Optimal pyoverdin-CPG composites for development of an optical biosensor to detect iron

Optical fluorescence-quenching-based biosensing cell is described and optimization of covalent binding of highly selective natural iron-chelating peptide secreted by bacteria is suggested. Pyoverdin biosynthesized by Pseudomonas monteilii and having 70% iron chelating activity was immobilized on amino alkylated controlled pore glass (CPG) and cross-linked with glutaraldehyde (2.5%, 28°C, 30 min). The pyoverdin-CPG immobilization was confirmed using fluorescence microscopic images (excitation range, 465–495 nm) for bright green fluorescence and by FTIR spectrum stretching at 3406.4 cm⁻¹ for amino group. The pyoverdin loading capacity of activated CPG matrix was 25 mg g⁻¹ of CPG and its rinsing analysis (leaking profile of the immobilized peptide vs. washing) detected negligible (2–3 μg) pyoverdin in the second wash.     

Detergent-compatible,organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: Purification and characterization

Proteases are now recognized as the most indispensable industrial biocatalyst owing to their diverse microbial sources and innovative applications. In the present investigation, a thermostable, organic solvent-tolerant, alkaline serine protease from Bacillus circulans MTCC 7942, was purified and characterized. The protease was purified to 37-fold by a three-step purification scheme with 39% recovery. The optimum pH and temperature for protease was 10 and 60°C, respectively. The apparent molecular mass of the purified enzyme was 43 kD as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The K_m and V_max values using casein-substrate were 3.1 mg/mL and 1.8 µmol/min, respectively. The protease remained stable in the presence of organic solvents with higher (>3.2) log P value (cyclohexane, n-octane, n-hexadecane, n-decane, and n-dodecane), as compared to organic solvents with lower (<3.2) log P value (acetone, butanol, benzene, chloroform, toluene). Remarkably, the protease showed profound stability even in the presence of organic solvents with less log P values (glycerol, dimethyl sulfate [DMSO], p-xylene), indicating the possibility of nonaqueous enzymatic applications. Also, protease activity was improved in the presence of metal ions (Ca²⁺, Mg²⁺, Mn²⁺); enhanced by biosurfactants; hardly affected by bleaching agents, oxidizing agents, and chemical surfactants; and stable in commercial detergents. In addition, a protease–detergent formulation effectively washed out egg and blood stains as compared to detergent alone. The protease was suitable for various commercial applications like processing of gelatinous film and as a compatible additive to detergent formulation with its operative utility in hard water.