Amidases bacterial

We also wanted to evaluate the disassembly of our dendritic system under physiological conditions. Thus, we synthesized a self-immolative AB6 dendron 32 with water-soluble tryptophan tail units and a phenylacetamide head as a trigger (Fig. 5.26) to evaluate disassembly in aqueous conditions. The phenylacetamide is selectively cleaved by the bacterial enzyme penicillin G amidase (PGA). The trigger was designed to disassemble through azaquinone methide rearrangement and cyclic dimethylurea elimination to release a phenol intermediate that will undergo six quinone methide elimination reactions to release the tryptophan tail units. 

Medicinal chemists are interested not only in hydrolysis of amides by mammalian amidases as exemplified above, but also in bacterial amidases as useful biosynthetic tools. Of particular interest is the enantioselective hydrolysis of chiral amides by various bacterial amidases. Some of these... 

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],... 

Hensel et al. have also reported the use of a-amino nitriles as substrates (Scheme 2.10). They discovered a number of new bacterial isolates with stereose-lechve nitrile hydratase achvity. A combination of stereoselechve nitrile hydratases and amidases was shown to be responsible for the production of phenylglycine 24 from nitrile 22 via amide 23. By investigating five isolates both (R)- and (S)-phenylglycine were produced in greater than 99% e.e. [12]. 

Fig. 1 Phylogenetic tree based on amino acid sequences of the polyamidase from Nocardia farcinica and other highly homologous bacterial amidases as well as amidases with substrate specificity for 6-aminohexanoate oligomers [20]...

Core structures of four B-lactam antibiotic families. The ring marked in each structure is the -lactam ring. The penicillins are susceptible to bacterial metabolism and inactivation by amidases and lactamases at the points shown. Note that the carbapenems have a different stereochemical configuration in the lactam ring that apparently imparts resistance to lactamases. Substituents for the penicillin and cephalosporin families are shown in Figures 43-2 and 43-6, respectively. 

The processes are based on whole bacterial cells. In the case of the pipecolic acid, an important building block for pharmaceutical chemistry, an S-selective amidase in Pseudomonas fluorescens cells, catalyses the reaction with high selectivity and the acid is obtained with an ee >99% (Scheme 6.27A). For the preparation of piperazine-2-carboxylic acid from the racemic amide a R- and a S-selective amidase are available. Utilising Klebsiella terrigena cells the S-enantiomer is prepared with 42% isolated yield and ee > 99%, while Burkholderia sp. cells catalyse the formation of the -enantiomer (ee=99%, Scheme 6.27 B). 

Pantothenic acid is largely excreted unchanged by mammals. Some phos-phopantetheine may also be excreted in the urine after administration of pantothenic acid, some of the label may be recovered in exhaled CO2. This is probably the result of intestinal bacterial metabolism, because many bacteria have pantothenase, a specific amidase that cleaves pantothenic acid to 8-alanine and pantoic acid. Pseudomonas species are capable of using pantothenic acid as their sole carbon source. 

The first generation process started with the chemical synthesis of the (R,S)-amide. There were several possible synthetic routes (Fig. 8) via the (R,S)-nitrile or (R,S)-acid [19, 20], A microbial screening program resulted in the isolation of several bacterial strains containing amidases that could specifically hydrolyze the (R)-amide. One of these strains, Comomonas acidivorans A 18 was particularly effective [21]. After the hydrolysis of the unwanted isomer the product (S)-2,2-dimethylcy-clopropane carboxamide was isolated from the bio-solution using a combination of salting-out and solvent extraction. This process had some intrinsic problems ... 

More than 100 members of the AS family of enzymes have been reported in the literature, but only for malonamidase (MAE2) (Shin et al., 2002) and C-terminal peptide amidase (PAM) (Labahn et al., 2002), two soluble bacterial enzymes, structural data are available. All three resolved structures of AS enzymes (FAAH, MAE2, and PAM) revealed a common core, consisting of a twisted p-sheet of 11 mixed strands, surrounded by a large number of a-helices (those of FAAH are shown in Fig. 4.3). Compared to other AS enzymes, which are mostly soluble proteins, FAAH displays two distinguished features i) integration into membranes, and ii) strong preference for hydrophobic substrates. 

Increased bacterial production of Amidases capable of hydrolyzing the 6-position amide to form 6-APA. 

All penicillins (Fignre 74) are composed of a thiazolidine ring attached to a beta-lactam, which in turn carries a free amide gronp (0=CNH) on which a substitution and an attachment (R) are made. In the case of benzylpenicillin, the R is a benzyl gronp. Penicillin may be metabolized by amidase to 6-aminopenicillanic acid, which has antibacterial activity, or by penicillinase (bacterial beta-lactamase), to penicilloic acid, which is devoid of antibacterial activity bnt is antigenic in natnre and acts as a sensitizing structure. The main sonrce of bacterial resistance to penicillin is in fact the prodnction of penicillinase by the microorganisms. 

