Enzymes penicillinase reaction

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. 

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). 

Optical sensors that are sensitive to pH and ammonia can save as a basis for the production of optical enzyme sensors [8]. Certain enzymes catalyse reactions with a variation in pH (penicillinase, cholinesterase, glucose oxidase or urease, etc.), or with liberation of ammonia (urease, glutaminase, amino acid oxidase) and are simply deposited onto the optical sensors. 

Like with primary amides (see Sect. 4.2.1), bacterial amidases can be useful for the transformation of secondary amides in drug synthesis. Bacterial amidases have been extensively studied in the presence of penicillins and other [i-lactam antibiotics, for which two hydrolysis reactions are possible. One of these is carried out by enzymes known as penicillinases or /3-lactamases that open the /3-lactam ring this aspect will be discussed in Chapt. 5. The second type of hydrolysis involves cleavage of the side-chain amide bond (4.47 to 4.48) and is carried out by an enzyme called penicillinacylase (penicillin amidohydrolase, EC 3.5.1.11). Both types of hydrolysis inactivate the antibiotic [29-31],... 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

Nafcillin (IV 3 to 6 g/24 hours in severe infections) is indicated for the treatment of infections due to penicillinase-producing staphylococci. It may be used to initiate therapy when a staphylococcal infection is suspected (see also Table 23). Like penicillins, nafcillin, inhibits the formation of cell walls and hence is bactericidal in nature. Penicillin binds to cellular receptors, now identified as transpeptida-tion enzymes, and, by binding to and inhibiting the transpeptidation reactions, the synthesis of cell wall pep-tidoglycan is interrupted. In addition, penicillin removes or inactivates an inhibitor of the lytic enzymes (autolysin). 

Derivatives of cellulose continue to be used as supports for new affinity chromatography media and for immobilization of enzymes. Trypsin has been immobilized on 4-aminobenzyl-cellulose. A penicillinase from Bacillus cereus has been immobilized by glutaraldehyde-mediated reaction with aminoethyl-cellulose. Tannin has been activated with cyanogen bromide and coupled with... 

Other, practically useful, enzyme electrodes include those based on urease and penicillinase. The penicillin electrode utilizes a pH electrode with immobilized penicillinase. It is widely used to monitor the penidllin content of fermentation reaction mixtures ... 

The accumulation of results presented so far suggested that the biosynthesis of the cephalosporins and penicillins was essentially the same in many respects and that, like the penicillins, the cephalosporins were probably derived from a-aminoadipylcysteinylvaline. The cephalosporin producing organisms that have been most studied to date (C. acremonium and S. clavuligerus) both also produce penicillin N. There are, therefore, two possible routes from the tripeptide to the cephem products, one by direct cyclisation of the tripeptide, the other by enzymic ring expansion of penicillin N. The first evidence that penicillin N could be converted enzymically to a cephalosporin was provided by Kohsaka and Demain 289) who demonstrated that lysed protoplasts of C. acremonium could convert penicillin N to another antibiotic or antibiotics which were destroyed by cephalosporinase but not by penicillinase. The product of this cell-free enzymic reaction was characterised as deacetoxycephalosporin C (297) by Yoshida etal. 290). These authors also showed that cell-free... 

Nishizawa et al. coated a microarray electrode with PPy and a penicillinase membrane. When the enzyme reaction occurred, conductivity changes were detected by the inaease in the current between two arrays at a constant applied voltage. A variety of other enzymes have been used in conducting polymer electrode sensors, and these have been reviewed usefully, by type of transducer, by Lange et Two other excellent reviews of conducting polymers for sensor applications that review protein applications include those by McQuade et al. and Guimard et al. 

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