Microorganisms enzyme extraction from

The following spectrophotometric methods are conceptually related to the POV however, they are intended for other purposes. HPLC-UVD at 234 nm was applied to detect lipid hydroperoxides, obtained in a set of experiments for assessing the effectiveness of lipoxygenase enzymes extracted from various microorganisms. The absorbance. A, measured at three different wavelengths is linearly correlated to the concentrations in xM units of lipid hydroperoxides, Clh, 7-oxocholesterol, Cqc and dienals, Cde according to the set of simultaneous equations 60. This method was used to track the Cu(II)-induced oxidation of LDL" °. 

Fig. 25.1 The three pathways for the preparation of natural flavours. The first two involve the extraction of the flavour or precursors from natural sources. The precursors can then be converted to the natural flavour by enzymes extracted from plants or microorganisms. The last method is the de novo synthesis of the flavour by microorganisms growing on simple substrates such as glucose and sucrose...

The characteristic flavor of various cheeses is primarily due to the enzymatic action of microbial flora contained in the curd. Enzymes extracted from these microorganisms and reacted with corresponding substrates may also produce a specific cheese flavor. The flavor produced may be economical and could be classified as "natural". 

The enzymes produced by these extremophiles, known as extremozymes, can function under extreme conditions. An illustrative list along with an indication of the extreme environments in which they can function is included in Table 20.1 (sources Kushner, 1978 Jones et al., 1983 Huber et al., 1989 Li et al., 1993 Davail et al., 1994 Adams et al., 1995). Enzymes extracted from these microorganisms have been tested for a variety of reactions and optimum temperatures have been found. Examples are enzymes from Pyrococcus furiosus for a- and p glucosidase, a-amylase, protease, and hydrogenase activities (Bryant and Adams, 1989 Costantino et al., 1990 Blumentals et al., 1990 Kegen et al., 1993 Laderman et al., 1993). 

This appears to be the first instance of omega oxidation of fatty acids in microorganisms it has been recognized for some time in the mammalian body (Deuel, 1957), and has been recently demonstrated with enzymes extracted from hog liver (Robbins, 1961). 

There are thousands of breweries worldwide. However, the number of companies using fermentation to produce therapeutic substances and/or fine chemicals number well over 150, and those that grow microorganisms for food and feed number nearly 100. Lists of representative fermentation products produced commercially and the corresponding companies are available (1). Numerous other companies practice fermentation in some small capacity because it is often the only route to synthesize biochemical intermediates, enzymes, and many fine chemicals used in minor quantities. The large volume of L-phenylalanine is mainly used in the manufacture of the artificial dipeptide sweetener known as aspartame [22389-47-0]. Prior to the early 1980s there was httle demand for L-phenyl alanine, most of which was obtained by extraction from human hair and other nonmicrobiological sources. 

Until about 1950, the predominant method of producing industrial enzymes was by extraction from animal or plant sources by 1993, this accounts for less than 10%. With the exception of trypsin, chymosin, papain [9001 -73-2J, and a few others, industrial enzymes are now produced by microorganisms grown in aqueous suspension in large vessels, ie, by fermentation (qv). A smaH (5%) fraction is obtained by surface culture, ie, soHd-state fermentation, of microorganisms (13). 

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). 

More than 3000 different enzymes have been extracted from animals, plants and microorganisms. Traditionally, they have been used in impure form since purification is expensive and pure enzymes may be difficult to store and use. There is usually an optimum temperature and pH for maximum activity of an enzyme. Outside these optimum conditions, activity may simply be held in check or the enzyme may become denatured , i.e. altered in such a way that activity is lost permanently, although some forms of denaturing are reversible. Many enzymes are also sensitive to transition-metal ions, the effect being specific to particular metal ions and enzymes. In some cases, certain metal ions are essential for the stability and/or activity of an enzyme. In other cases, metal ions may inhibit the activity of an enzyme. Similarly, certain organic compounds can act as enzyme inhibitors or activators. 

The second development for which IGT is known world-wide is their work on biodesulfurization. The IGT intellectual property package, developed by Kilbane s group, includes two microorganisms, R. rhodochrous strain ATCC 53968 (IGTS8) and B. sphaericus strain ATCC 53969 as well as the enzymes derived from them and cell-free extracts. The biocatalysts and their use were protected in a series of eight patents (plus one US equivalent) and though in some patents a process is claimed, the main emphasis is on biocatalyst. A summary of these patents and the comparison with the early patents of EBC was given in Chapter 3. The patents were entitled ... 

The relation between microbial diversity and soil functions is poorly understood because we cannot measure easily the microbial diversity, even if we can detect unculturable microorganisms by molecular techniques (Nannipieri et al. 2003). In addition, the present assays for measuring microbial functions determine the overall rate of entire metabolic processes, such as respiration, or specific enzyme activities, without identifying the active microbial species involved. The recent advances in RNA extraction from soil might permit us to determine active species in soil (Griffiths et al. 2000 Hurt et al. 2001). Further advances in understanding require us to determine the composition of microbial communities and microbial functions in microhabitats. 

Vitamin Bj2 is converted to co-enzyme B12 by extracts from microorganisms supplemented with ATP Coenzyme B12 is associated with many biochemical reactions-... 

From this point on there are numerous ways of proceeding with the analysis. Crude extracts can be made from different mutant cells. The extract from one mutant can be used to complement the extract from another mutant in tryptophan synthesis, which can lead to an assay for a particular enzyme carried by one mutant that is missing in the other mutant. The goal at this juncture is to purify each enzyme of the pathway so that the properties of the enzymes and the reactions they catalyze can be individually scrutinized. All of the tryptophan enzymes have been isolated from E. coli. Studies of other systems indicate a remarkable similarity for the operation of this pathway in different microorganisms and plants. 

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