Ruchira Mukherji

A new role for penicillin acylases: Degradation of acyl homoserine lactone quorum sensing signals by Kluyvera citrophila penicillin G acylase

Use of penicillin acylases for the production of semi-synthetic penicillins is well-known. Escherichia coli penicillin G acylase (EcPGA) has been extensively used for this purpose; however, Kluyvera citrophila penicillin G acylase (KcPGA) is assumed to be a better substitute, owing to its increased resilience to extreme pH conditions and ease of immobilization. In the present article we report a new dimension for the amidase activity of KcPGA by demonstrating its ability to cleave bacterial quorum sensing signal molecules, acyl homoserine lactones (AHL) with acyl chain length of 6-8 with or without oxo-substitution at third carbon position. Initial evidence of AHL degrading capability of KcPGA was obtained using CV026 based bioassay method. Kinetic studies performed at pH 8.0 and 50 °C revealed 3-oxo-C6 HSL to be the best substrate for the enzyme with V(max) and K(m) values of 21.37+0.85 mM/h/mg of protein and 0.1+0.01 mM, respectively. C6 HSL was found to be the second best substrate with V(max) and K(m) value of 10.06+0.27 mM/h/mg of protein and 0.28+0.02 mM, respectively. Molecular modeling and docking studies performed on the active site of the enzyme support these findings by showing the fitting of AHLs perfectly within the hydrophobic pocket of the enzyme active site.

Novel glycolipids synthesized using plant essential oils and their application in quorum sensing inhibition and as antibiofilm agents.

Essential oils (EOs) form an important part of traditional medicine so their anti-microbial and, in the recent past, antiquorum sensing activity has been well studied. However it is likely that due to their hydrophobic nature and reduced solubility in aqueous environments full potential of their activity cannot be realized. hence it is only rational to formulate a process to make these molecules more polar in nature. The present paper reports synthesis of sophorolipids using 12 different essential oils as substrates, thus providing surfactant-like properties to these EOs. The synthesis protocol makes the use of Candida bombicola ATCC 22214 as producer organism. The production process required 7 days of incubation at 28°C and 180 rpm. Preliminary characterization of the synthesized essential oil sophorolipids (EOSLs) was performed using thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR). Additionally, essential oils that were incapable of mediating quorum sensing inhibition (QSI) on their own became potent quorum sensing inhibitors upon conversion into their corresponding EOSLs. Antibiofilm potential of these EOSLs was also demonstrated using V. cholerae as test organism. Use of essential oils as substrates for glycolipid synthesis has not been attempted previously, and hence this is the first report.

Fluorescent sophorolipid molecular assembly and its magnetic nanoparticle loading: a pulsed laser process

We report realization of strong green fluorescence in a fully biocompatible highly spherical mesoscale molecular assembly of sophorolipid created by pulsed UV laser processing of a water-based dispersion of sophorolipid. Remarkably, the said fluorescence is totally absent in the original molecule of sophorolipid and is thus the property of the pulsed laser induced modifications in the molecule and the assembly. We have separately examined the consequences of laser irradiation for glucose and oleic acid components which form the sophorolipid. This fluorescence character appears to be driven by the oleic acid component while the assembly process is assisted by the glucose component. Importantly the laser synthesized mesostructures can be easily redispersed in an aqueous medium after being dried and can also be loaded with magnetic nanoparticles (magnetite) for inducing the hyperthermia effect.

Biological activity of sophorolipids and their possible use as antiviral agents

Sophorolipids are surface active glycolipids consisting of a hydrophilic sophorose unit and a hydrophobic portion composed of a fatty acid tail. Crude sophorolipid sample contains both acidic and lactonic forms of sophorolipid with different degrees of acetylation and varying lengths of the fatty acid chains depending on the substrates used in the production process. Carboxylic end in the acidic form of the fatty acid is free, whereas in the lactonic form, it is internally esterified. Sophorolipids show different physicochemical properties with wide range of applications for each structural compound. Lactonic form of sophorolipids shows surface tension reducing ability and biological activity, whereas the acidic form possesses better foam forming ability and higher solubility. Presence of acetyl groups gives hydrophilic nature to the sophorolipids which promotes its antiviral and cytokine-stimulating properties. The aim of this review is to explore and suggest the plausibility of sophorolipids as therapeutic and prophylactic agents for the treatment of viral diseases.

Selective Imaging of Quorum Sensing Receptors in Bacteria Using Fluorescent Au Nanocluster Probes Surface Functionalized with Signal Molecules

Fluorescent ultrasmall gold clusters decorated with bacterial quorum sensing signal molecules, acyl homoserine lactone, are synthesized. These fluorescent probes are found to have emission in the near-infrared spectral region advantageous for bioimaging. Imaging studies using different strains of bacteria with and without acyl homoserine lactone receptors with the aid of confocal microscopy have shown that the probe interacts preferentially with cells possessing these receptors. This indicates that, with appropriate surface functionalization, the Au clusters can be used for receptor specific detection with enhanced selectivity.

Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases.

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 β-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.