@article { ISI:000258819000009, title = {Characterization of smallest active monomeric penicillin V acylase from new source: a yeast, rhodotorula aurantiaca (NCIM 3425)}, journal = {Process Biochemistry}, volume = {43}, number = {9}, year = {2008}, month = {SEP}, pages = {961-967}, publisher = {ELSEVIER SCI LTD}, type = {Article}, address = {THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND}, abstract = {

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.

}, keywords = {Intracellular enzyme, N-terminal sequencing, Penicillin V acylase, Purification, Rhodotorula aurantiaca, Substrate specificity}, issn = {1359-5113}, doi = {10.1016/j.procbio.2008.04.024}, author = {Kumar, Atul and Prabhune, Asmita and Suresh, C. G. and Pundle, Archana} } @article { ISI:000208979401243, title = {Permeabilization effect of organic solvents on cell bound penicillin v acylase activity of Erwinia aroideae (DSMZ 30186)}, journal = {Journal of Biotechnology}, volume = {136}, number = {S}, year = {2008}, month = {OCT}, pages = {S321}, publisher = {ELSEVIER SCIENCE BV}, type = {Meeting Abstract}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, issn = {0168-1656}, doi = {10.1016/j.jbiotec.2008.07.1932}, author = {Kumar, Atul and Pundle, Archana} } @article { ISI:000267662600010, title = {Effect of cultural conditions and media constituents on production of penicillin V acylase and CTAB treatment to enhance whole-cell enzyme activity of rhodotorula aurantiaca (NCIM 3425)}, journal = {Applied Biochemistry and Biotechnology}, volume = {157}, number = {3}, year = {2009}, month = {JUN}, pages = {463-472}, publisher = {HUMANA PRESS INC}, address = {999 RIVERVIEW DRIVE SUITE 208, TOTOWA, NJ 07512 USA}, abstract = {

Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic beta-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 degrees C. Fructose was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170 and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic detergent. Whole-cell activity was found to be doubled (204\%) on treatment of 0.01 g dry weight of cells with 50 mu g/ml solution of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.

}, keywords = {Atomic force microscopy, Intracellular enzyme, Penicillin Vacylase, Permeabilization, Rhodotorula aurantiaca}, issn = {0273-2289}, doi = {10.1007/s12010-008-8294-0}, author = {Kumar, Atul and Singh, Sanjay and Poddar, Pankaj and Prabhune, Asmita and Pundle, Archana} } @article { ISI:000264899000012, title = {Effect of organic solvents on cell-bound penicillin V acylase activity of erwinia aroideae (DSMZ 30186): a permeabilization effect}, journal = {Journal of Molecular Catalysis B-Enzymatic}, volume = {57}, number = {1-4}, year = {2009}, month = {MAY}, pages = {67-71}, publisher = {ELSEVIER SCIENCE BV}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, abstract = {

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.

}, keywords = {dielectric constant, Erwinia aroideae, Hydrophobicity, Organic solvents, Penicillin V acylase, Permeabilization}, issn = {1381-1177}, doi = {10.1016/j.molcatb.2008.06.018}, author = {Kumar, Atul and Pundle, Archana} } @article { ISI:000271538700006, title = {Rhodotorula aurantiaca penicillin V acylase: active site characterization and fluorometric studies}, journal = {Journal of Photochemistry and Photobiology B-Biology}, volume = {97}, number = {2}, year = {2009}, month = {NOV}, pages = {109-116}, publisher = {ELSEVIER SCIENCE SA}, address = {PO BOX 564, 1001 LAUSANNE, SWITZERLAND}, abstract = {

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.

}, keywords = {active site residues, Fluorometric studies, Penicillin V acylase, Rhodotorula aurantiaca}, issn = {1011-1344}, author = {Kumar, Atul and Gowda, Nagaraj M. and Gaikwad, Sushama M. and Pundle, Archana} } @article { ISI:000307537300043, title = {Characterization of a new bacillus cereus ATUAVP1846 strain producing penicillin V acylase, and optimization of fermentation parameters}, journal = {Annals of Microbiology}, volume = {62}, number = {3}, year = {2012}, month = {SEP}, pages = {1287-1293}, publisher = {SPRINGER}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

Penicillin acylases are involved chiefly in the industrial production of semisynthetic penicillins, which remain the most widely used group of antibiotics. We have isolated a new bacterial strain ATUAVP1846 that produces penicillin V acylase (PVA). Phylogenetic analysis using 16S rRNA sequencing showed 99.37\% homology with Bacillus cereus. Maximum PVA production was observed with B. cereus ATUAVP1846 at 30A degrees C, pH 7 after 24 h fermentation time under submerged conditions. Highest enzyme productivity was achieved using sucrose as carbon source, and tryptone and ammonium hydrogen phosphate as nitrogen sources. Minimal medium containing 0.4\% glucose and 0.3\% ammonium hydrogen phosphate was found to be optimal for maximum PVA production from B. cereus ATUAVP1846. The crude enzyme from B. cereus ATUAVP1846 was partially purified using ammonium sulfate fractionation and showed highest enzymatic activity in the hydrolysis of penicillin V at 40A degrees C and pH 6. The crude enzyme preparation also showed unique substrate specificity, preferring ampicillin and cephalexin over penicillin V.

}, keywords = {16S rRNA, Bacillus cereus, isolation, Ntn hydrolase, Optimization, Penicillin acylase}, issn = {1590-4261}, doi = {10.1007/s13213-011-0374-7}, author = {Sunder, Avinash Vellore and Kumar, Atul and Naik, Neha and Pundle, Archana V.} }