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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.

Saxena R, Singh R - Biomed Res Int (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Amity Institute of Microbial Biotechnology, Amity University, Sector 125, Noida, Uttar Pradesh 201303, India.

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.

No MeSH data available.


Related in: MedlinePlus

(a) Peptide mass spectra of the tryptic digested peptides as obtained from MALDI TOF mass spectrometry. (b) Annotated MS/MS spectra of fragmentation of 5 peptides in MS/MS that produce mostly y and b ions. The parent m/z values and the sequence of the identified peptides are indicated.
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fig3: (a) Peptide mass spectra of the tryptic digested peptides as obtained from MALDI TOF mass spectrometry. (b) Annotated MS/MS spectra of fragmentation of 5 peptides in MS/MS that produce mostly y and b ions. The parent m/z values and the sequence of the identified peptides are indicated.

Mentions: The MALDI TOF MS analysis of the tryptic digested enzyme generated a spectrum of peaks representing the m/z ratio of each peptide fragment (Figure 3(a)). Automated analysis of the spectra was performed by software Protein Prospector Auto MS-Fit. Five major peptides were further analyzed on nanospray TOFMS/MS which fragmented the peptides into y and b ions, indicating the best matching peptide sequences (Figure 3(b)).


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.

Saxena R, Singh R - Biomed Res Int (2015)

(a) Peptide mass spectra of the tryptic digested peptides as obtained from MALDI TOF mass spectrometry. (b) Annotated MS/MS spectra of fragmentation of 5 peptides in MS/MS that produce mostly y and b ions. The parent m/z values and the sequence of the identified peptides are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4352737&req=5

fig3: (a) Peptide mass spectra of the tryptic digested peptides as obtained from MALDI TOF mass spectrometry. (b) Annotated MS/MS spectra of fragmentation of 5 peptides in MS/MS that produce mostly y and b ions. The parent m/z values and the sequence of the identified peptides are indicated.
Mentions: The MALDI TOF MS analysis of the tryptic digested enzyme generated a spectrum of peaks representing the m/z ratio of each peptide fragment (Figure 3(a)). Automated analysis of the spectra was performed by software Protein Prospector Auto MS-Fit. Five major peptides were further analyzed on nanospray TOFMS/MS which fragmented the peptides into y and b ions, indicating the best matching peptide sequences (Figure 3(b)).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Amity Institute of Microbial Biotechnology, Amity University, Sector 125, Noida, Uttar Pradesh 201303, India.

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.

No MeSH data available.


Related in: MedlinePlus