Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated Il-lactamases. However, inhibition data for these three inhibitors with a wide range of different plasmid-mediated P-lactamases have not yet been compared under the same experimental conditions. A number of groups have inferred that clavulanic acid inhibits extended-spectrum TEM and SHV P-lactamases, but inhibition data have rarely been published. In this study, the 50%o inhibitory concentrations of these three ,-lactamase inhibitors for 35 plasmid-mediated P-lactamases have been determined. Of these 35 P-lactamases, 20 were extendedspectrum TEM-or SHV-derived I-lactamases. The other 15 enzymes were conventional-spectrum P-lactamases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the 35 plasmid-mediated I-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent than sulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negative plasmid-mediated I-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showed that clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrum enzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extendedspectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the two groups of P-lactamases. There were no significant differences between the overall activities of tazobactam and clavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes, although differences in their inhibition profiles were observed.I-lactamases are plasmid-encoded or chromosomally encoded bacterial enzymes which hydrolyze 3-lactam antibiotics.Plasmid-mediated ,-lactamases can transfer rapidly between bacterial genera and consequently pose a major threat to the successful use of ,-lactam agents. More than 60 different types of plasmid-encoded ,-lactamases have been characterized, and for the purpose of this work, the enzymes have been classified as either conventional-spectrum or TEM-and SHVderived extended-spectrum enzymes. The conventional-spectrum ,-lactamases are from Bush groups 2a, 2b, 2c, and 2d, and extended-spectrum TEM and SHV enzymes are all from group 2b' (4).The conventional-spectrum enzymes include enzymes such as TEM-1 and SHV-1, which do not confer resistance to cephalosporins such as ceftazidime and cefotaxime. A recent survey of 802 gram-negative clinical isolates showed that TEM-1 and SHV-1 were responsible for mediating P-lactam resistance in 17% of clinical isolates (37). Other conventionalspectrum enzymes which are often found in clinical isolates include the OXA and PSE P-lactamases (1). The most common conventional-spectrum plasmid-mediated 13-lactamase found in gram-positive bacteria is the penicillinase produced by the majority of Staphylococcus aureus clinical isolates (26).Most of the extended-spectrum pl...

Mesophilic nitrile-degrading enzymes are widely dispersed in the Bacteria and lower orders of the eukaryotic kingdom. Two distinct enzyme systems, a nitrilase catalyzing the direct conversion of nitriles to carboxylic acids and separate but cotranscribed nitrile hydratase and amidase activities, are now well known. Nitrile hydratases are metalloenzymes, incorporating FeIII or CoII ions in thiolate ligand networks where they function as Lewis acids. In comparison, nitrilases are thiol-enzymes and the two enzyme groups have little or no apparent sequence or structural homology. The hydratases typically exist as alpha beta dimers or tetramers in which the alpha- and beta-subunits are similar in size but otherwise unrelated. Nitrilases however, are usually found as homomultimers with as many as 16 subunits. Until recently, the two nitrile-degrading enzyme classes were clearly separated by functional differences, the nitrile hydratases being aliphatic substrate specific and lacking stereoselectivity, whereas the nitrilases are enantioselective and aromatic substrate specific. The recent discovery of novel enzymes in both classes (including thermophilic representatives) has blurred these functional distinctions. Purified mesophilic nitrile-degrading enzymes are typically thermolabile in buffered solution, rarely withstanding exposure to temperatures above 50 degrees C without rapid inactivation. However, operational thermostability is often increased by addition of aliphatic acids or by use of immobilized whole cells. Low molecular stability has frequently been cited as a reason for the limited industrial application of "nitrilases"; such statements notwithstanding, these enzymes have been successfully applied for more than a decade to the kiloton production of acrylamide and more recently to the smaller-scale production of nicotinic acid, R-(-)-mandelic acid and S-(+)-ibuprofen. There is also a rapidly growing catalog of other potentially useful conversions of complex nitriles in which the regioselectivity of the enzyme coupled with the ability to achieve high conversion efficiencies without detriment to other sensitive functionalities is a distinct process advantage.

