Munir A. Anwar

Partial and complete alteration of surface charges of carboxymethylcellulase by chemical modification: thermostabilization in water-miscible organic solvent

Abstract Carboxymethylcellulase was purified from Aspergillus niger to homogeneity level. The native and subunit molecular weights were found to be 42 and 45 kDa, respectively. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of dimethylamine hydrochloride (DIMAM) and ethylenediamine dihydrochloride (EDAM) as nucleophiles. The amino groups of DIMAM were further modified by acetic anhydride for the complete elimination of surface charges (double modification, DM). The specificity constants (V max /K m ) of EDAM (1.1), DIMAM (1.2) and DM-A (1.0) were increased as compared with native enzyme (0.16). Partial neutralization of surface negative charges (DIMAM), reversal of surface negative charges (EDAM) and complete neutralization of negative plus positive charges (DM-A), of CMCase had a drastic effect on the thermostability determined in the aqueous buffer, pH 5.2. Gibbs activation free energies of denaturation ( ΔG* ) of native, EDAM, DIMAM and DM-A at 80°C were 110, 107, 102 and 103 kJ mol −1 , respectively, whereas enthalpy of denaturation ( ΔH* ) of native, EDAM, DIMAM and DM-A at 80°C were 143, 144, 213 and 197 kJ mol −1 , respectively. The entropies of denaturation (ΔS*) of native, EDAM, DIMAM and DM-A at 80°C were 91, 105, 316 and 265 Jmol −1 K -1 , respectively, indicating highly disordered conformations of all the transition states of modified CMCases. On the other hand, the thermostability of doubly modified CMCase in 50% (v/v) aqueous dioxan (DM-D) was dramatically increased with concomitant activation with increasing temperatures up to 95°C as compared with the native CMCase under similar conditions. The analysis of thermodynamic parameters revealed that ΔS* of denaturation became negative implying a highly ordered transition state of DM-D in the presence of a solvent of higher hydrophobicity. We wish to propose that at least monomeric enzymes could be made significantly more thermostable in water-miscible organic solvents by totally converting the charged surface groups to non-polar ones by double modification .

Analysis of the dibenzothiophene metabolic pathway in a newly isolated Rhodococcus spp.

Out of 17 samples collected from diverse environments, 110 bacterial isolates of varied characteristics were screened for their dibenzothiophene-desulphurizing activity. A single isolate, Eu-32, originating from a soil sample taken from the roots of a eucalyptus tree, displayed dibenzothiophene-desulphurizing activity. This isolate metabolized dibenzothiophene to 2-hydroxybiphenyl (2-HBP), as detected by HPLC, and was also able to use other organic sulphur compounds as a sole sulphur source. Based on morphological, biochemical and molecular studies, it was found that the organism belongs to the genus Rhodococcus, with a maximum of 95% identity to species in this genus for the partial sequence of the 16S rRNA gene. Isolate Eu-32 could desulphurize 0.2 mM dibenzothiophene to 2-HBP in 72 h at a temperature of 30 degrees C and pH 7.0. The structure and molecular mass of metabolites produced from dibenzothiophene desulphurization were identified by GC-MS, and two sulphur-free products, 2-HBP and biphenyl, were detected in ethyl acetate extract. It was concluded that isolate Eu-32 is a unique desulphurizing biocatalyst that desulphurizes dibenzothiophene through an extended, sulphur-specific degradation pathway with the selective cleavage of C-S bonds.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of Pakistan and their industrial importance

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99% 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97%, 98% and 99% 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.

Technical communication: Determination of cuprous ions in bacterial leachates and for environmental monitoring

A sensitive and precise spectrophotometric method has been developed for the determination of copper(I) in bacterial leach liquors produced by the action of Thiobacillus ferrooxidans and T. thiooxidans on copper ores. In this method bicinchoninic acid (BCA) has been used as the chromogenic reagent which produces a stable purple complex with Cu(I) which was found to obey Beers Law and with λmax at 560 nm. The coloured complex has a molar extinction coefficient (ε) value of 6.6 × 103 l mol−1 cm−1; specific absorptivity (α) value of 0.104 ml−1 g cm−1 and the Sandell sensitivity (S) value was 0.0096 μg cm2. Optimal conditions for development of coloration/sensitivity were determined. Interferences due to cations and anions were investigated and various masking agents for alleviating their inhibition were studied. The method has been found very useful in determining ratios of Cu(I) to Cu(II) in bacterial leach liquors and should play a significant role in determining the reaction mechanisms of biological leaching and for environmental monitoring.