinum, Trichothecium roseum Gauemannomyces graminis, Saprolegnia parasitica

Eicosanoid Various PGs and PG-like substances, depending on the PUFA used PGA, E, F, and PG and PGE2-like substances PGE2, PGF2a PGA2, PGB2, PGE2

Various PGs and PG-like substances depending on the PUFA used PGF2a and its isomers, 3-HETE, PGE2, aspirin-sensitive metabolites, a-pentanor-PGF2a-g-lactone isomers PGE2, PGF2a

(18R)-HETE, (19R)-HETE, 17, 18-DiHETE, trihydroxy acids

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Table 2 The occurrence of prostanoids in the algae (Lamacka and Sajbidor, 1995) Alga

Eicosanoid

Euglena gracilis, Gracilaria verrucosa, G. lichenoids Laurencia hybrida, Murrayella periclados, Platysiphonia miniata, Cottoniella filamentosa, Gracilariopsis lemaneiformis, Constantinea simplex M. periclados, C. simplex, Gr. lemaneiformis M. periclados, Pl. miniata, C. filamentosa Farlowia molis, C. simplex, Gr. lemaneiformis C. simplex, Laminaria sinclairii, Laminaria setchellii, Laminaria saccharia

PGE2, PGF2a Hybridalactone, (12S)-HETE

(12S)-HEPE (6E)-LTB4, ethyl ester, Hepoxilin B3 (12R, 13S)-DiHETE and (12R, 13S)-DiHEPE Constanolactone A and B, (15S)-HETE, (15S)-HEPE

reduce the need for strong oxidizers, extreme temperature ( 20C), and many side products. Among many fungi searched for LOX, COX, and the epoxidase pathway, only the Oomycota family and yeast from the genus Dipodascopsis have shown COX activity (Van Dyk et al., 1994). It was noted that in all cases studied that LOX activity was associated with vegetative growth, while COX activity was associated with sexual reproduction. Lamacka and Sajbidor (1995) have shown various bacteria and fungi synthesizing PGs in Table 1, and occurrence of some prostanoids in algae in Table 2. The presence of some PGs in protozoans is also known. Tetrahymena pyriformis contain PGE2, PGB, and PGF2a with a molar ratio of 62.8:34:2.7, respectively. PGs were also detected in Entamoeba histolytica and in Acanthamoeba castellani (Lamacka and Sajbidor, 1995). The literature brings out the obvious possibility of microbial production of PGs but no study reporting large-scale production by these organisms; more research and optimization of the processes still remains to be exploited. 3.3. Enzymatic production of PGs The main difficulties of PG biotechnology are being overcome by using mammalian enzymes more effectively by optimizing physicochemical parameters such as temperature, pH, ionic strength, concentration and type of substrates and products, enzyme cofactors, and bioengineering parameters such as reactor type, reaction kinetics, agitation, aeration, etc. (Lamacka and Sajbidor, 1997). The first thing needed to be known is the arachidonate cascade enzymes, their reaction factors, and biotechnological possibilities.

4. PGH synthase (COX) On an economic basis, it can be argued that COX is the most important enzyme of the world, since its catalytic products account for US$3–5 billion in annual pharmaceutical sales. It is the rate-limiting enzyme of prostanoid biosynthesis (Smith and Marnett, 1991). COX (EC 1.14.99.1) is a homodimer of 70-kDa polypeptides, each subunit containing an iron(III)protoporphyrin IX. This haeme group is required for both catalytic activities of the enzyme

