02265nas a2200241 4500008004100000022001400041245011000055210006900165260007300234300000800307490000700315520137800322653001601700653002001716653001801736653002501754653002501779100002901804700002601833700001901859700002001878856012501898 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 uhttp://library.ncl.res.in/content/penicillin-v-acylase-pectobacterium-atrosepticum-exhibits-high-specific-activity-and-002265nas a2200277 4500008004100000022001400041245007800055210006900133260009200202300001200294490000700306520130400313653001601617653002001633653001701653653001901670653001801689653001901707653001501726653001501741100002901756700002801785700002601813700003501839856011301874 2016 eng d a0738-855100aPenicillin acylases revisited: importance beyond their industrial utility0 aPenicillin acylases revisited importance beyond their industrial a4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLANDbTAYLOR & FRANCIS LTDcMAR a303-3160 v363 a

It is of great importance to study the physiological roles of enzymes in nature; however, in some cases, it is not easily apparent. Penicillin acylases are pharmaceutically important enzymes that cleave the acyl side chains of penicillins, thus paving the way for production of newer semi-synthetic antibiotics. They are classified according to the type of penicillin (G or V) that they preferentially hydrolyze. Penicillin acylases are also used in the resolution of racemic mixtures and peptide synthesis. However, it is rather unfortunate that the focus on the use of penicillin acylases for industrial applications has stolen the spotlight from the study of the importance of these enzymes in natural metabolism. The penicillin acylases, so far characterized from different organisms, show differences in their structural nature and substrate spectrum. These enzymes are also closely related to the bacterial signalling phenomenon, quorum sensing, as detailed in this review. This review details studies on biochemical and structural characteristics of recently discovered penicillin acylases. We also attempt to organize the available insights into the possible in vivo role of penicillin acylases and related enzymes and emphasize the need to refocus research efforts in this direction.

10abeta-lactam10acholoyl glycine10ain vivo role10aNtn hydrolases10apathogenicity10aquorum sensing10aregulation10asignalling1 aAvinash, Vellore, Sunder1 aPundle, Archana, Vishnu1 aRamasamy, Sureshkumar1 aSuresh, Cheravakkattu, Gopalan uhttp://library.ncl.res.in/content/penicillin-acylases-revisited-importance-beyond-their-industrial-utility-002534nas a2200265 4500008004100000022001400041245013800055210006900193260000800262300001400270490000800284520159300292653003701885653001801922653001701940653002401957653002101981100002902002700002202031700002602053700002402079700001402103700002002117856013102137 2017 eng d a0175-759800aPenicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa0 aPenicillin V acylases from gramnegative bacteria degrade Nacylho cMAR a2383-23950 v1013 a

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.

10aN-acylhomoserine lactone acylase10aNtn hydrolase10apathogenesis10aPenicillin Vacylase10aQuorum quenching1 aSunder, Avinash, Vellore1 aUtari, Putri, Dwi1 aRamasamy, Sureshkumar1 avan Merkerk, Ronald1 aQuax, Wim1 aPundle, Archana uhttp://library.ncl.res.in/content/penicillin-v-acylases-gram-negative-bacteria-degrade-n-acylhomoserine-lactones-and-attenuate02146nas a2200193 4500008004100000022001400041245014800055210006900203260000900272300001600281490000600297520140000303100002101703700002901724700003001753700002601783700002001809856012301829 2020 eng d a2470-134300aProcess development for 6-aminopenicillanic acid production using lentikats-encapsulated escherichia coli cells expressing penicillin V acylase0 aProcess development for 6aminopenicillanic acid production using cNOV  a28972-289760 v53 a

Penicillin V acylase (PVA, EC 3.5.1.11) hydrolyzes the side chain of phenoxymethylpenicillin (Pen V) and finds application in the manufacture of the pharmaceutical intermediate 6-aminopenicillanic acid (6-APA). Here, we report the scale-up of cultivation of Escherichia coli whole cells expressing a highly active PVA from Pectobacterium atrosepticum and their encapsulation in polyvinyl alcohol-poly(ethylene glycol) Lentikats hydrogels. A biocatalytic process for the hydrolysis of 2% (w/v) Pen V was set up in a 2 L reactor using the Lentikats-immobilized whole cells, with a customized setup to enable continuous downstream processing of the reaction products. The biocatalytic reaction afforded complete conversion of Pen V for 10 reaction cycles, with an overall 90% conversion up to 50 cycles. The bioprocess was further scaled up to the pilot-scale at 10 L, enabling complete conversion of Pen V to 6-APA for 10 cycles. The 6-APA and phenoxy acetic acid products were recovered from downstream processing with isolated yields of 85-90 and 87-92%, respectively. Immobilization in Lentikats beads improved the stability of the whole cells on storage, maintaining 90-100% activity and similar conversion efficiency after 3 months at 4 degrees C. The robust PVA biocatalyst can be employed in a continuous process to provide a sustainable route for bulk 6-APA production from Pen V.

1 aSawant, Amol, M.1 aSunder, Avinash, Vellore1 aVamkudoth, Koteswara, Rao1 aRamasamy, Sureshkumar1 aPundle, Archana uhttp://library.ncl.res.in/content/process-development-6-aminopenicillanic-acid-production-using-lentikats-encapsulated