Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp

• Published in: Process biochemistry (1991) 2006-09, Vol.41 (9), p.2002-2009
• Main Authors: Patel, Rajesh K., Dodia, Mital S., Joshi, Rupal H., Singh, Satya P.
• Format: Article
• Language: English
• Subjects: Alkaline protease; Bacillus sp; Haloalkaliphiles; Protein denaturation; Protein folding; Salt tolerance
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2006.04.016

An extracellular alkaline protease from a novel haloalkaliphilic bacterium (Ve1) was purified to the homogeneity, with a molecular weight of 30–32 kDa. The bacterium was related to Bacillus pseudofirmus on the basis of 16S rRNA gene sequencing. The enzyme was active in the range of pH 8.5–12 with the optimum at 10–11. The requirement of the salt for enzyme catalysis was increased on increasing temperature and a shift in temperature optima from 37 to 55 °C was evident in the presence of 2% salt. The enzyme was highly stable at 37 °C and retained 50% activity at 45 °C. However, the enzyme was quite unstable at temperatures beyond 55 °C. However, the enzyme was quite stable at higher temperature in the presence of NaCl and CaCl 2. The enzyme was stable with different surfactants; in-fact, SDS and Triton X-100 were slightly stimulatory. The activity was affected by mono and divalent cations to varying extent. The protease was sensitive to urea denaturation and the renaturation of denatured protein under in vitro conditions was affected by different factors. While protein concentration played pronounced role in the renaturation by dialysis (89% renaturation); pH, salt and redox conditions did not affect the renaturation significantly. The study on this enzyme assumes significance in the light of dual extremities of pH and salt coupled with moderate temperature stability.