One-step purification and characterization of an alkaline protease from haloalkaliphilic Bacillus sp.
An alkaline protease producer haloalkaliphilic bacteria (isolate Vel) was isolated from west coast of India. It was related to Bacillus pseudofirmus on the basis of 16S r RNA gene sequencing, lipid profile and other biochemical properties. The protease secreted by this bacteria was purified 10-fold with 82% yield by a single step method on Phenyl Sepharose 6 Fast Flow column. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 29 000 Da. The Km and Vmax towards caseinolytic activity were found to be 2 mg ml(-1) and 289.8 microg min(-1), respectively. The enzyme was active over the pH range of 8.5-12.0, the optimum being 10-11.0. The purified enzyme when kept at 45 degrees C and 50 degrees C for 40 min retained 92% and 85% protease activity, respectively. Effect of NaCl concentration on protease activity showed that the enzyme was slightly inhibited with high concentration of salt. The proteolytic activity was inhibited by PMSF, suggesting that the enzyme may belong to serine type protease. Interestingly, the activity was slightly enhanced with SDS (0.1%) and Triton X-100 (0.1%) but remained unaffected by Tween 80 (0.1%). The activity was affected by metal ions to varying extent. While Mn2+, Zn2+ and Mg2+ had no significant effect on protease activity, the enzyme was activated with Ca2+ (1 mM) and Cu2+ (5 mM). The stability of the enzyme in the presence of detergent components and surfactants is particularly attractive for its application in detergent industries.
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Anshu
A
Gupta
Ipsita
I
Roy
R K
RK
Patel
S P
SP
Singh
S K
SK
Khare
M N
MN
Gupta
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D013696:Temperature
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010450:Endopeptidases; C087822:alkaline protease