Enzymes penicillinase

Directions for preparing a potentiometric biosensor for penicillin are provided in this experiment. The enzyme penicillinase is immobilized in a polyacrylamide polymer formed on the surface of a glass pH electrode. The electrode shows a linear response to penicillin G over a concentration range of 10 M to 10 M. 

Mifflin and associates described a membrane electrode for the quantitative analysis of penicillin in which the enzyme penicillinase is immobilized in a polyacrylamide gel that is coated on a glass pH electrode. The following data were collected for a series of penicillin standards. 

Penicillin can likewise be determined by using the enzyme penicillinase to destroy the penicillin with production of hydrogen ions which can be determined using a normal glass pH electrode. Many other organic materials can be determined by similar procedures.34,35... 

As a simple model for the enzyme penicillinase, Tutt and Schwartz (1970, 1971) investigated the effect of cycloheptaamylose on the hydrolysis of a series of penicillins. As illustrated in Scheme III, the alkaline hydrolysis of penicillins is first-order in both substrate and hydroxide ion and proceeds with cleavage of the /3-lactam ring to produce penicilloic acid. In the presence of an excess of cycloheptaamylose, the rate of disappearance of penicillin follows saturation kinetics as the cycloheptaamylose concentration is varied. By analogy to the hydrolysis of the phenyl acetates, this saturation behavior may be explained by inclusion of the penicillin side chain (the R group) within the cycloheptaamylose cavity prior to nucleophilic attack by a cycloheptaamylose alkoxide ion at the /3-lactam carbonyl. The presence of a covalent intermediate on the reaction pathway, although not isolated, was implicated by the observation that the rate of disappearance of penicillin is always greater than the rate of appearance of free penicilloic acid. 

Like other semisynthetic penicillins, methicillin exhibits an antibacterial effect similar to that of benzylpenicillin. The main difference between methicillin and benzylpenicillin is that it is not inactivated by the enzyme penicillinase, and therefore it is effective with respect to agents producing this enzyme (staphylococci). It is used for infections caused by benzylpenicillin-resistant staphylococci (septicemia, pneumonia, empyemia, osteomyelitis, abscesses, infected wounds, and others). Synonyms of this drug are cinopenil, celbenin, staphcillin, and others. 

Microbial inhibition tests are extremely sensitive for -lactam antibiotics, primarily penicillin, but mostly are more than 100-fold less sensitive for other commonly used antibacterials such as macrolides, sulfonamides, tetracyclines, or chloramphenicol (4, 5). Therefore, inhibition tests usually classify residues as belonging to the -lactam group. Antibiotics other than -lactams and sulfonamides can be detected by use of the enzyme penicillinase and aminobenzoic acid, respectively (1, 6). 

The natural penicillins, primarily G and V, have a relatively narrow spectrum. They act mostly on gram-positive organisms. The fact that proper selection of precursors could lead to new variations in the penicillin side chain offered the first source of synthetic penicillins. Penicillin V, derived from a phenoxy-acetic acid precursor, attracted clinical use because of its greater acid tolerance, which made it more useful in oral administration. Also, the widespread use of penicillin eventually led to a clinical problem of penicillin-resistant staphylococci and streptococci. Resistance for the most part involved the penicillin-destroying enzyme, penicillinase, which attacked the beta-lactam structure of the 6-aminopenicillanic acid nucleus (6-APA). Semisynthetic penicillins such as ampicillin and carbenicillin have a broader spectrum. Some, such as methicillin, orafi-cillin, and oxacillin, are resistant to penicillinase. In 1984, Beecham introduced Augmentin, which was the first combination formulation of a penicillin (amoxicillin) and a penicillinase inhibitor (clavulanic acid). Worldwide production of semisynthetic penicillins is currently around 10,000 tons/year, the major producers are Smith Kline Beecham, DSM, Pfizer, and Toyo Jozo. 

Certain bacteria use the enzyme penicillinase to decompose penicillin and render it inactive. The Michaelis-Menten constants for this enzyme and substrate are = 5 X 10 mol L and k2 = lXl0 s.- ... 

The existence of rogue strains of bacteria that were resistant to antibiotics was nothing new. As mentioned earlier in the chapter, by the early 1960s, 10% of the strains of Staphylococcus aureus produced penicillin-destroying enzymes (penicillinases) and were thus resistant to the first-generation... 

