Microbial cell-free extracts

Chakrabarty has extensively reviewed the biodegradation of pesticides (J ). Table I shows the results of several studies on the enzymatic activity of microbial cell-free extracts for pesticide degradation. Clearly, there is substantial evidence to suggest that enzymes might be used in the development of biotechnology for use in degradation of pesticides. 

Table I. Enzymatic Activity of Microbial Cell-Free Extracts For Pesticide Degradation...

Biocatalysis Chemical reactions mediated by biological systems (microbial communities, whole organisms or cells, cell-free extracts, or purified enzymes aka catalytic proteins). 

All of the principles and ideas covered in the previous section may be translated directly to the use of microorganisms as tools in the production of compounds of plant biosynthetic or biodegradative importance. Just as one finds microbial systems to be of value in preparing metabolites in mammalian systems, it may be possible to use microbial transformations to prepare derivatives of alkaloids that might be found rarely or only in very small quantities in plants. In this way, abundant prototype alkaloids may be used as microbial transformation substrates to provide a range of metabolites. As in the mammalian case, metabolism studies using plant tissues, tissue cultures, or cell-free extracts may be conducted in parallel with microbial metabolic systems. Metabolites common to both would be prepared in quantity by relatively simple fermentation scale-up methods. 

The 1-pro-7 -hydrogen is lost on oxidizing geraniol with a cell-free extract from Cannabis sativa (Vol. 7, p. 9, ref. 96), asymmetric microbial reduction of ( )-citronellal to (-)-citronelloI is reported, and callus cultures of Nicotiana tabacum selectively hydroxylate linalool, dihydrolinalool, and the derived acetates at the -methyl group [e.g. to give (59)]. ... 

Enzyme Preparations used in food processing are derived from animal, plant, or microbial sources (see Classification, below). They may consist of whole cells, parts of cells, or cell-free extracts of the source used, and they may contain one active component or, more commonly, a mixture of several, as well as food-grade diluents, preservatives, antioxidants, and other substances consistent with good manufacturing practices. 

Indolmydn.—Previous evidence on the biosynthesis of indolmycin (88) in Strepto-myces griseus cultures accords with the pathway shown in Scheme 4. The first two steps in the pathway have been carried out using cell-free extracts of 5. griseus - and recent work has led to the isolation of two enzymes which can effect these transformations. The first, tryptophan transaminase, catalysed the pyridoxal phosphate-dependent transamination of L-tryptophan, but not D-trptophan, and in common with some other microbial transaminases, a-ketoglutarate was an efficient amino-group acceptor. L-Phenylalanine, tyrosine, and 3-methyltryptophan (this compound inhibited enzyme function) also underwent transamination. 

Biocatalysis covers a broad range of scientific and technical disciplines, which are geared to develop biocatalysts and biocatalytic processes for practical purposes. The natural pool of biocatalysts is extremely diverse and includes whole cells of microbial, plant or animal origin, as well as cell-free extracts and enz3rmes derived from these sources. The wide range of catalytic power offered by nature remains, however, largely imexplored. Currently, only a very small fraction of the known biocatalysts are actually being applied on a commercial scale. For example, of the approximately 4,000 known enzymes, about 400 are available commercially, but only about 40 are actually used for industrial applications. 

Over the past few years, an impressive array of epoxide hydrolases has been identified from microbial sources. Due to the fact that they can be easily employed as whole-cell preparations or crude cell-free extracts in sufficient amounts by fermentation, they are just being recognized as highly versatile biocatalysts for the preparation of enantiopure epoxides and vicinal diols. The future will certainly bring an increasing number of useful applications of these systems to the asymmetric synthesis of chiral bioactive compounds. As for all enzymes, the enantioselectivity of... 

Laboratory studies have established the capacity of many microorganisms to degrade or to transform nitrogenous compounds under aerobic or anaerobic conditions. The results obtained from these investigations which involved pime cult ires, or cell-free extracts of microorganisms are important, because they may be predictive of their environmental fate. However, these studies do not reproduce the conditions found in nature where these organisms are exposed to a mixture of compounds and interact with other microbial communities. 

Microbial systems show similarities to the animal systems, and Hamid and Smith [36] have clearly demonstrated the involvement of a microsomal enzyme system in aflatoxin degradation in A. flavus. The degradation of aflatoxins by cell>free extracts of A flavus was enhanced by NADFH... 

