Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids

In this study, we demonstrate the application of multiple functional properties of proteins generated through coupling of residue-specific and site-specific incorporation method. With green fluorescent protein (GFP) as a model protein, we constructed multifunctional GFP through sitespecific incorporation of L-3,4-dihydroxyphenylalanine (DOPA) and residue-specific incorporation of (2S, 4S)-4- fluoroproline (4S-FP) or L-homopropargylglycine (hpg). Fluorescence analysis revealed a conjugation efficiency of approximately 20% for conjugation of DOPA-containing variants GFPdopa, GFPdp[4S-FP], and GFPdphpg onto chitosan. While incorporation of 4S-FP improved protein folding and stability, hpg incorporation into GFP allowed conjugation with fluorescent dye/polyethylene glycol (PEG). In addition, the modification of GFPhpg and GFPdphpg with PEG through Cu(I)-catalyzed click reaction increased protein thermal stability by about two-fold of the wild-type GFP.     

Biotransformation of aromatic and heterocyclic amides by amidase of whole cells of Rhodococcus sp. MTB5: Biocatalytic characterization and substrate specificity

In this study, an amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50?mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50?°C and 50?mM, respectively. The enzyme was quite stable for more than 6?h at 30?°C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50?mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120?min, respectively, using 5?mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383?mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest that Rhodococcus sp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value.     

Binding to syntenin-1 protein defines a new mode of ubiquitin-based interactions regulated by phosphorylation

Syntenin-1 is a PDZ domain-containing adaptor that controls trafficking of transmembrane proteins including those associated with tetraspanin-enriched microdomains. We describe the interaction of syntenin-1 with ubiquitin through a novel binding site spanning the C terminus of ubiquitin, centered on Arg(72), Leu(73), and Arg(74). A conserved LYPSL sequence in the N terminus, as well as the C-terminal region of syntenin-1, are essential for binding to ubiquitin. We present evidence for the regulation of this interaction through syntenin-1 dimerization. We have also established that syntenin-1 is phosphorylated downstream of Ulk1, a serine/threonine kinase that plays a critical role in autophagy and regulates endocytic trafficking. Importantly, Ulk1-dependent phosphorylation of Ser(6) in the LYPSL prevents the interaction of syntenin-1 with ubiquitin. These results define an unprecedented ubiquitin-dependent pathway involving syntenin-1 that is regulated by Ulk1.     

Chitosan and its derivatives for gene delivery

Gene delivery can particularly be used for the treatment of diseases by the insertion of genetic materials (DNA and RNA) into mammalian cells either to express new proteins or to prevent the expression of existing proteins. Chitosan, a natural polymer is nontoxic, biocompatible, and biodegradable and it is used as a support material for gene delivery. However, practical use of chitosan has been mainly limited to its unmodified forms, and thus modified chitosans can be used for the wide range of biomedical applications including the interaction and intracellular delivery of genetic materials. In this context, this review paper provides the recent development on chitosan derivatives available for gene delivery.     

Metabolic Characterization of cold active Pseudomonas, Arthrobacter, Bacillus, and Flavobacterium spp. from Western Himalayas

Himalayan soils undergo dramatic temporal changes in their microclimatic properties. The soil habitats in the high altitude cold habitats of Himalayas are little explored with respect to bacterial diversity and metabolic potentials of the bacterial species. Soil habitat in Western Himalayas is dominated by the genera of Pseudomonas, Arthrobacter, Bacillus, and Flavobacterium. Strains were found to be diverse in their metabolic potentials to utilize different carbon sources by growing them on media containing 114 different sole carbon sources. Bacillus sp. STL9 was supported by the lowest number (12.3%) of the carbon sources while growth was observed in 73.7% of the carbon sources tested for the Pseudomonas sp. SPS2. Carbohydrates appeared to be preferred carbon sources for these Himalayan isolates followed by amino acids and proteins. These microbes also produced various extra-cellular hydrolytic enzymes having biotechnological potentials, lipase being the one secreted by most strains (85.7%) followed by β-galactosidase (42.8%). Antibiotic resistance profiling for 85 different antibiotics has also been described.     

Rediscovery of Lichen Genus Psorotheciopsis Rehm for India

The genus Psorotheciopsis Rehm recorded by Cunningham from Calcutta in 1879 has been rediscovered for India after a gap of 135 years. The taxonomic characters of Psorotheciopsis patellarioides (Rehm) Rolf Santesson are provided to facilitate the identification of the species. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)