title: MALDI-TOF MS and CD spectral analysis for identification and structure prediction of a purified, novel, organic solvent stable, fibrinolytic metalloprotease from Bacillus cereus B80.
abstract: The ability to predict protein function from structure is becoming increasingly important; hence, elucidation and determination of protein structure become the major steps in proteomics. The present study was undertaken for identification of metalloprotease produced by Bacillus cereus B80 and recognition of characteristics that can be industrially exploited. The enzyme was purified in three steps combining precipitation and chromatographic methods resulting in 33.5% recovery with 13.1-fold purification of enzyme which was detected as a single band with a molecular mass of 26 kDa approximately in SDS-PAGE and zymogram. The MALDI-TOF MS showed that the enzyme exhibited 70-93% similarity with zinc metalloproteases from various strains Bacillus sp. specifically from Bacillus cereus group. The sequence alignment revealed the presence of zinc-binding region VVVHEMCHMV in the most conserved C terminus region. Secondary structure of the enzyme was obtained by CD spectra and I-TASSER. The enzyme kinetics revealed a Michaelis constant (Km) of 0.140 μmol/ml and Vmax of 2.11 μmol/min. The application studies showed that the enzyme was able to hydrolyze various proteins with highest affinity towards casein followed by BSA and gelatin. The enzyme exhibited strong fibrinolytic, collagenolytic, and gelatinolytic properties and stability in various organic solvents.
authors: Amity Institute of Microbial Biotechnology, Amity University, Sector 125, Noida, Uttar Pradesh 201303, India.
authors: Rajshree
authors: R
authors: Saxena
authors: Amity Institute of Microbial Biotechnology, Amity University, Sector 125, Noida, Uttar Pradesh 201303, India.
authors: Rajni
authors: 0000-0002-2862-7011
authors: R
authors: Singh
mesh_terms: D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism; D002942:Circular Dichroism / Q000379:methods*; D003094:Collagen / Q000378:metabolism; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005342:Fibrinolysis; D005780:Gelatin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D012997:Solvents / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D013997:Time Factors
chemical_list: D002364:Caseins; D009930:Organic Chemicals; D010455:Peptides; D012997:Solvents; D012965:Sodium Chloride; D005780:Gelatin; D003094:Collagen; D045726:Metalloproteases