ABSTRACT
Aims: To identify predominant microorganisms in dish washing scrubbers collected from ten different sources. Study Design: Collection of dish wash scrubbers from different sources, pour plating of appropriately prepared dish wash scrub suspensions and subsequent isolation and identification of predominant isolates. Assessment of antibiotic susceptibility of the selected isolates by disc diffusion assay. Place and Duration of Study: April, 2018 - June 2018. Methodology: A total of 10 dish wash scrubbers (synthetic green scrubber pads free from any anti-bacterial preservatives belonging to the same brand) were collected from various sources. Appropriately prepared dish wash scrub suspensions in peptone water were pour plated on Plate Count Agar (PCA) and MacConkey agar. Predominant colonies selected from the plates based on the colony morphology were subjected to Grams staining, catalase, oxidase, indole, citrate, urease tests and genotypic identification by 16S ribosomal RNA sequencing. The identified isolates were tested for their susceptibility to eight antibiotics by disc diffusion method. Results: Irrespective of the sample source, most of the dish wash scrubbers sampled harbored similar types of colonies. From the colonies obtained two of them were identified by 16S rRNA sequencing and subsequent blasting as Klebsiella pneumoniae and Acinetobacter radioresistens. The isolates were deposited in the NCBI database with accession numbers MK032217 (Klebsiella pneumoniae RSV02) and MK032134 (Acinetobacter radioresistens RSV 01). These isolates were tested for their susceptibility to different antibiotics and Acinetobacter radioresistens RSV 01 was found to be more antibiotic susceptible than Klebsiella pneumoniae RSV02. Conclusion: Observations of this study confirm the potential role of dish wash scrubbers as vehicle for potential pathogens and their ability to act as cross contaminating agents in food processing environments.
ABSTRACT
The role of sugars, polyhydroxy compounds, phenylacetic acid and 6-aminopenicillanic acid in stabilization of immobilized penicillin G acylase (IMPGA) was studied. The loss in the activity of IMPGA at 50 degrees C, 2 h, after incorporation of sucrose and mannitol at 0.1 M concentration was 16 and 18% respectively; the loss in the activity of the enzyme under these conditions in the absence of stabilizing agents was 40%.
Subject(s)
Carbohydrates/pharmacology , Enzyme Stability , Enzymes, Immobilized/chemistry , Hot Temperature , Penicillin Amidase/chemistry , Polymers/pharmacologyABSTRACT
Penicillin V acylase from Fusarium sp. SKF 235 culture filtrate was purified to homogeneity. The enzyme was a glycoprotein and composed of single polypeptide chain with molecular weight of 83,200 Daltons. The pH and temperature optima were 6.5 and 55 degrees C, respectively. The KM for penicillin V was 10 mM but the enzyme was inhibited by penicillin V at concentrations above 50 mM. Products of reaction, 6-aminopenicillanic acid and phenoxyacetic acid inhibited the enzyme competitively and noncompetitively with Ki values of 18 mM and 45 mM, respectively. The enzyme specifically hydrolyzed penicillin V, cephalosporanic acid V and penicillin V sulphoxide. Other phenoxy acetyl amides studied were not hydrolysed. It is proposed that phenoxyacetyl moiety alone is not recognized by the penicillin V acylase and in addition, the beta-lactam structure contributes in formation of enzyme-substrate complex.
Subject(s)
Amidohydrolases/analysis , Binding, Competitive , Cephalosporins/metabolism , Culture Media , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/chemical synthesis , Fusarium/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Molecular Weight , Penicillanic Acid/analogs & derivatives , Penicillin Amidase/antagonists & inhibitors , Penicillin V/analogs & derivatives , Phenoxyacetates/chemistry , Substrate Specificity , TemperatureABSTRACT
Cephalosporin acylases have application in the production of 7-aminocephalosporanic acid which forms a key raw material for the preparation of semisynthetic injectable cephalosporins. The enzymes are of industrial importance and hyperproducing genetically engineered strains have been constructed. Different aspects of these enzymes such as subunit structure, post translational modification, primary structure, substrate specificity and their importance in pharmaceutical industry are discussed.
Subject(s)
Amino Acid Sequence , Cephalosporins/chemical synthesis , Fermentation , Penicillin Amidase/chemical synthesisABSTRACT
Enzymatic parameters such as pH, temperature and substrate concentration were studied for the hydrolysis of 7-PADCA by penicillin G acylase. Optimum pH and temperature were 8.0 and 50 degrees C, respectively. Km value of soluble and immobilized enzyme for 7-PADCA was 2.3 x 10(-5) M and 7.5 x 10(-5) M, respectively. At 7-PADCA concentration of 5% and an IME: 7-PADCA ratio of 1:2.5, the hydrolysis was complete in 110 min.
Subject(s)
Cephalosporins/metabolism , Enzymes, Immobilized , Hydrogen-Ion Concentration , Hydrolysis , Penicillin Amidase/metabolism , TemperatureABSTRACT
The growth of Candida sp. 115 was investigated on the constituents of penicillin G hydrolysis reaction mixture. Neither penicillin G nor 6-aminopenicillanic acid was degraded or utilised for growth. The yeast accepted phenylacetic acid, sodium acetate and glucose as growth substrates. Phenylacetic acid was metabolised via p-hydroxy phenylacetic acid, which was the only accumulated metabolite. The enzymes responsible for hydroxylation of phenylacetic acid were induced by phenylacetic acid and sodium acetate.
Subject(s)
Acetic Acid , Acetates/metabolism , Candida/growth & development , Culture Media , Glucose/metabolism , Hydrolysis , Penicillin G/metabolism , Phenylacetates/metabolismABSTRACT
Fermentation parameters for the production of penicillin G acylase by Escherichia coli NCIM 2400 have been evaluated. The bacterium produced the enzyme intracellularly when grown in nutrient broth containing PAA. PAA stimulated the enzyme synthesis by 8-10 fold and reduced the lag period. The optimum concentration of PAA for induction was 20 mM and addition of PAA prior to inoculation gave maximum production of PGA. Glucose, lactose, sorbitol, acetate and lactate even at 0.1% concentration catabolically repressed the enzyme formation. Peptone was the best utilised 'N' source for the enzyme production. Phosphate and yeast extract were found to be essential for both the growth and for enzyme biosynthesis. Temperature between 22-24 degrees C was optimum and under ideal condition E. coli NCIM 2400 produced 0.45-0.55 U/ml of penicillin G acylase.
Subject(s)
Amidohydrolases/biosynthesis , Escherichia coli/enzymology , Fermentation , Hydrogen-Ion Concentration , Hydrolysis , Penicillin Amidase/biosynthesis , Penicillin G/metabolism , Phenylacetates/pharmacologyABSTRACT
Binding of penicillin amidase from E. coli 436 to aniline-, benzylamine- and phenylethylamine-Sepharose was studied. Binding of the enzyme to aniline-Sepharose was exclusively due to hydrophobic interactions. Benzylamine-Sepharose binds the enzyme due to affinity interactions in the absence of ammonium sulphate and due to hydrophobic interactions in the presence of ammonium sulphate. A conformational change in the penicillin amidase molecule due to ammonium sulphate there by exposing the side chain binding site as a hydrophobic core is suggested.