@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.} } @article { ISI:000300202400005, title = {Purification and characterization of YxeI, a penicillin acylase from Bacillus subtilis}, journal = {International Journal of Biological Macromolecules}, volume = {50}, number = {1}, year = {2012}, month = {JAN}, pages = {25-30}, publisher = {ELSEVIER SCIENCE BV}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, abstract = {

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent Km of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93 x 10(2) M-1 and 2.51 x 10(5) M-1, respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride. (C) 2011 Elsevier B.V. All rights reserved.

}, keywords = {Bacillus subtilis, Conjugated bile acid hydrolases, Ntn hydrolase, Penicillin acylase}, issn = {0141-8130}, doi = {10.1016/j.ijbiomac.2011.09.018}, author = {Rathinaswamy, Priya and Gaikwad, Sushama M. and Suresh, C. G. and Prabhune, Asmita and Brannigan, James A. and Dodson, Guy G. and Pundle, Archana V.} } @article { ISI:000323584400008, title = {Structural modelling of substrate binding and inhibition in penicillin V acylase from pectobacterium atrosepticum}, journal = {Biochemical and Biophysical Research Communications}, volume = {437}, number = {4}, year = {2013}, month = {AUG}, pages = {538-543}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, abstract = {

Penicillin V acylases (PVAs) and bile salt hydrolases (BSHs) have considerable sequence and structural similarity; however, they vary significantly in their substrate specificity. We have identified a PVA from a Gram-negative organism, Pectobacterium atrosepticum (PaPVA) that turned out to be a remote homolog of the PVAs and BSHs reported earlier. Even though the active site residues were conserved in PaPVA it showed high specificity towards penV and interestingly the penV acylase activity was inhibited by bile salts. Comparative modelling and docking studies were carried out to understand the structural differences of the binding site that confer this characteristic property. We show that PaPVA exhibits significant differences in structure, which are in contrast to those of known PVAs and such enzymes from Gram-negative bacteria require further investigation. (C) 2013 Elsevier Inc. All rights reserved.

}, keywords = {Bile salt hydrolase, docking, Homology modelling, Inhibition, Pectobacterium, Penicillin acylase}, issn = {0006-291X}, doi = {10.1016/j.bbrc.2013.06.109}, author = {Avinash, Vellore Sunder and Panigrahi, Priyabrata and Suresh, C. G. and Pundle, Archana V. and Ramasamy, Sureshkumar} } @article { ISI:000362964900007, title = {Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases}, journal = {Journal of Industrial Microbiology \& Biotechnology}, volume = {42}, number = {11}, year = {2015}, month = {NOV}, pages = {1493-1506}, publisher = {SPRINGER HEIDELBERG}, address = {TIERGARTENSTRASSE 17, D-69121 HEIDELBERG, GERMANY}, abstract = {

Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in beta-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.

}, keywords = {Alkalistable, beta-lactam antibiotics, Consensus approach, Disulfide bridge, Ion pairs, Penicillin acylase, Thermostability}, issn = {1367-5435}, doi = {10.1007/s10295-015-1690-x}, author = {Panigrahi, Priyabrata and Chand, Deepak and Mukherji, Ruchira and Ramasamy, Sureshkumar and Suresh, C. G.} }