Biocatalyst biosynthesis

For bioprocessing purposes, increase in the stability of biocatalysts is quite often achieved by immobilization of cells or enzymes [57-60], This technology is an attractive alternative to the use of expensive free enzymes and cofactors, and can coordinate multistep enzymatic processes into a single operation. Furthermore, fermentative biosynthesis of cephamycin C using immobilized cells of S. clavuli-gerus NRRL 3585 was accomplished by Freeman and Aharonowitz [61], Jensen et al. [62] reported on the immobilization of P-lactam synthesizing enzymes from the same wild-type culture. None of these early studies used penicillin substrates other than the normal intermediate penicillin N, such as penicillin G. 

Despite this limitation, in vitro biosynthesis using purified biocatalysts promises to be a simple and inexpensive means to access a wealth of complex natural product structures under clean and controlled conditions. The versatility of this technology can be significantly enhanced by the application of genetic engineering to create novel proteins. 

Wasteful processes can be made more economical and environmentally compatible by applying new green technologies. For example, the use of biosynthesis or catalytic transformations, with biocatalysts or other catalysts that are highly selective, which occur under mild reaction conditions is possible they save energy and provide high structural selectivity, thereby reducing byproducts. 

Fig. 2.18 Simplified scheme of the cycle/aUocation of magnesium in a green plant. Mg is involved - inter aha - in making peptide bonds, in tricarboxylate cycle, hydrolysis of molecules and binding of CO (ribulose-bisphosphatecarboxylase/oxidase or PEP carboxylase in plants which also employs Mg or Mtf+ in some plants (Kai et al. 2003)). Reaction steps in which Mg takes part as biocatalyst are marked by broken lines/arrows. Citrate and other intermediates of the tricarboxylate cycle, particularly malate, are employed by higher plants for extraction of essential metals, including Mg, Fe and Mn (thus the closed loop) from soil via and by means of the roots. This closed loop depicts a manner of autocatalysis. Amino acids which are required for protein biosynthesis are produced by reductive amination from tricarboxylate cycle intermediates and other 2-oxoadds which likewise eventually...

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

While only enzymes involved in pikromycin biosynthesis have been discussed in this section, they represent excellent examples for application of secondary metabolite pathway enzymes to produce novel natural products through chemoenzymatic approaches. In the upcoming sections, additional enzymes involved in structurally diverse natural product biosyntheses will be included to further examine biocatalysts functioning in drug discovery and development. 

Even in the case it should be possible to separate ribozyme activity from the ribosome or to isolate an in vitro selected ribozyme that can catalyze the same type of peptide bond formation as a ribosome, however such a biocatalyst seem does not to be suitable for simple practical use rather than using a chemical coupling reagent. In principle, this conclusion is also valid for the nonribosomal poly- or multienzymes which are involved in the biosynthesis of peptide antibiotics[7Z. Up to now, they have only found application in the synthesis field of cyclosporin, gramicidin S, special P-lactam antibiotics and analogs. 

As is evident from the work that is reviewed here, the application of biocatalysts in preparative carbohydrate synthesis has continued to expand and diversify in recent years. Of particular note is the reengineering of biocatalyst properties (e.g., substrate specificity, stability) by the process of directed evolution. Significant progress in the study of the enzymatic biosynthesis of complex carbohydrate has been made with the development in protein purification, molecular genetics, and new methods of enzymological analysis. Bioinformatics provides a large number of putative candidates for carbohydrate-active enzymes. The combined... 

Thus, the reduced form of cytochrome P-450 functions as the oxygenactivating biocatalyst of a wide variety of mixed-function oxidations by vertebrate tissues effecting biosynthesis and catabolism of steroid hormones, bile acid formation, and the metabolism of drugs and other xeno-biotics (16). Since reduced pyridine nucleotides do not react directly with hemoproteins, the hydroxylase systems must include components that mediate the electron transport from TPNH to cytochrome P-450. There also must be distinctive diflFerences in composition causing the substrate specificity of the oxygenations. 

For the biosynthesis of a secondary metabolite at least one enzyme is required, changing the primary metabolite into a (specific) secondary product. However, in most cases more enzymes are involved, e.g. the number of enzymes for the biosynthesis of vinblastine, starting from tryptophan and geranyl diphosphate is higher than 25. Plants are thus not only a rich source of complex, biological active, fine-chemicals, but are probably an even more rich source of biocatalysts. Its potential in bioconversions is recognized [2]. 

The multistep chemical conversion of penicillin G to 7-ADCA (Fig. 16) has recently been replaced by a 2-step biosynthesis (Fig. 18). This is a major step forward to shorten the industrial synthesis of cephalosporins and this has already been implemented. The 7-N-adipoyl-ADCA is obtained directly through fermentation with a modified Penicilium chrysogenum followed by a simple enzymatic removal of the amino substituent. Again, various reagents such as silylat-ing agents, phosphor halides, pyridine and DMF, and some halogenated solvents have been replaced by biocatalysts in aqueous medium. 

Combinatorial biosynthesis can be practically realized by (i) chemical modification by biocatalysts (biotransformation), (ii) mutasynthesis, (iii) combinatorial metabolism in hybrids, and (iv) activation of silent metabolism (Figure 2.11). 

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