For the enzymic analysis of polysaccharide structure, the hydrolytic enzymes are the most useful, and little attention will be paid in this article to other types of enzyme. No consideration at all will be accorded enzymes acting on non-carbohydrate moieties. For diis reason, heteropolysaccharide-peptide and lipopolysaccharide complexes (such as those from bacterial cell-walls) are not discussed, as satisfactory treatment of these would also require mention of other types of enzyme, such as amidases for coverage of this subject, an authoritative text and several reviews - may be consulted. Other types of carbohydrate-containing macromolecule, such as glycosaminoglycans ( mucopolysaccharides ) and glycoproteins, for which it is relatively easy to restrict consideration to the polysaccharide portion, are discussed, but only with reference to their carbohydrate moieties. 

Nakagawa Y, Hasegawa A, Hiratake J et al. (2007) Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity mechanistic implication for amide hydrolysis by serine hydrolases. Protein Eng Des Sel 20(7) 339-346 Nardini M, Lang DA, Liebeton K et al. (2000) Crystal stracture of Pseudomorms aeruginosa lipase in the open conformation. The prototype for family LI of bacterial lipases. J Biol Chem 275 31219-31225... 

The putrefactive bacteria, which obviously must have acted in former times as they do at present, secrete proteolytic enzymes as well as amidases. In succession, these various enzymes exerted their action on the nitrogenous materials of marine animals to give finally, among numerous derivatives, volatile fatty adds, of which some were endowed with a rotatory power. These optically active substances, mixed with fats which had resisted bacterial decomposition, and subjected to the combined action of a high temperature and a strong pressure, formed the natural petroleums. 

The study of bacterial amidases has led the writer to seek a method for recovering and utilizing the residual nitrogen of various industries. This problem is concerned with two distinct propositions (i) the transformation of organic nitrogen, such as that contained in the waste liquors from distilleries or in the scum from sugar-refineries, into anunonia and volatile fatty... 

In the experiments with yeast, it is the amidases in reserve inside the cells which act. Necessarily, we are led to use large quantities in order to obtain results. To obviate this inconvenience, which would render the process not very practical, bacteria are utilized that are capable of secreting amidases in abundance. The bacterial q>ecies chosen must be well determined, for not all the ammonia ferments lend themselves to the exacting work required by industry. Moreover, it is for many reasons indi nsable that the bacterium adopted shall be acclimated to the conditions of the environment so that the formation and the secretion of its catalysts shall be favored as much as possible. In fact, bacterial activity producing ammonia is alwa) very slow. The bacteria are very sensitive to their own products, and the deamidization which they cause is never... 

We see then that the production of ammonia depends not only on the bacterial species, but also on the concentration of the medium in nitrogenous substances. Furthermore, it is also related to the temperature. For actions of short duration, the optimum is 55°. Thus, in a fermentation of 24 hours duration, we obtain from 40 to 50 per cent more ammonia at this ten erature than at 40°. But the results observed in longer fermentations are more favorable at the temperature of 40° than at that of 55°. We can then conclude that the production and the secretion of amidases are favored by quite high temperatures, but that in the long run this weakens the bacteria. However, this sensitiveness to the concentration of the medium and to the elevation of the temperature disappears with acclimation. 

TABLE III. Distribution of Taurocholate Amidase and Cholate-7-dehydroxylase Among Various Bacterial Genera... 

Recently, the potential of bacterial enzymes for the synthesis of aromatic, optically active amides, and carboxylic acids firom racemic nitriles was evaluated. An enantiomer-selective amidase, active on several 2-aryl and 2-aryloxy propionamides, was identifided and purified from Brevibacterium sp. strain R312 [145]. A nitrilase, found in Acinetobacter sp. strain AK226 and able to hydrolyze efihciently both aromatic and aliphatic nitriles, was reported to hydrolyze racemic nitriles to optically active 2-aryl propionic acids [146]. Enzyme system of Rhodococcus butanica could be successfully adapted for the kinetic resolution of a-arylpropionitriles resulting in the formation of (R)-... 

In 2003, Griengl s group reported the hydrolysis of cyanohydrins by treatment with bacterial cells of Rhodococcus erythropolis NCIMB 11540, which have a highly active nitrile hydratase/amidase enzyme system. In this manner, (R)-2-chloromandelic acid and (R)-2-hydroxy-4-phenylbutyric acid, two important pharmaceutical intermediates, could be prepared in high optical and chemical yield after short reaction times (3 and 1.5 h, respectively) (Scheme 3.43). 

Kfen, V., and Martmkova, L. (2006) Immobilization of fungal nitrilase and bacterial amidase - two enzymes working in accord. Biocatal. Biotransform., 24, 414-418. 

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