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.     

Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1

The chromophore conformations of the red and far red light induced product states "Pfr" and "Pr" of the N-terminal photoreceptor domain Cph1-N515 from Synechocystis 6803 have been investigated by NMR spectroscopy, using specific 13C isotope substitutions in the chromophore. 13C-NMR spectroscopy in the Pfr and Pr states indicated reversible chemical shift differences predominantly of the C(4) carbon in ring A of the phycocyanobilin chromophore, in contrast to differences of C15 and C5, which were much less pronounced. Ab initio calculations of the isotropic shielding and optical transition energies identify a region for C4-C5-C6-N2 dihedral angle changes where deshielding of C4 is correlated with red-shifted absorption. These could occur during thermal reactions on microsecond and millisecond timescales after excitation of Pr which are associated with red-shifted absorption. A reaction pathway involving a hula-twist at C5 could satisfy the observed NMR and visible absorption changes. Alternatively, C15 Z-E photoisomerization, although expected to lead to a small change of the chemical shift of C15, in addition to changes of the C4-C5-C6-N2 dihedral angle could be consistent with visible absorption changes and the chemical shift difference at C4. NMR spectroscopy of a 13C-labeled chromopeptide provided indication for broadening due to conformational exchange reactions in the intact photoreceptor domain, which is more pronounced for the C- and D-rings of the chromophore. This broadening was also evident in the F2 hydrogen dimension from heteronuclear 1H-13C HSQC spectroscopy, which did not detect resonances for the 13C5-H, 13C10-H, and 13C15-H hydrogen atoms whereas strong signals were detected for the (13)C-labeled chromopeptide. The most pronounced 13C-chemical shift difference between chromopeptide and intact receptor domain was that of the 13C4-resonance, which could be consistent with an increased conformational energy of the C4-C5-C6-N2 dihedral angle in the intact protein in the Pr state. Nuclear Overhauser effect spectroscopy experiments of the 13C-labeled chromopeptide, where chromophore-protein interactions are expected to be reduced, were consistent with a ZZZssa conformation, which has also been found for the biliverdin chromophore in the x-ray structure of a fragment of Deinococcus radiodurans bacteriophytochrome in the Pr form.     

Enhanced decolorization of sulfonated azo dye methyl orange by single and mixed bacterial strains AK1, AK2 and VKY1

Abstract

Methyl orange, a sulfonated azo dye having various industrial applications was decolorized by three bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1. The effect of various factors such as dye concentration, pH, temperature and NaCl concentration on decolorization was investigated. At 200?mg/L methyl orange concentration, the strains AK1, AK2 and VKY1 exhibited maximum decolorizing potential of 93, 95 and 96%, respectively, at temperature 35?°C and pH 7.0 within 18?h of incubation. These strains decolorized the dye over a wide range of pH (5–10), temperature (15–55?°C), and NaCl concentration (5–20?g/L). Further, these strains decolorize up to 800?mg/L concentrations of methyl orange within 24?h. The dye decolorization efficiency was further increased by using different consortia of these three strains which could decolorize the dye completely within 12?h of incubation. The cell-free extracts of the strains AK1, AK2 and VKY1 grown on methyl orange exhibited the azoreductase activity of 0.4794, 1.56 and 1.01?µM/min/mg protein, respectively. HPLC and FTIR analysis of the dye decolorized sample indicated the formation of 4-aminobenzenesulfonic acid and N,N-dimethyl-p-phenylenediamine as breakdown products of azo bond. The high decolorization potential of these bacterial strains individually and in consortia has potential application in remediation of dye effluent.     

Comparative evaluation of label-free SINQ normalized spectral index quantitation in the central proteomics facilities pipeline

Normalized spectral index quantification was recently presented as an accurate method of label‐free quantitation, which improved spectral counting by incorporating the intensities of peptide MS/MS fragment ions into the calculation of protein abundance. We present SINQ, a tool implementing this method within the framework of existing analysis software, our freely available central proteomics facilities pipeline (CPFP). We demonstrate, using data sets of protein standards acquired on a variety of mass spectrometers, that SINQ can rapidly provide useful estimates of the absolute quantity of proteins present in a medium‐complexity sample. In addition, relative quantitation of standard proteins spiked into a complex lysate background and run without pre‐fractionation produces accurate results at amounts above 1 fmol on column. We compare quantitation performance to various precursor intensity‐ and identification‐based methods, including the normalized spectral abundance factor (NSAF), exponentially modified protein abundance index (emPAI), MaxQuant, and Progenesis LC‐MS. We anticipate that the SINQ tool will be a useful asset for core facilities and individual laboratories that wish to produce quantitative MS data, but lack the necessary manpower to routinely support more complicated software workflows. SINQ is freely available to obtain and use as part of the central proteomics facilities pipeline, which is released under an open‐source license.     

Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine–serine-rich (RS) domains of SR- and SR-like proteins

The Fe(II) and 2-oxoglutarate dependent oxygenase Jmjd6 has been shown to hydroxylate lysine residues in the essential splice factor U2 auxiliary factor 65 kDa subunit (U2AF65) and to act as a modulator of alternative splicing. We describe further evidence for the role of Jmjd6 in the regulation of pre-mRNA processing including interactions of Jmjd6 with multiple arginine–serine-rich (RS)-domains of SR- and SR-related proteins including U2AF65, Luc7-like protein 3 (Luc7L3), SRSF11 and Acinus S′, but not with the bona fide RS-domain of SRSF1. The identified Jmjd6 target proteins are involved in different mRNA processing steps and play roles in exon dependent alternative splicing and exon definition. Moreover, we show that Jmjd6 modifies splicing of a constitutive splice reporter, binds RNA derived from the reporter plasmid and punctually co-localises with nascent RNA. We propose that Jmjd6 exerts its splice modulatory function by interacting with specific SR-related proteins during splicing in a RNA dependent manner.     

Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation

N-Glycosylation starts in the endoplasmic reticulum (ER) where a 14-sugar glycan composed of three glucoses, nine mannoses, and two N-acetylglucosamines (Glc(3)Man(9)GlcNAc(2)) is transferred to nascent proteins. The glucoses are sequentially trimmed by ER-resident glucosidases. The Glc(3)Man(9)GlcNAc(2) moiety is the substrate for oligosaccharyltransferase; the Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) intermediates are signals for glycoprotein folding and quality control in the calnexin/calreticulin cycle. Here, we report a novel membrane-anchored ER protein that is highly conserved in animals and that recognizes the Glc(2)-N-glycan. Structure determination by nuclear magnetic resonance showed that its luminal part is a carbohydrate binding domain that recognizes glucose oligomers. Carbohydrate microarray analyses revealed a uniquely selective binding to a Glc(2)-N-glycan probe. The localization, structure, and binding specificity of this protein, which we have named malectin, open the way to studies of its role in the genesis, processing and secretion of N-glycosylated proteins.     

The Conformational Properties of the Glc3Man Unit Suggest Conformational Biasing within the Chaperone-assisted Glycoprotein Folding Pathway

A major puzzle is: are all glycoproteins routed through the ER calnexin pathway irrespective of whether this is required for their correct folding? Calnexin recognizes the terminal Glcα1-3Manα linkage, formed by trimming of the Glcα1-2Glcα1-3Glcα1-3Manα (Glc₃Man) unit in Glc₃Man₉GlcNAc₂. Different conformations of this unit have been reported. We have addressed this problem by studying the conformation of a series of N-glycans; i.e. Glc₃ManOMe, Glc₃Man₄,₅,₇GlcNAc₂ and Glc₁Man₉GlcNAc₂ using 2D NMR NOESY, ROESY, T-ROESY and residual dipolar coupling experiments in a range of solvents, along with solution molecular dynamics simulations of Glc₃ManOMe. Our results show a single conformation for the Glcα1-2Glcα and Glcα1-3Glcα linkages, and a major (65%) and a minor (30%) conformer for the Glcα1-3Manα linkage. Modeling of the binding of Glc₁Man₉GlcNAc₂ to calnexin suggests that it is the minor conformer that is recognized by calnexin. This may be one of the mechanisms for controlling the rate of recruitment of proteins into the calnexin/calreticulin chaperone system and enabling proteins that do not require such assistance for folding to bypass the system. This is the first time evidence has been presented on glycoprotein folding that suggests the process may be optimized to balance the chaperone-assisted and chaperone-independent pathways.     

Production of an oligosaccharide-specific cellobiohydrolase from the thermophilic fungus <Emphasis Type="Italic">Thielavia terrestris</Emphasis>

Objectives

To express and determine the hydrolytic activity of a cellobiohydrolase (TTCBH6B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris.

Results

Ttcbh6B encodes a protein of 507 amino acid residues with a predicted molecular mass of 54 kDa. TTCBH6B contains a familial 6-glycosyl hydrolase catalytic domain and a type I carbohydrate-binding module. TTCBH6B was expressed and purified to homogeneity but the purified enzyme was inactive against Avicel. It could, however, digest Celluclast-treated Avicel producing cellobiose (0.27 μmol min^?1 mg^?1). To determine the substrate preferences of TTCBH6B, oligosaccharides of varying numbers of subunits were generated by acid hydrolysis of Avicel and fluorescently tagged. Peaks corresponding to oligosaccharides containing three to six glucose units were reduced to cellobiose after addition of TTCBH6B.

Conclusion

TTCBH6B is active against shorter oligosaccharides rather than polysaccharides.