Optimization of parameters that affect the activity of the alkaline protease from halotolerant bacterium, bacillus acquimaris VITP4, by the application of response surface methodology and evaluation of the storage stability of the enzyme

Background: It was previously shown that the activity of a serine protease from a moderately halotolerant Bacillus aquimaris VITP4 strain is active in a wide range of pH and temperatures and could be modulated by the presence of the divalent metal ions

Objectives: In the present study, a quantitative analysis was done in order to explore the parameters that are contributing to the protease activity

Materials and Methods: Changes in the secondary structure of the enzyme was determined by circular dichroism analysis. The conditions for the optimal activity was investigated by Response Surface Methodology. Stability of the enzyme was determined by thermal inactivation experiments

Results: The initial one-factor-at-a-time experiments have indicated that the activity of the enzyme could be enhanced not only by the presence of low concentrations of NaCl but also by divalent metal ions, such as Ca[2+], Mn[2+] and Cu[2+]. A clear dependence of the activity to the secondary structure of the enzyme could be established using circular dichroism spectroscopy. In the next level of optimization, four factors; viz. pH, temperature, concentration of Ca[2+], and Mn[2+] were used to optimize the conditions required for the maximal activity of the enzyme by Response Surface Methodology, and the data could be explained using quadratic model. Under optimal condition of 43[degree]C, pH 8.0, 8.2 mM Ca[2+], and 4.3 mM Mn[2+] a 1.5 times enhancement in the enzyme activity could be achieved. The storage stability of the enzyme under these selected conditions has indicated a non-linear relation between the conditions for the enzymatic activity as well as stability. However, the condition for the maximal stability [267+/-18 min] has corresponded to that of the optimal conditions for the maximal activity

Conclusions: This study, for the first time, has explored the possibility of using statistical methods for identifying the optimal conditions for alkaline protease activity isolated from the halotolerant Bacillus aquimaris VITP4

Isolation and optimization of poly β hydroxybutyrate producing cyanobacterial strains.

Cyanobacteria have many unexploited potential for natural products with a huge variability in structure and biological activity. Under stress conditions they are reported to produce biopolymers like poly-β-hydroxybutyrate (PHB), which can be produced intracellularly.Cyanobacteria are capable of synthesizing small amount of poly-β-hydroxybutyrate (PHB) under nitrogen and phosphorous starvation conditions. High performance liquid chromatography (HPLC) analysis revealed that about percentage PHB of the cell dry weight (CDW) was accumulated under mixotrophic culture condition. However, Nile red stained cells showed the presence of large quantities of granules in the cell cytoplasm when viewed under fluorescent microscope. The qualitative observation was in contrast to the quantitative HPLC analysis which suggested that the fluorescent granules are PHB. Among the ten different isolates, three strains showed the accumulation of PHB. The amount of PHB produced after 10 days in the depleted conditions are 1.182 mg/l for spirulina, 2.322 mg/l for anabena and 3.741 mg/l. The maximum PHB producer was further studied in detail. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis. The present study shows increased PHB accumulation in different species by nitrogen and phosphorous depleted condition and pH concentration in the growth media.