Nandan Singh Darmwal

Simultaneous production of alkaline lipase and protease by antibiotic and heavy metal tolerant Pseudomonas aeruginosa

An efficient bacterial strain capable of simultaneous production of lipase and protease in a single production medium was isolated. Thirty six bacterial strains, isolated from diverse habitats, were screened for their lipolytic and proteolytic activity. Of these, only one bacterial strain was found to be lipase and protease producer. The 16S rDNA sequencing and phylogenetic analyses revealed that strain (NSD‐09) was in close identity to Pseudomonas aeruginosa. The maximum lipase (221.4u2009U/ml) and protease (187.9u2009U/ml) activities were obtained after 28 and 24u2009h of incubation, respectively at pH 9.0 and 37u2009°C. Castor oil and wheat bran were found to be the best substrate for lipase and protease production, respectively. The strain also exhibited high tolerance to lead (1450u2009µg/ml) and chromium (1000u2009µg/ml) in agar plates. It also showed tolerance to other heavy metals, such as Co+2, Zn+2, Hg+2, Ni+2 and Cd+2. Therefore, this strain has scope for tailing bioremediation. Presumably, this is the first attempt on P. aeruginosa to explore its potential for both industrial and environmental applications.

Oxidant and solvent stable alkaline protease from Aspergillus flavus and its characterization

The increase in agricultural practices has necessitated the judicious use of agricultural wastes into value added products. In this study, an extracellular, organic solvent and oxidant stable, serine protease was produced by Aspergillus flavus MTCC 9952 under solid state fermentation. Maximum protease yield was obtained when the strain was grown under wheat bran and corn cob mixture (1:1) incubated for 48 h at pH 9.0 and temperature 37°C with 50% of initial moisture content. The partially purified enzyme showed wide range of pH optima (8.0-12.0) and pH stability (7.0-12.0), whereas, optimum temperature was 40°C and was stable over a wide range of temperature 30-45°C. The protease was extremely stable towards several organic solvents. The enzyme retained 80% of its original activity in the presence of non ionic and ionic surfactants and 100% with 10% H 2O2 after 1 h of incubation at 30°C. In addition, the enzyme showed excellent compatibility with some commercial powder detergents. The compatibility of our protease with several detergents, oxidants and organic solvents suggests its possible use in detergent industry and peptide synthesis.

An oxidant and organic solvent tolerant alkaline lipase by P. aeruginosa mutant: downstream processing and biochemical characterization

An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45°C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0–11.0 and temperature range 35–55 °C. The metal ions Ca2+, Mg2+ and Na+ tend to increase the enzyme activity, whereas, Fe2+ and Mn2+ ions resulted in discreet decrease in the activity. Divalent cations Ca+2 and Mg+2 seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca+2 (5 mM) the optimum temperature shifted from 45°C to 55°C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40–70% of its original activities. The values of Km and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL−1 and 5000 μg.mL−1.min−1, respectively.

Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L−1; starch, 15.0 g.L−1; triton-X-100, 0.93 mL.L−1; incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL−1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R2) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.