Two novel metabolites, SB 212021 and SB 212305, have been isolated from a Streptomyces and shown to have molecular formulae of C15H10N2O5 and C20H17N3O8S, respectively. The structures were deduced by a combination of NMR techniques and mass spectral fragmentation patterns and shown to be novel membersof the phenazine group of antibiotics. In the absence of added zinc, both compounds had IC50's of 1^75^for the Bacteroides fragilis 262 CfiA and Xanthomonas maltophilia h-l metallo-/Mactamases. The compounds also inhibited ACEwith IC50's of 55 and 68 fiM, respectively. Mode of action studies illustrate that the compounds inihibit some metalloenzymes by chelation of the active site metal ion. They exhibit poor antibacterial activity.Metallo/Mactamases, whilst not as prevalent in nature as their serine counterparts, do constitiute a possible threat to /Mactam chemotherapy due to the unavailability of effective inhibitors.Although only produced by a limited number of organisms they are potentially transferable by plasmids and could therefore pose a wider threat1*. The search for inhibitors of this enzyme is thus aimed at identifying structural types from natural product screening that could provide leads for chemical modification/synthesis.We report on the isolation of two novel phenazines, designated SB 212021 (1) and SB 212305 (2), produced by an unidentified Streptomyces sp. and found to inhibit zinc-dependent metallo-/Mactamase from Bacillus cereus. This paper describes the isolation, physico-chemical properties, structure determination and biological activity of SB 212021 and SB 212305. Materials and Methods Fermentation ConditionsThe unidentified Streptomyces sp. was maintained as a vegetative cell suspension stored in glycerol 10% under liquid nitrogen. Vegetative cell and spore suspension (1 ml) was used to inoculate seed medium (100ml) containing 1.5% agar. The seed stage medium consisted of yeast extract (Oxoid) 0.5%, malt extract (Oxoid) 1.0%, glycerol 1.0% and peptone soya (Oxoid) 0.5% dissolved in distilled water and adjusted to pH 6.5 before sterilisation in an autoclave at 121°C for 15 minutes. Four spiked 500 ml flasks, each containing 60 ml of seed stage mediumas defined above, were inoculated with 3 plugs from a culture plate. The inoculated flasks were incubated on a gyratory shaking table at 240 rpm for 60 hours at 28°C and after this time were used to inoculate 70 spiked 500ml flasks, each containing 60ml of seed stage medium as defined above (inoculum level ca. 5%, 3ml/flask).These inoculated flasks were shaken at 240rpm for 4.5 days at 28°C. The harvested broth was

Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M(r), 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying kcat/KM values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced kcat, while orthosubstitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65 degrees C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50 degrees C, 2.5 h at 60 degrees C, 13 min at 70 degrees C, and less than 3 min at 80 degrees C.

Summary: The first known report of the isolation of thermophilic bacteria which produce nitrile-degrading enzymes is presented. One of the strains isolated was studied in detail. Strain Dac521, classified as Bacillus pallidus, was capable of growth on acetonitrile, benzonitrile, propionitrile, acetamide, benzamide and propionamide as the sole carbon and nitrogen source in minimal nutrient media. The strain produced separate aliphatic-nitrile (e.g. acetonitrile)- and aromatic-nitrile (e.g. benzonitrile)-degrading activities. Acetonitrile-degrading activity was produced constitutively and enzyme production was not enhanced by the addition of substrate. Under conditions where benzonitrile was the sole carbon and nitrogen source in minimal nutrient media, acetonitrile-degrading enzyme activity was completely inhibited and benzonitrile-degrading activity was induced. Growth on substrates as sole carbon and nitrogen sources, together with the substrate specificity of cell-free extracts, suggested that acetonitrile and benzonitrile degradation may have occurred via nitrile hydratase and nitrilase pathways, respectively. Both the acetonitrile- and benzonitrile-degrading enzyme systems were significantly more thermostable in whole-cell preparations and cell-free extracts compared to their mesophilic counterparts.