Apart from that of E. coli, the only microbial system for deoxyribonu-cleotide synthesis which has been studied in detail is that of L. leichmannii. Prior to the definitive biochemical studies described below, nutritional experiments had demonstrated that the vitamin Bu requirement of L. leichmannii was involved in an essential way in the biosynthesis of deoxyribonucleotides. Blakley and Barker 24) showed that cell-free extracts of this microorganism catalyzed the reduction of cytidylate to deoxycyti-dylate and showed also that this reaction required a vitamin B12 derivative and NADP. 

The conversion of D-glucose (17) into D-fructose (9) by a microbial enzyme (Scheme 5) was first reported in 1957 when Marshall and Kooi found glucose isomerase activity in cell-free extracts of Pseudomonas hydrophila (91. This enzymatic activity was enhanced in the presence of arsenate. Soon thereafter, other arsenate-requiring enzymes were isolated from Aerobacter sp. as well as Escherichia freundii [10]. Enzymes required arsenate when D-glucose or D-fructose was the substrate but not when the corresponding 6-phosphates 11 and 12 were offered. Purification of the arsenate-dependent principle component from Escherichia intermedia allowed the conclusion that the enzyme was a glucose 6-phosphate isomerase (EC 5.3.1.9) that was able to isomerise free D-glucose when it was complexed with arsenate [11]. 

It is not known if m-tyrosine can be converted to l-DOPA by a cell-free extract of a species susceptible to m-tyrosine. If so, would the process be highly efficient in vivol Synthetic proherbicides, such as diclofop-methyl [11], that are inactive at the molecular target site are much more effective when applied to intact plants than the active molecule to which they are converted in vivo. This is due to superior cuticular and cellular uptake of the proherbicide. Some potent natural phytotoxins from microbial origin, such as hydantocidin and 2,5-anhydro-D-glucitol, are protoxins [170-171]. 

Some carcinogens are inactive in microbial assay systems because they require metabolic activation by mammalian enzymes to the ultimate active form. This activation can be duplicated, to some extent, by incorporating into the microbial system cell-free extracts derived from mammalian tissues. A convenient source is the liver of rats induced with the polychlorinated biphenyl Aroclor 1254. 

In fact, successful deracemization was not achieved when using a cell-free extract of the Alcaligenes cells, which showed NADH-dependent (R)-selective ADHs active in the oxidation reaction, coupled to a NADH-dependent (S)-selective ADH for the reduction reaction. Instead, it was easily achieved when using microbial whole cells in the biooxidation reaction or when combining the same cell-free extract with a NADPH-dependent ADH. In both cases, possible short circuits between the two cofactor regeneration systems were therefore avoided, thanks to the compartmen-talization of one of the involved biocatalysts in the cells or to the use of enzymes with different cofactor specificity. 

Lebeau et al. (2002) investigated the sorption of cadmium by viable microbial cells that were free or immobilized in alginate beads by incubating the bacteria in a liquid soil extract medium at pH 5 7 and Cd concentrations of 1 to 10 mg L-1. The percentage of Cd biosorbed reached a maximum (69%) at low Cd concentrations and neutral pH. Thus, the effectiveness of bacteria, inoculated into metal-contaminated soils, would largely depend on the concentration of the metal and its distribution between the biomass and the medium. 

Fig. 23.1 Microbial routes from natural raw materials to and between natural flavour compounds (solid arrows). Natural raw materials are depicted within the ellipse. Raw material fractions are derived from their natural sources by conventional means, such as extraction and hydrolysis (dotted arrows). De novo indicates flavour compounds which arise from microbial cultures by de novo biosynthesis (e.g. on glucose or other carbon sources) and not by biotransformation of an externally added precursor. It should be noted that there are many more flavour compounds accessible by biocatalysis using free enzymes which are not described in this chapter, especially flavour esters by esterification of natural alcohols (e.g. aliphatic or terpene alcohols) with natural acids by free lipases. For the sake of completeness, the C6 aldehydes are also shown although only the formation of the corresponding alcohols involves microbial cells as catalysts. The list of flavour compounds shown is not intended to be all-embracing but focuses on the examples discussed in this chapter... 

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