Preparative-scale fermentation

Preparative-scale fermentation of papaveraldine, the known benzyliso-quinoline alkaloid, with Mucor ramannianus 1839 (sih) has resulted in a stereoselective reduction of the ketone group and the isolation of S-papaverinol and S-papaverinol M-oxide [56]. The structure elucidations of both metabolites were reported to be based primarily on ID and 2D NMR analyses and chemical transformations [56]. The absolute configuration of S-papaverinol has been determined using Horeau s method of asymmetric esterification [56]. The structures of the compounds are shown in Fig. 7. 

Clark et al. [53] subjected primaquine to metabolic studies using microorganisms. A total of 77 microorganisms were evaluated for their ability to metabolize primaquine, of these, 23 were found to convert primaquine to one or more metabolites (thin-layer chromatography analysis). Preparative scale fermentation of primaquine with four different microorganisms resulted in the isolation of two metabolites, identified as 8-(3-carboxy-l-methylpropylamino)-6-methoxyquinoline and 8-(4-acetamido-l-methylbutylamino)-6-methoxyquinoline. The structures of the metabolites were proposed, based primarily on a comparison of the 13C NMR spectra of the acetamido metabolite and the methyl ester of the carboxy metabolite with that of primaquine. The structures of both metabolites were confirmed by direct comparison with authentic samples. 

For the screening, 25 microbial cultures, obtained from the University of Mississippi Department of Pharmacognosy culture collection, were used. Microbial bioconversion studies of sarcophine (45) showed that it can be metabolized by several fungi species. Preparative-scale fermentation with Absidia glauca American-type culture collection (ATCC) 22752, Rhizopus arrhizus ATCC 11145, and R. stolonifer ATCC 24795 resulted in the isolation... 

Microbial bioconversion studies of manzamine A and ent-8-hydroxymanzamine A have shown that they can be metabolized by several microbial species.76 Preparative-scale fermentation of manzamine A with Fusarium oxysporium f. gladioli ATCC 11137 resulted in the isolation of the known ircinal A (61) as a major metabolite. Preparative-scale fermentation of enf-8-hydroxymanzamine A with Nocardia sp. ATCC 11925 and Fusarium oxysporium ATCC 7601 resulted in the isolation of the new major metabolite 12,34-oxamanzamine F (62). The latter metabolite showed no cytotoxicity against different cell lines (>10 pg/ml). 

Preparative-scale Fermentation When the control studies have proven that a microorganism is performing a desired metabolism, preparative-scale fermentations are then conducted for the isolation, purification, and structural elucidation of greater quantities of the metabolite(s) produced. 

Based on TLC analyses and control studies, only Streptomyces spectabilis ATCC 27465 (grown in medium 0) was shown to be capable of complete conversion of 3-methoxysampangine into two more polar compounds, designated 3-MeOSAMMl and 3-MeOSAMM2. Limitations in time and starting material for 3-methoxysampangine did not allow for preparative-scale fermentation studies. 

It is needless to mention that this is only a tentative identification of those metabolites and further work has to be done in the synthesis of the starting material and the preparative-scale fermentation to produce enough quantities of those metabolites for structural elucidation and other biological studies. 

Figure 5.8 Typical industrial-scale fermentation equipment as employed in the biopharmaceutical sector (a). Control of the fermentation process is highly automated, with all fermentation parameters being adjusted by computer (b). Photographs (a) and (b) courtesy of SmithKline Beecham Biological Services, s.a., Belgium. Photograph (c) illustrates the inoculation of a laboratory-scale fermenter with recombinant microorganisms used in the production of a commercial interferon preparation. Photograph (c) courtesy of Pall Life Sciences, Dublin, Ireland...

Recently an isotope dilution method has been reported O for assaying neomycin sulphate. However, it is first necessary to prepare 14C-labelled neomycin sulphate. This is accomplished by adding l4C-labelled glucose to a small-scale fermentation of 5. nad-iat. l4C-labelled neomycin can then be extracted by solvent-extraction or by ion-exchange chromatography. 

Although the use of an epoxide hydrolase was already claimed for the industrial synthesis of L- and meso-tartaric acid in 1969 [51], it was only recently that applications to asymmetric synthesis appeared in the hterature. This fact can be attributed to the limited availabihty of these biocatalysts from sources such as mammals or plants. Since the production of large amounts of crude enzyme is now feasible, preparative-scale applications are within reach of the synthetic chemist. For instance, fermentation of Nocardia EHl on a 701-scale afforded > 700 g of lyophilized cells [100]). 

The fermentation methods used to prepare L-phenylalanine, threonine, lysine, and cysteine are discussed in detail in Chapter 3. The adaptation of these methods to prepare unnatural amino acids, such as the use of transaminases, is also discussed in that chapter. One of the large-scale amino acids, L-glumatic acid, which is often sold as its monosodium salt, is not covered because its preparation by fermentation is long established.3... 

Several biotechnological synthetic methods for D-pantothenic add and its precursor D-pantolactone have been developed over the past 15 years. Although all have reached preparative scale and might result in cost-effective production processes, they differ considerably in their process characteristics - for example educts and space-time yields, especially when a fermentation and biotransformation are compared. Compared with the chemical process, the biotechnological processes reduce waste production and provide the possibility of a more environmentally friendly yet still competitive means of production of D-pantothenic acid. 

Lactic acid is prepared by fermentation on the industrial scale. It is used in dyeing and calico printing. Antimony lactate finds application as a mordant. 

A preparative-scale biotransformation of sampangine was performed with Beauvaria bassiana ATCC 7159 using the standard two-stage fermentation procedure. This afforded a pure red metabolite (33 mg, 9.7% yield) (Fig. (5)). 

Some years ago, we started a project aimed at the development of simple and efficient enzymatic methods for the synthesis of biologically-active carbohydrates on a preparative scale. The use of enzymes as catalysts for the synthesis of carbohydrates has many potential advantages, such as enabling selective, stereo specific syntheses with a minimum of reaction steps under mild conditions and in aqueous solutions in which carbohydrates are highly soluble. Moreover, many enzymes now can be produced in quantity by fermentation and can be immobilized and reused. 

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