Metabolism of a substrate by an enzyme to form a compound that irreversibly inhibits that enzyme. Penicillinase inhibitors, such as clavulanlc acid and 5ul-bactam, are suicide inhibitors. 

The best known realizations are optodes for penicillin [206, 209, 210], urea [211], and glucose [212]. A very sophisticated version of these sensors [213] uses functionalized optical nbers to which biotin is covalently bound. To this affinant, using the biotin-avidin system, the enzyme (penicillinase, esterase, urease) is connected, itself covalently bearing the indicator. The slopes of the calibration graphs for these optodes depend on the surrounding pH value and the buffer capacity however, linear responses are obtained for 0.1-10 mM substrate concentrations. 

Beta-lactamases Bacterial enzymes (penicillinases, cephalosporinases) that hydrolyze the beta-lactam ring of certain penicillins and cephalosporins... 

It has been observed that the hydrolysis of penicillin with dilute alkali or with the enzyme penicillinase) yields penicilloic acid (a diearboxylie acid), which readily eliminates a molecule of carbon dioxide to form penilloic acid, thereby suggesting that a carboxyl group is present in the P-position with regard to a negative group. 

Mechansim of Action. The drug is speeifieally resistant to inactivation by the presenee of the enzyme penicillinase found in staphylococci. It has been observed to induee penicillinase formation which specifically restrains its usage in the control, management and treatment of penicillin G-sensitive infections. 

Because of the usual lag time between sample collection and analysis at the laboratory, sample storage is an important step. The potential effects of physicochemical factors such as oxidation, proteolysis, and precipitation and biological factors that include microbiological and enzymatic reactions need to be considered when storing samples. For example, the production of the enzyme penicillinase, which is capable of reducing the concentration of penicillin in kidney tissue stored at 4°C, has been reported in some studies. Preservation can be achieved through the addition of enzyme inhibitors (e.g., piperonylbutoxide inhibits cytochrome P450). 

Cephalosporin C, which contains a dihydrothiazine-j8-lactam ring system, shows at least 10% of the activity of cephalosporin N against Staph, aureus and induces the formation of the enzyme penicillinase by Staph, aureus and Bacillus cereus. This may be attributed to the fact that the stereochemistry of a major part of the cephalosporin C nucleus is similar to that of 6-aminopenicillanic acid. The precise factors which determine the ability of a substance to act as an inducer of penicillinase are clearly different from those which make the substance a good substrate of the enzyme. The /9-lactam ring in cephalosporin C is insensitive to penicillinase. However, the affinity of a substance for a penicillinase varies with the source of the enzyme. Cephalosporin C acts as a competitive inhibitor of the action of penicillinase from Bacillus cereus, but not from Staph, aureus, on benzylpenicillin. In contrast, the V-phenylacetyl derivative of 7-aminocephalosporanic acid is a powerful inhibitor of the staphylococcal enzyme12... 

Bacteria adapt rapidly so that mutations can arise that alter the receptor sites, making it impossible for penicillin to bind to them. There are other bacteria that have the capacity to make the enzyme penicillinase. This is a (3-lactamase enzyme that catalyzes the hydrolysis of the (3-lactam ring, thereby destroying the activity of the penicillin. So, for example, penicillin G is now ineffective against Staphylococcus aureus. 

Some bacteria are resistant to penicillin, because they produce an enzyme, penicillinase, that destroys the /3-lactam ring in the antibiotic. Synthesis of analogs afforded a partial solution to this problem. Ultimately, however, it became necessary to turn to antibiotics with completely different modes of action. Erythromycin, produced by a strain of Streptomyces bacteria first found in soil samples in the Philippines in 1952, functions in a distinct manner. It is a large ring lactone that interferes with the bacterial ribosome, its cell-wall protein S5mthesis factory. Although erythromycin is unaffected by penicillinase, bacteria resistant to it have developed over the decades since its introduction into the antibiotic arsenal. 

The widespread use of penicillin eventually led to a clinical problem of penicillin-resistant staphylococci and streptococci. Resistance for the most part involved the penicillin-destroying enzyme, penicillinase, which attacked the beta-lactam structure of the 6-amino-penicillanic acid nucleus (6-APA). 

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