• Journal of Applied Biological Chemistry
• /
• v.53 no.3
• /
• pp.132-138
• /
• 2010

A gene encoding a putative alanine racemase in B. pseudomycoides was cloned and expressed in Escherichia coli BL21(DE3) using a pET-21 vector harbouring 6xHistidine tag. Affinity purification of the recombinant alanine racemase with a nickel resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 46 kDa. The enzyme was the most active toward L-alanine and secondly D-alanine, implying that the enzyme is an alanine racemase. D-cysteine significantly inhibited the enzyme activity and also L-cysteine to a lesser extent. The enzyme was considerably activated by addition of pyridoxal-5'-phosphate (PLP), showing that 73% increase in activity was observed at 0.3 mM, compared to control. The enzyme was the most active at pH 9.0 and more stable at alkaline pHs than acidic pHs.

• Journal of Applied Biological Chemistry
• /
• v.53 no.4
• /
• pp.195-201
• /
• 2010

A gene encoding an alanine racemase in B. pseudomycoides was cloned and one (Cys316) or both of two cysteines (Cys316 and Cys365) was (were) substituted with alanine. The cysteine (-) alanine racemases were expressed in E. coli BL21 (DE3) using a pET-21 vector. The expressed enzymes were purified through affinity chromatography using 6xHis ligand. The purified enzymes all showed major one bands by SDS-PAGE analysis, corresponding to 46 kDa. The cysteine (-) alanine racemases as well as the wild type enzyme showed alanine racemase activities, indicating that the enzyme is an alanine racemase and the cysteines in the enzyme may not be involved in the catalysis and/or substrate binding. Thermal stabilities of Cys (-) alanine racemases decreased considerably and half-lives were 26 (wild type), 21 (C316A) and 18 min (C316-365A), respectively at $60^{\circ}C$ pH 8.0, suggesting that cysteine is considerably contributive to the thermal stability of the alanine racemase.

• Journal of Soil and Groundwater Environment
• /
• v.19 no.2
• /
• pp.38-43
• /
• 2014

In this study, we evaluated the methylene blue (MB) adsorption potential of non-treated and UV-C pretreated bacterial biomass from aqueous solution. The UV-C pretreatment denature the biomass and has increased overall functional groups when compared to non-treated biomass. The biosorbent was exposed to various pH, biomass dose, and contact time. The results showed that the dried and UV-C pretreated biomass effectively removed MB within 30 min. Dried and UV-C pretreated biomass of Bacillus pseudomycoides JH 2-2 showed a adsorption of 858.2 and 1072.4 mg/g at optimum conditions (pH: 9.0, contact time: 30 min, biomass dose: 1 g/L). Similarly, dried and UV-C pretreated biomass of Citrobacter freundii JH 11-2 showed an adsorption 868.3 and 954 mg/g at optimum conditions (pH: 9.0, contact time: 10 min, biomass dose: 1.5 g/L). The changes in the functional groups of UV-C pretreated biomass could be responsible for enhanced adsorption of MB. The results obtained have shown that non-treated and UV-C pretreated biomass has a high adsorption capacity for MB dye and can be used as a low-cost biosorbent in wastewater treatments.