Two steps purification, biochemical characterization, thermodynamics and structure elucidation of thermostable alkaline serine protease from Nocardiopsis alba strain OM-5.

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50-80 °C were in the range of 2.50 × 10-3 to 5.50 × 10-3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased ß-sheets content.

Biochemical, thermodynamic and structural characteristics of a biotechnologically compatible alkaline protease from a haloalkaliphilic, Nocardiopsis dassonvillei OK-18.

This report describes purification strategies, biochemical properties and thermodynamic analysis of an alkaline serine protease from a marine actinomycete, Nocardiopsis dassonvillei strain OK-18. The solvent tolerance, broad thermal-pH stability, favourable kinetics and thermodynamics suggest stability of the enzymatic reaction. The enzyme was active in the range of pH 7-12 and 37-90 °C, optimally at pH 9 and 70 °C. The deactivation rate constant (Kd), half-life (t½), enthalpy (ΔH*), entropy (ΔS*), activation energy (E) and change in free energy (ΔG*) suggested stability and spontaneity of the reaction. ß-Sheets as revealed by the Circular dichroism (CD) spectroscopy, were the major elements in the secondary structure of the enzyme, while Fourier-transform infrared spectroscopy (FTIR) indicated the presence of amide I and amide II. Based on the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis, the amino acid sequence had only 38% similarity with other proteases of Nocardiopsis strains, suggesting its novelty. The Ramachandran Plot revealed the location of the amino acid residues in the most favored region. The blood de-staining, gelatin hydrolysis, silver recovery and deproteinization of crab shells established the biotechnological potential of the enzyme.