Activity of Amidases

In all the previous e q)eriments the formation of ammonia was of very little importance. Furthermore, the enzyme had a very limited activity, and it has not been possible to analyze the products formed with the exception of the ammonia. It follows that the chemical reaction induced by these enzymes could not be estabKshed experimentally. The principal difficulty in this kind of investigation lies in the fact that the alkaline medium necessary for the activity of amidase very easily becomes infected. It is also -to be noted that the amidases are intracellular enzymes, and that they are obtained with great difficulty in the liquids. Furthermore, these enzymes are considerably weakened by antiseptics. 

I hi monohusic jicids (Icanuiiizc under the influence of amiduse, and are transformed into ammonia. s dth. IO.v.  

Betain is transformed under tlie iiitliiein e of amida-e into the acetate of triin tliylamine  

spiiranin, t)ie di umidi/afion bettiiis with the. itiiide ftroup and entLs with tlie same result as with a .parti, mid The molecular simplitk ution ha.s also hern oiisiTXTd with Klulaniii iu id  

All thesit transformations are the result of a reduction. TTht hydrofjfcn neces.sary omes from a de(om Misition I wafer. ITiis hitter reaction ewlcnlly (aimot fake phne ex ej t in the [ re.scncc of easily oxidi/aihle suhstaiiies whiih appropriate stej) hy. step the oxyj en lilH rated. 

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Amidase (bacteria) HA at 10-30 mM is an activator of amidase-induced hydrolysis of acetamide (enhancing fccat) 52, 53... 

There are two pathways for the degradation of nitriles (a) direct formation of carboxylic acids by the activity of a nitrilase, for example, in Bacillus sp. strain OxB-1 and P. syringae B728a (b) hydration to amides followed by hydrolysis, for example, in P. chlororaphis (Oinuma et al. 2003). The monomer acrylonitrile occurs in wastewater from the production of polyacrylonitrile (PAN), and is hydrolyzed by bacteria to acrylate by the combined activity of a nitrilase (hydratase) and an amidase. Acrylate is then degraded by hydration to either lactate or P-hydroxypropionate. The nitrilase or amidase is also capable of hydrolyzing the nitrile group in a number of other nitriles (Robertson et al. 2004) including PAN (Tauber et al. 2000). 

Figure 8.7 Transformation of rra 5-3-aryl-2-methyloxiranecarbonitriles using the combined NHase and amidase activities of Rhodococcus sp. AJ270...

Amidases can be found in all kinds of organisms, including insects and plants [24], The distinct activities of these enzymes in different organisms can be exploited for the development of selective insecticides and herbicides that exhibit minimal toxicity for mammals. Thus, the low toxicity in mammals of the malathion derivative dimethoate (4.44) can be attributed to a specific metabolic route that transforms this compound into the nontoxic acid (4.45) [25-27]. However, there are cases in which toxicity is not species-selective. Indeed, in the preparation of these organophosphates, some contaminants that are inhibitors of mammalian carboxylesterase/am-idase may be present [28]. Sometimes the compound itself, and not simply one of its impurities, is toxic. For example, an insecticide such as phos-phamidon (4.46) cannot be detoxified by deamination since it is an amidase inhibitor [24],... 

Esters, amides, hydrazides, and carbamates can all be metabolized by hydrolysis. The enzymes, which catalyze these hydrolytic reactions, carboxylesterases and amidases, are usually found in the cytosol, but microsomal esterases and amidases have been described and some are also found in the plasma. The various enzymes have different substrate specificities, but carboxylesterases have amidase activity and amidases have esterase activity. The two apparently different activities may therefore be part of the same overall activity. 

Plettner E., DeSantis G., Stabile M. and Jones J. B. (1999) Modulation of esterase and amidase activity of subtilisin Bacillus lentus by chemical modification of cysteine mutants. J. Am. Chem. Soc. 121, 4977-4981. 

Previous efforts have failed to identify an enzyme with robust Ceph C amidase activity. Some glutaryl-7-ACA acylases can directly convert Ceph C to 7-ACA, but they do so with very poor efficiency and have not been considered for a single-enzyme manufacturing process.30-33 Nonetheless, glutary 1-7-AC A acylases with measurable activity on Ceph C are classified as cephalosporin C acylases. Mutagenesis approaches such as ePCR have been used in an attempt to improve the activity of these enzymes on Ceph C, but only marginal improvements in the desired activity have... 

The serine hydrolase family is one of the largest and most diverse classes of enzymes. They include proteases, peptidases, lipases, esterases, and amidases and play important roles in numerous physiological and pathological process including inflammation [53], angiogenesis [54], cancer [55], and diabetes [56]. This enzyme family catalyzes the hydrolysis of ester, thioester, and amide bonds in a variety of protein and nonprotein substrates. This hydrolysis chemistry is accomplished by the activation of a conserved serine residue, which then attacks the substrate carbonyl. The resulting covalent adduct is then cleaved by a water molecule, restoring the serine to its active state [57] (Scheme 1). 

Pressure activation and inactivation is investigated for several enzymes like penicillin amidase (E.coli), glutamate dehydrogenase (P. woesei) and lipase (Rhizopus arrhizus) in the pressure range between 1 bar and 4000 bar. In dependance of pressure and temperature the enzymes are acivated or inactivated and hence their enantioselectiviy can be directed. The activation of the enzymes corresponds to a decrease in the KM value which results in a higher substrate affinity. ... 

The nitrile group is a versatile building block, in particular since it can be converted into acids or amides. It undergoes hydrolysis but requires relatively harsh reaction conditions. Nature provides two enzymatic pathways for the hydrolysis of nitriles. The nitrilases convert nitriles directly into acids, while the nitrile hy-dratases release amides. These amides can then be hydrolysed by amidases (see also above). Often nitrile hydratases are combined with amidases in one host and a nitrile hydratase plus amidase activity can therefore be mistaken as the activity of a nitrilase (Scheme 6.32). A large variety of different nitrilases and nitrile hydratases are available [100, 101] and both types of enzyme have been used in industry [34, 38, 94]. 

Fournand, D., Bigey, R, and Arnaud, A. 1998. Acyl transfer activity of an amidase from Rhodococcus sp. strain R312 Formation of a wide range of hydroxamic acids. Applied and Environmental Microbiology, 64 2844—52. 

The nitrile-converttng enzymes in R. rhodochrous J1 strain exhibit high versatUily against a variety of aromatic and heteroaromatic nitriles [5, 15, 23] with the only formation of amide, this strain being deficient of amidase activity toward aromatic amides. 

The operational thermal stability of enzymes can be easily evaluated in experiments carried out in a CSMR fed with a saturating substrate concentration, while varying the temperature but maintaining all the other parameters constant. Each enzyme of the cascade system was tested by feeding the CSMR with the appropriate substrate. The kinetic characterization of amidase-catalyzed reactions in runs fed with a nitrile was hampered by the fact that the intracellular enzyme works in cascade with nitrile hydratase. The concentration of amide, produced in situ in the first step, varied with the time and reaction conditions and did not assure the differential conditions needed for an accurate analysis, the amide being completely converted by amidase in some runs. Hence, amidase activity was characterized independently by feeding the reactor with amide as the substrate [35]. 

Figure 7.9 The activation of indoxacarb to an active insecticide by an esterase/amidase.

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