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(Rowley et al., 1998). There are two or three oligosaccharides attached as Man9(GlcNAc)2 and Man6(GlcNAc)2 per protein molecule. There are four glycosylation sites at Asn-68, Asn-104, Asn-144, and Asn-410. Ser-530 is the site of aspirin inactivation near the active site (Smith and Marnett, 1991). Two isoforms of COX exist, COX-1 and COX-2. COX-1 is a constitutive enzyme functioning in basal or housekeeping PG synthesis, whereas COX-2 is an inducible enzyme, which is associated with inflammatory processes. Both isoforms have similar Vmax and Km values for arachidonate, and undergo suicide inactivation. COX-1 requires a hydroperoxide initiator and, hence, it is proposed that cellular PG synthesis is regulated by the level of intracellular hydroperoxide. COX-2 is, however, initiated by considerably lower levels of hydroperoxide than COX-1, which could provide a biochemical basis for differential control of PG synthesis by the two isoforms. COX isoforms are located within both endoplasmic reticulum (ER) and the nuclear envelope (NE) (Rowley et al., 1998). The best substrates for the COX reactions are 8,11,14-eicosatrienoic acid (di-homo-glinolenic acid) and 5,8,11,14-eicosatetraenoic acid (AA) with a Km of 2–10 mM, and a turnover number of 1400 min 1. On the other hand, docosahexaenoic acid is a potent competitive inhibitor with a Ki of about 5 mM. Other n 6 fatty acids were reported to be poor substrates of the enzyme. Glutathione is an electron donor in the COX reaction (Smith and Marnett, 1991). The first reaction is a bis-oxygenation of arachidonate producing PGG2, and the second reaction is the peroxidase reaction, a two-electron reduction of the 15-hydroperoxyl group of PGG2 to form PGH2. PGH synthase is believed to interact with arachidonate having a kink in the carbon chain due to rotation about the C-9/C-10 bond. The enzyme first abstracts the pro-S hydrogen from C-13. A molecule of O2 is added at C-11 from the solvent site. Serial cyclization of the incipient 11-peroxyl radical yields an endoperoxide with aliphatic chains trans to one another. A second O2 is then added at C-15, and finally, reduction of peroxyl radicals produces PGG2 with the stereochemistry of a natural product (Rowley et al., 1998; Gross, 1997). Dynamics have been shown to play critical roles in the mechanism and inhibition of COX-1 and COX-2. Many inhibitors of both isoforms are time dependent and the enzymes are allosterically activated by AA (Swinney et al., 1998). The peroxidase activity has been copurified and coimmunoprecipitated with COX activity of PGH synthase. Peroxidase activity reduces PGG2 to PGH2, the final product of COX activity. A haeme prosthetic group is required for this activity as well as a hydroperoxide. Many nonsteroidal anti-inflammatory drugs such as aspirin, ibuprofen, flurbiprofen, indomethacin, and meclofenamate inhibit the COX activity, but not the hydroperoxidase activity (Smith and Marnett, 1991; Rowley et al., 1998). In addition, a substrate-induced inactivation mechanism has been suggested (Varfolomeyev and Mevkh, 1993). It operates in parallel with involvement of a few intermediates of the catalytic conversion of substrates into PGH. The principal intermediates are some peroxidates of the reaction. It is evident that endlessly high concentrations of oxygen and of an electron donor do not totally prevent enzyme inactivation. Thus, PGH synthase is a ‘suicide’ enzyme. The enzyme is fairly stable in solutions of free enzyme at 70C for several months, but it rapidly inactivates in the presence of oxygen. Fortunately, this inactivation can be protected by adding cofactors (i.e. tryptophan, hydroquinone, phenol). Usually, it makes 103 catalytic turnovers and thereafter is inactivated (Smith and Marnett, 1991).

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An acetone–pentane preparation of PG synthase from sheep seminal vesicles has been described, and some properties of the preparations were studied (Wallach and Daniels, 1971). It has been shown that triethanolamine, imidazole, and Tris buffers inhibited the enzyme, but not as well as Na-EDTA. The optimal level of glutathione (GSH) addition was determined as 2.5  10 4 to 10 3 M. The optimum pH range was 7.5–8.5, and reaction rate was the same when run in either air or oxygen, but rapid stirring was most important presumably for adequate gas exchange. The enzyme lost its initial activity at 50C, 55C, and 60C by 25%, 38%, and 82%, respectively. Increasing the amount of acetone–pentane powder in solution increased the product sixfold; then a no-effect point was reached. Decanoic acid inhibits the reaction, whereas caprylic, nonanoic, undecanoic, and lauric acids do not. Further experiments have shown that the inhibitor was not formed in the absence of substrate, but formed in the presence of substrate and glutathione. 4.1. PG endoperoxide-metabolizing enzymes PG endoperoxide-metabolizing enzymes are tissue-selective to produce a particular type of PG or TX that regulates the function of the tissue with its specific biological activity (CurtisPrior, 1988). Three of them are the most important metabolic enzymes. 4.1.1. PGH–PGD isomerase It catalyzes the isomerization of 9,11-endoperoxide to 9-keto and 11a-hydroxyl groups. It is highly unstable, and its half-life is about 20 h at 0C. Inactivation can be suppressed by storage at 80C. For activity, reduced glutathione and a pH of 5.5–7.0 is needed (CurtisPrior, 1988; Lamacka and Sajbidor, 1997). 4.1.2. PGH–PGE isomerase It catalyzes the isomerization of 9,11-endoperoxide moiety to 9a-hydroxyl and 11-keto groups. It requires cofactors (glutathione) and an optimum pH of 6. At neutral pH, about 50% of the activity is lost after 24 h at 4C (Curtis-Prior, 1988; Lamacka and Sajbidor, 1997). 4.1.3. PGF2 reductase The conversion of PGH2 to PGF2a is a reductive cleavage of the 9,11-endoperoxide bound catalytically by this enzyme. It does not require cofactors, and it is relatively thermostable (Curtis-Prior, 1988; Lamacka and Sajbidor, 1997). 4.2. Some factors affecting enzyme activity The pH range of the COX complex is between 7.5 and 8.5. The optimum pH for sheep seminal PGE2-synthase complex was reported as 8.0. Similarly, optimum temperature for the PG synthase complex has been reported to be around 37C, which represents body temperature. Most of the enzymes are inactivated rapidly at higher temperatures. Some buffers (triethanolamine, imidazole, and Tris) are absolute inhibitors of the enzyme, whereas the best buffer is EDTA. Oxygen is an absolute requirement for the reaction. Stirring, agitation, or

