Production of enzymes

Enzymes are now produced for a variety of applications, going from bulk high tonnage processes in which the enzymes are considered as commodities (Hodgson 1994) to small-scale applications for refined uses and research where enzymes are considered specialties (Thomas et al. 2002). Level of production and type of application define the kind of process for its production. Specialty enzymes to be used in medicine and health-care products are usually required in high levels of purity 

The production process can be divided into four stages  

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Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

A -Pyrroline has been prepared in low yield by oxidation of proline with sodium hypochlorite (71), persulfate (102), and periodate (103). A -Pyrroline and A -piperideine are products of enzymic oxidation via deamination of putrescine and cadaverine or ornithine and lysine, respectively (104,105). This process plays an important part in metabolism and in the biosynthesis of various heterocyclic compounds, especially of alkaloids. 

Increased production of enzymes required for energy-yielding reactions and for synthesis of RNA and proteins... 

The biotransformation process has been improved by significant advances in biochemical engineering advances in genetic and protein engineering, microbiological manipulations for the production of enzymes, and the use of biocatalysts in immobilized form and large-scale purification methods. 

E. coli for example, it is used for the production of enzymes as additives in detergent solutions. Proteins are secreted into cultivation media and are usually biologically active. 

Simply on the basis of the normal composition of marine organisms, we would expect proteins and peptides to be normal constituents of the dissolved organic carbon in seawater. While free amino acids might be expected as products of enzymic hydrolysis of proteins, the rapid uptake of these compounds by bacteria would lead us to expect that free amino acids would normally constitute a minor part of the dissolved organic pool. This is precisely what we do find the concentration of free amino acids seldom exceeds 150 xg/l in the open ocean. It would be expected that the concentration of combined amino acids would be many times as great. There have been relatively few measurements of proteins and peptides, and most of the measurements were obtained by measuring the free amino acids before and after a hydrolysis step. Representative methods of this type have been described [245-259]. Since these methods are basically free amino acid methods, they will be discussed next in conjunction with those methods. 

According to Albersheim and coworkers,10 the mechanism of induction of pathogeny is associated with an interaction, between the pathogen and the cell-wall saccharides of the host, which influences the production of enzymes degrading its cell walls. The production of enzyme is regulated both by the pathogen and the host. 

P. A. Benkovic, M. Hegazi, B. A. Cunningham and S. J. Benkovic, Biochemistry 18, 830 (1979) use continuous monitoring of inorganic phosphate product of enzyme catalyzed hydrolysis of fructose biphosphate. The pH change monitored by phenol red results from an acid, base adjustment of the liberated phosphate. This is a detailed valuable, if complicated, account. 

Now we come to the question what is the basis of the antibacterial action of the sulfa drugs Many useful drugs in human medicine are enzyme inhibitors, small molecules that frequently bear a structural resemblance to the substrates or products of enzyme-catalyzed reactions. So it is with the sulfonamides. 

Kang, M.J., Tholey, A., Heinzle, E. Quantification of low molecular mass substrates and products of enzyme catalyzed reactions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass. Spectrom. 2000, 14, 1972-1978. 

The involvement of isobutylhydroxylamine, (CH3)2CHCH2NH—OH (4), and of HA (NH2OH) in the biosynthesis of the antibiotics valanimycin (5) and nebularine (6), respectively, has been demonstrated in Streptomyces species (see Section n.B). In the case of nebularine, HA is released in the final step of its production by enzymatically induced deamination of adenosine, while the isobutylhydroxylamine is a precursor for the biosynthesis of valanimycin. In cyanobacterium, the presence of free and bound HA was demonstrated to be a product of enzyme-mediated glutamine oxidation ... 

Nuclear magnetic resonance (NMR) measurements are usually considered to be slow processes, but recent advances in the design of flow-through NMR cells have allowed the method to be applied in combinatorial chemistry 97). These technological improvements were applied to the development of two NMR-based high-throughput ee assays for evaluating the products of enzyme- or transition metal-catalyzed reactions 98). 

The use of MALDI-MS for the measurement of low molecular mass compounds is widely accepted now [61], but quantification remains problematic. The main problem is the inhomogeneous distribution of the analytes within the matrix [62]. This leads to different amounts of ions and therefore to different signal intensities at various locations of a sample spot. The simplest and most effective way to overcome this problem is the use of an appropriate internal standard [63]. The use of deuterated compounds with a high molecular similarity to the analyte as internal standards leads to a linear correlation between relative signal intensities and relative amount of the compound to be quantified (Fig. 4b) [64]. Using this approach it is possible to quantitate substrates and products of enzyme catalyzed reactions. Two examples were shown recently by Kang and coworkers [64, 65]. The first was a lipase catalyzed reaction which produces 2-methoxy-N-[(lR)-l-phenylethyl]-acetamide (MET) using rac-a-... 

Singleton, V. L., Salgues, M., Zaya, J., and Trousdale, E. (1985). Caftaric acid disappearance and conversion to products of enzymic oxidation in grape must and wine. Am.. Enol. Vitic. 36, 50-56. 

The use of enzymes for the manufacture of leather played a major role for the industrial scale production of enzymes. For the preparation of hides and skins for tanning, the early tanners kept the dehaired skins in a warm suspension of the dungs of dogs of birds. Wood was the first in 1898 to show that the bating action of the unpleasant dungs was caused by the enzymes (pepsin, trypsin, lipase) which they contained. In the context... 

Guanosine 5 -(a-D-rhamnopyranosyl pyrophosphate) (23) was shown to be a product of enzymic reduction of the mannosyl ester (17) in the presence of a plant extract93 or an enzyme preparation from an unidentified strain of Gram-negative bacteria.94,95 In the latter example, the 6-deoxy-D-talosyl ester, presumably 24, is also produced. 

Oligosaccharide derivatives of uridine 5 -pyrophosphate are probably present in higher plants. Cellobiose and a disaccharide composed of a glucose and an arabinose were identified among products of enzymic hydrolysis of a uridine diphosphate sugar fraction from larch wood.20... 

The advent of recombinant DNA technology has allowed the isolation of genes and expression of proteins that are found in biologic tissues in exceedingly small quantities. This has permitted large-scale production of enzymes such as tissue plasminogen activator (i.e., tPA, alteplase) that cannot be extracted from tissues in quantities required for therapeutic use. Table 9.2... 

Production of enzymes to degrade the antibiotic, e.g. novel (3-lactamases, extended-spectrum p-lactamases, or aminoglycoside-modifying enzymes. 

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