Enzymes extraction from bacteria

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

Note that plasmids exist as circular dsDNA when they occur in bacteria, that is, when they are replicated and transcribed in viw. When plasmids arc extracted from bacteria they also occur as drcular dsDNA, but they can easily be converted to linear dsDNA in a test tube for the purpose of receiving inserts. Special enzymes called restriction enzymes recognize specific and rarely occurring sequences of base pairs and catalyze the hydrolysis of the dsDNA at a specific point within or near the sequence. In this way, a plasmid consisting of several thousand base pairs can be cut at one site only and converted to a single piece of linear dsDNA,... 

Amino acids find applications as ingredients of infusion solutions for parenteral nutrition and individually for treatment of specific conditions. They are obtained either by fermentation processes similar to those used for antibiotics or in cell-free extracts employing enzymes isolated from bacteria (Table 25.2). Details of the many and varied processes reported in the literature will be found in the appropriate references in the Further reading section at the end of the chapter. 

It has been demonstrated that a variety of enzymes exhibited enhanced mechanical and chemical stability when immobilized on a solid support, producing a biocatalyst. Munnecke first immobilized a pesticide detoxification extract from bacteria by absorption on glass beads. The absorbed extract retained activity for what was then a remarkable full day. Wood and co-workers, using isocyanate-based polyurethane foams (Hypol ), found that a number of enzymes unrelated to OP hydrolysis could be covalently bound to this polymer. Later, Havens and Rase immobilized a parathion hydrolase. Furthermore, Turner observed that polyurethane foams are excellent adsorption materials for OP such as pesticide vapors. ... 

Although the formation of p-aminobenzoic acid (36) (Fig. 7.12) can be explained by amination and loss of pyruvate from w<7-chorismic acid, enzyme extracts from Enterobacter aerogenes and two Streptomyces species contain p-amino-benzoate synthase and /5< -chorismate synthase activity. Kinetic data suggest that synthesis of p-aminobenzoic acid occurs from chorismic acid (Johanni et al., 1989). p-Aminobenzoic acid is important in the formation of folic acid in fungi and bacteria (Haslam, 1974). 

The problem of pyrimidine degradation by microorganisms has been studied by enrichment culture techniques, whereby bacterial strains are grown which utilize pyrimidines as their source of nitrogen and carbon. Studies have been carried out with intact cells as w ell as with purified enzymes obtained from bacteria. Wang and Lampen demonstrated that pyrimidines were converted to barbituric acid by cell-free extracts... 

None of the commercially available enzyme preparations are identical. Different species of E. coU give enzymes that vary in chemical constitution, and biological properties, such as rate of clearance. The difference becomes even more marked when the enzyme is extracted from bacteria of different species. 

Cytochrome c can easily be extracted from tissue particles by dilute salt solutions. It was isolated by Keilin and Hartree in 1930 and shown to contain a porphyrin ring structure. In 1933 Zeilen and Reuter established that cytochrome c was a heme (iron-porphyrin) protein. Slightly different forms of cytochrome a were distinguished in yeast and bacteria by Keilin in 1934 and the different properties of cytochrome a and a3 by Tamiya et al. in 1937. The identity of cytochrome 03, the enzyme which activates oxygen with Warburg s atmungsferment, was proposed by Keilin in 1939. Cytochrome a/a3 was renamed cytochrome oxidase by Malcolm Dixon (1939). The oxidation route then offered was ... 

Bacterial electrodes [11, 31, 33, 46, 48, 49, 60] In this type of electrode, a suspension of suitable bacteria is placed between the sensor proper and a dialysis membrane that prevents passage of high-molecular substances (see fig. 8.3). The sensor is usually a gas probe. In the simple types of bacterial electrode, the determinand is converted by a suitable strain of bacteria into a product sensed by the gas probe. Thus it is possible to determine arginine [46], glutamine [48],/.-aspartic acid [31],/.-histidine [60] and nitrate [33]. Hybrid bacterial - enzyme electrodes contain both a bacterial strain and a suitable enzyme. For example, an extract from ivingas Neurospora chossa can be used as a source of NAD nucleosidase and an Escherichia coli culture as a source of nicotinamide deaminase, so that the electrode responds to NAD [49] as a result of the series of reactions... 

It is important to identify and measure the concentrations of a number of compounds in a mixture simultaneously for several reasons. First, among related compounds there may exist precursors of active ones, and pathways of pheromone synthesis may be elucidated. This is true for steroids in the human axilla. Nixon etal. (1988) determined the concentration of five steroids extracted from axillary hair of adult men aged 18 to 40 years. The relationships in concentrations between the two ketones 5Q -androst-16-en-3-one and 4,16-androstadien-3-one suggest that axillary bacteria reduce the former to the latter with the aid of the enzyme 4-ene-5a-reductase. Humans have a low olfactory threshold for several 16-androstenes, and the fact that some men have large quantities of 16-androstenes (Nixon etal., 1988) is biologically suggestive. 

Together with biochemical reactions catalyzed by enzymes of yeasts and bacteria, chemical reactions also occur between molecules already present in the must, those gradually extracted from the grape solids during fermentation, those derived from metabolism and, possibly, also those released by the wood. For many of them, the temperature and dissolved oxygen parameters related to technological operations of the winery can have dramatic effects and the quality of the final wine depends on the type and intensity of reactions taking place. 

These carbohydrates may be added to all kinds of foods, such as cereals, cakes, biscuits, and health drinks. They can be extracted from things like chicory root or produced from sugar by the action of specific enzymes. A little FOS is also to be found in bananas, leeks, and wheat, and the other prebiotics also occur naturally, but no fruit or vegetable by itself can supply the 5 g of oligosaccharides needed daily to boost the good bacteria. Indeed the normal person s diet contains only about 2 g of these carbohydrates. 

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