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passing oxygen gas through the reaction mixture can assure good availability of enough oxygen for the reaction. Nicotinamide, cytochrome c, Na-ATP, and FAD as cofactors, reduced glutathione as electron donor, ascorbic acid, catechol, hydroquinone, and phenol at low concentrations as antioxidants have been reported as activators of the reaction. Also, in order to trigger the PGH synthase, about 10 nM of lipid hydroperoxides were required. The best substrates for the enzyme complex are AA, and eicosa-8,11,14-trienoic acid. On the other hand, n 3 series of fatty acids are poor substrates. At higher substrate concentrations, the reaction proceeded asymptotically within 15 min, whereas at low concentrations (0.5 or 0.25 mg/ml), the reaction prolonged and yield improved (Lamacka and Sajbidor, 1997; Smith and Marnett, 1991; Wallach and Daniels, 1971).

5. Applications of enzymes in PG production Acetone–pentane powder of sheep seminal fluid in a batch process was used to synthesize the PGs. With adequate care taken, recycling of the enzyme from the reaction mixture by centrifugation can increase the yield as suggested from the results of the study (Wallach and Daniels, 1971). As a batch process, tissue homogenates containing the enzymes were added to suitable mediums, pH adjusted to 7.5–8.0, and incubated at 37C for 2–14 h. Then, extraction was done with inorganic solvents. Finally, the extract was purified by chromatography. Rapid inactivation of the enzyme is the major problem encountered (Lamacka and Sajbidor, 1997). Prostaglandin synthase complex has successfully immobilized on DEAE-Sephadex, but its activity is lost at subsequent cycles (Lamacka and Sajbidor, 1997). Photocross-linkable gel immobilization has been used, and by the fifth cycle, the yield decreased to 30% of the initial yield. This may be explained by the accumulation of the inhibitors within the gel (Ahern et al., 1983). Also, the enzyme has been immobilized on insoluble polyelectrolyte complexes, and 40–70% of initial activity was shown to be present with an increased thermal stability (Lamacka and Sajbidor, 1997). Different methods of enzyme immobilization will be needed for improved synthesis. A semibatch process (Sada et al., 1989) has been investigated for the PG synthesis. Substrate is gradually added to keep the level of inhibitors lower. This process has yielded 3.5-fold higher total PGE2 in comparison with the batch process giving a concentration of 1.0 mg/cm3. A continuous-flow bioreactor with a hollow fiber membrane of polyacrylonitrile has been used for continuous bioproduction, but the reaction time increased from 20 min to 100 min, and yield decreased 10% over batch process due to rapid deactivation of the enzyme (Lamacka and Sajbidor, 1997).

6. Conclusion Enzymatic oxidation products of essential n 3 fatty acids have shown to be functionally very important molecules in biological systems. Prostaglandins, a class of those molecules,

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are needed for the market with reasonable cost, availability, and purity for medical, pharmaceutical, and research uses. Different methods of biotechnological PG production have been investigated, but still none of them can compete with chemical synthesis, which is basically very costly. More research is needed on transgenic animals and microorganisms to biologically produce PGs in higher amounts and for lower price. Similarly, enzymatic production of PGs is another means of production in great demand. Enzyme immobilization and stabilization is needed to prevent rapid inactivation, which is the major problem. Continuous removal of product from the reaction medium while keeping the system unaffected may be a solution that can be resolved by bioreactor and system designs. Also, enzyme engineering can have a role in making the PG cascade enzymes more active and stable for biotechnological applications. There is a very important need for the biotechnological and cheaper production of PGs with higher purity and availability.

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