02327nas a2200253 4500008004100000022001400041245013200055210006900187260009900256300001200355490000700367520135500374653002501729653002601754653002501780653001701805653002701822653002601849100001601875700002101891700001901912700002001931856012201951 2008 eng d a1359-511300aCharacterization of smallest active monomeric penicillin V acylase from new source: a yeast, rhodotorula aurantiaca (NCIM 3425)0 aCharacterization of smallest active monomeric penicillin V acyla aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLANDbELSEVIER SCI LTDcSEP a961-9670 v433 a

An intracellular monomeric penicillin V acylase (PVA) of 36,000 Da exhibiting pI of 4.19, purified from newly identified yeast source, Rhodotorula aurantiaca (NCIM 3425). The enzyme was purified by hydrophobic interaction chromatography. The enzyme showed optimal activity at 45 degrees C and retained 80% activity after incubation at 45 degrees C and pH 5.5 for I h. The enzyme showed maximum activity at pH 5.5 and was very stable between pH 5.5-6.5 with optimum stability at pH 6.0. It exhibited 50% of its original activity after 30 min incubation at 60 degrees C. Enzyme hydrolyzed substrates with benzyl side chain but preferred penicillin V as primary substrate. N-terminally located serine supports the fact that it belongs to Ntn (N-terminal nucleophile)-hydrolase superfamily. The initial ten amino acid residues of R. aurantiaca PVA were identical to the initial sequence of NADH dehydrogenase (EC 1.6.99.3): however the enzyme lacks dehydrogenase activity. EGTA, EDTA, hexane and ethyl acetate stabilized the activity where as small chain alcohols inhibited it. 1,4-Dioxane, THF (tetrahydrofurane), phenol and benzyl alcohol severely inhibited enzyme activity while BME and DTT increased it. Tween 80 and Tween 20 highly enhanced the activity where as SIDS and Triton X-100 inhibited it. (C) 2008 Elsevier Ltd. All rights reserved.

10aIntracellular enzyme10aN-terminal sequencing10aPenicillin V acylase10aPurification10aRhodotorula aurantiaca10aSubstrate specificity1 aKumar, Atul1 aPrabhune, Asmita1 aSuresh, C., G.1 aPundle, Archana uhttps://library.ncl.res.in/content/characterization-smallest-active-monomeric-penicillin-v-acylase-new-source-yeast-001925nas a2200229 4500008004100000022001400041245013500055210006900190260007300259300001000332490000700342520105100349653002401400653002101424653001901445653002101464653002501485653002101510100001601531700002001547856012801567 2009 eng d a1381-117700aEffect of organic solvents on cell-bound penicillin V acylase activity of erwinia aroideae (DSMZ 30186): a permeabilization effect0 aEffect of organic solvents on cellbound penicillin V acylase act aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAY a67-710 v573 a

Erwinia aroideae (DSMZ 30186) is a potential microbial culture to produce intracellular penicillin V acylase (PVA). The whole cell PVA activity was improved by permeabilization with various organic solvents. The cell-bound PVA activity showed an eightfold increase upon treatment with chloroform (5 mu L/mg(dry) (biomass)) for 10 min and diethyl ether (10 mu L/mg(dry) (biomass)) for 45 min. Hexane, toluene, ethyl acetate and dichloromethane enhanced the enzyme activity up to two-, six-, four- and two-fold, respectively; whereas, PVA activity declined drastically on permeabilization with acetone, pyridine and alcohols. The physicochemical properties of the organic solvents used for permeabilization were correlated with the change in activity. It was found that solvents with high hydrophobicity (log P > 0.68) and lower dielectric constant (< 9) were relatively effective in increasing PVA activity. These results allow systematic selection of suitable solvent for best performance. (c) 2008 Elsevier B.V. All rights reserved.

10adielectric constant10aErwinia aroideae10aHydrophobicity10aOrganic solvents10aPenicillin V acylase10aPermeabilization1 aKumar, Atul1 aPundle, Archana uhttps://library.ncl.res.in/content/effect-organic-solvents-cell-bound-penicillin-v-acylase-activity-erwinia-aroideae-dsmz-002493nas a2200229 4500008004100000022001400041245010300055210006900158260006900227300001200296490000700308520163100315653002501946653002501971653002501996653002702021100001602048700002302064700002502087700002002112856013102132 2009 eng d a1011-134400aRhodotorula aurantiaca penicillin V acylase: active site characterization and fluorometric studies0 aRhodotorula aurantiaca penicillin V acylase active site characte aPO BOX 564, 1001 LAUSANNE, SWITZERLANDbELSEVIER SCIENCE SAcNOV a109-1160 v973 a

Penicillin V acylase (PVA), a member of newly evolved Ntn-hydrolase superfamily, is a pharmaceutically important enzyme to produce 6-aminopenicillanic acid. Active site characterization of recently purified monomeric PVA from Rhodotorula aurantiaca (Ra-PVA), the yeast source, showed the involvement of serine and tryptophan in the enzyme activity. Modification of the protein with serine and tryptophan specific reagents such as PMSF and NBS showed partial loss of PVA activity and substrate protection. Ra-PVA was found to be a multi-tryptophan protein exhibiting one tryptophan, in native and, four in its denatured condition. Various solute quenchers and substrate were used to probe the microenvironment of the putative reactive tryptophan through fluorescence quenching. The results obtained indicate that the tryptophan residues of Ra-PVA were largely buried in hydrophobic core of the protein matrix. Quenching of the fluorescence by acrylamide was collisional. Acrylamide was the most effective quencher amongst all the used quenchers, which quenched 71.6% of the total intrinsic fluorescence of the protein, at a very less final concentration of 0.1 M. Surface tryptophan residues were found to have predominantly more electropositively charged amino acids around them, however differentially accessible for ionic quenchers. Denaturation led to shift lambda(max) from 336, in native state, to 357 nm and more exposed to the solvent, consequently increase in fluorescence quenching with all quenchers. This is an attempt towards the conformational studies of Ra-PVA. (C) 2009 Elsevier B.V. All rights reserved.

10aactive site residues10aFluorometric studies10aPenicillin V acylase10aRhodotorula aurantiaca1 aKumar, Atul1 aGowda, Nagaraj, M.1 aGaikwad, Sushama, M.1 aPundle, Archana uhttps://library.ncl.res.in/content/rhodotorula-aurantiaca-penicillin-v-acylase-active-site-characterization-and-fluorometric-002266nas a2200241 4500008004100000022001400041245011000055210006900165260007300234300000800307490000700315520137800322653001601700653002001716653001801736653002501754653002501779100002901804700002601833700001901859700002001878856012601898 2015 eng d a0141-813000aPenicillin V acylase from pectobacterium atrosepticum exhibits high specific activity and unique kinetics0 aPenicillin V acylase from pectobacterium atrosepticum exhibits h aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcAUG a1-70 v793 a

Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria. (C) 2015 Elsevier B.V. All rights reserved.

10aCooperative10aEnzyme kinetics10aGram-negative10aPenicillin V acylase10aSubstrate inhibition1 aAvinash, Vellore, Sunder1 aRamasamy, Sureshkumar1 aSuresh, C., G.1 aPundle, Archana uhttps://library.ncl.res.in/content/penicillin-v-acylase-pectobacterium-atrosepticum-exhibits-high-specific-activity-and-0