Shafinaz Shahir

Biodegradation of 4-aminobenzenesulfonate by Ralstonia sp. PBA and Hydrogenophaga sp. PBC isolated from textile wastewater treatment plant

A co-culture consisting of Hydrogenophaga sp. PBC and Ralstonia sp. PBA, isolated from textile wastewater treatment plant could tolerate up to 100 mM 4-aminobenzenesulfonate (4-ABS) and utilize it as sole carbon, nitrogen and sulfur source under aerobic condition. The biodegradation of 4-ABS resulted in the release of nitrogen and sulfur in the form of ammonium and sulfate respectively. Ninety-eight percent removal of chemical oxygen demand attributed to 20 mM of 4-ABS in cell-free supernatant could be achieved after 118 h. Effective biodegradation of 4-ABS occurred at pH ranging from 6 to 8. During batch culture with 4-ABS as sole carbon and nitrogen source, the ratio of strain PBA to PBC was dynamic and a critical concentration of strain PBA has to be reached in order to enable effective biodegradation of 4-ABS. Haldane inhibition model was used to fit the degradation rate at different initial concentrations and the parameters μ(max), K(s) and K(i) were determined to be 0.13 h⁻¹, 1.3 mM and 42 mM respectively. HPLC analyses revealed traced accumulation of 4-sulfocatechol and at least four unidentified metabolites during biodegradation. This is the first study to report on the characterization of 4-ABS-degrading bacterial consortium that was isolated from textile wastewater treatment plant.

Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase

The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, kcat and kcat/Km higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.

Identification of genes involved in the 4-aminobenzenesulfonate degradation pathway of Hydrogenophaga sp. PBC via transposon mutagenesis

Genes involved in the 4-aminobenzenesulfonate (4-ABS) degradation pathway of Hydrogenophaga sp. PBC were identified using transposon mutagenesis. The screening of 10,000 mutants for incomplete 4-ABS biotransformation identified four mutants with single transposon insertion. Genes with insertions that impaired the ability to utilize 4-ABS for growth included (1) 4-sulfocatechol 1,2-dioxygenase β-subunit (pcaH2) and 3-sulfomuconate cycloisomerase involved in the modified β-ketoadipate pathway; (2) 4-aminobenzenesulfonate 3,4-dioxygenase component (sadA) involved in aromatic ring hydroxylation; and (3) transposase gene homolog with a putative cis-diol dehydrogenase gene located downstream. The pcaH2 mutant strain accumulated brown metabolite during growth on 4-ABS which was identified as 4-sulfocatechol through thin layer chromatography and HPLC analyses. Supplementation of wild-type sadA gene in trans restored the 4-ABS degradation ability of the sadA mutant, thus supporting the annotation of its disrupted gene.

Cloning and functional analysis of the genes coding for 4-aminobenzenesulfonate 3,4-dioxygenase from Hydrogenophaga sp. PBC.

The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.

Determination of tamoxifen and its metabolites in human plasma by nonaqueous capillary electrophoresis with contactless conductivity detection

A new approach for the quantification of tamoxifen and its metabolites 4‐hydroxytamoxifen, N‐desmethyltamoxifen, and 4‐hydroxy‐N‐desmethyltamoxifen (endoxifen) in human plasma samples using NACE coupled with contactless conductivity detection (C4D) is presented. The buffer system employed consisted of 7.5 mM deoxycholic acid sodium salt, 15 mM acetic acid, and 1 mM 18‐crown‐6 in 100% methanol. The complete separation of all targeted compounds (including endoxifen racemate) could be achieved within 6 min under optimized conditions. The proposed method was validated and showed good linearity in the range from 100 to 5000 ng/mL with correlation coefficients between 0.9922 and 0.9973, LODs in the range of 25–40 ng/mL, and acceptable reproducibility of the peak area (intraday RSD 2.2–3.1%, n = 4; interday (3 days) RSD 6.0–8.8%, n = 4). The developed method was successfully demonstrated for the quantification of tamoxifen and its metabolites in human plasma samples collected from breast cancer patients undertaking tamoxifen treatment.

Draft Genome Sequence of Arsenic-Resistant Microbacterium sp. Strain SZ1 Isolated from Arsenic-Bearing Gold Ores

ABSTRACT Microbacterium sp. strain SZ1 isolated from gold ores of a Malaysia gold mine was found to be highly resistant to arsenic. Here, we report the draft genome sequence of SZ1, which may provide further insights into understanding its arsenic resistance mechanism. In this draft genome, a complete set of ars operons and two additional scattered ars genes were encoded.

Covalent Immobilization of Tyrosinase onto Multi-walled Carbon Nanotubes and Its Potential Use In Phenol Biosensing

The possibility of modifying the surface properties of multi-walled carbon nanotubes (MWCNTs) has stimulated increasing interest in their application as components in biosensors. In this sense, it is possible to employ surface modified MWCNTs as support to immobilize biomaterials such as enzymes. In this study the enzyme tyrosinase was immobilized onto functionalized MWCNTs (fMWCNTs) via covalent bonding and activity of immobilized tyrosinase was measured via electrochemical detection of phenol. MWCNTs were first treated with sulphuric acid and nitric acid with ratio 1 : 3 at 70oC to introduce carboxylated groups (-COOH). The carboxyl moieties were then activated by treatment with a cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to enable tyrosinase immobilization via amide bonding. FTIR spectra of tyrosinase immobilized fMWCNTs showed the presence of peaks attributing to aliphatic C-N (1382 cm-1) and amide carbonyl (1639 cm-1) vibrations which confirmed successful covalent immobilization of tyrosinase onto fMWCNTs. Electrochemical measurements using tyrosinase-fMWCNTS-CPE revealed increasing limiting current values of reduction peak with increasing phenol concentrations at -200mV. The appearance of the reduction current indicates that the immobilization process retained the biological activity of the covalently bonded tyrosinase on fMWCNTs surface. This study has demonstrated the potential of using MWCNTs as support for enzyme immobilization and their application in biosensor technology.

Covalent immobilization of tyrosinase onto commercial multi-walled carbon nanotubes and its effect on enzymatic activity

Multi-walled carbon nanotubes (MWCNTs) exhibit unique structural, electrical, mechanical, electrochemical, and chemical properties [1]. Moreover, the possibility of modifying their surface properties through different methods has stimulated increasing interest in their application as components in biosensors. In this sense, it is possible to employ carbon nanotubes as support to immobilize enzymes.