1. Comparative Proteomics of Intracellular and Extracellular Proteins from Bacillus subtilis and Bacillus licheniformis
Adeyanju M. M.1, Somiari R. I.2, Russell S.2, Mayko F.2, Daini O.A.1 and Adebawo, O.O.1
1Department of Biochemistry, Obafemi Awolowo College of Health Sciences,
Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria.
2 ITSI-Biosciences, Johnstown Pennsylvania, United States of America.
INTRODUCTION
The capacity of selected Bacillus strains to produce and secrete large quantities of extracellular enzymes has placed them among the most important industrial enzyme producers (Schallmey et al., 2004). However, environmental factors influence the set of proteins produced by members of this group. While extensive studies have been reported on this genus, not much information is available on the local strains in Nigeria despite its industrial potentials. In this study we compared using proteomics technique the intracellular and extracellular proteins from Bacillus subtilis and B. licheniformis. This was with a view to identifying important industrial proteins and the optimum temperature of production as well as the versatility of the organisms.
OBJECTIVES
Isolation and identification of the major proteins present in B. subtilis and B. licheniformis
Quantifying the relative abundance of the selected proteins in the two bacteria
Determining the organism with higher versatility out of B. subtilis and B. licheniformis
CONCLUSION
In conclusion, the B. subtilis and B. licheniformis isolates investigated in the present study are veritable strains that could be used as starter culture for the production of important industrial proteins. The B. licheniformis holds promise as a more versatile strain given the variety and quantity of proteins produced.
RESULTS
The results of the intracellular and extracellular protein concentrations revealed the mean values of 96.24±0.28 and 53.13±0.16 μg/mL for B. subtilis at 37oC while ±0.20 and 82.78±0.10 μg/mL were obtained for B. licheniformis at same temperature. The intracellular and extracellular protein concentrations for B. licheniformis at 50oC were observed to be ±0.24 and 33.35±0.22 μg/mL respectively. The results revealed that the values of protein concentrations for B. licheniformis were higher than those of Bacillus subtilis at 37oC as presented in Table 1.
From the mass spectrometry spectrum and database search, the identified proteins are as presented in Tables 3 to 5.
METHODOLOGY
Reactivation and Sub-culturing of Bacillus licheniformis and Bacillus subtilis
Extraction and Precipitation of Proteins from the organisms
Total Protein Concentration Determination employing the method of Bradford (1976)
Separation of Intracellular Proteins by 2D-DIGE
Protein Expression Analysis using 2D-DeCyder Software
Scanning Using Typhoon Variable Mode Imager
Separation of Extracellular Proteins by 1D SDS-PAGE
In-gel Tryptic Digestion and Extraction of Proteins following the method of Shevchenko, Wilm, Vorm and Mann (1996).
Separation of peptide fragments using Linear Trapped Quadrupole (LTQ XL) Mass Spectrometer
Protein Identification by species-specific databases for Bacillus licheniformis and Bacillus subtilis downloaded from National Center for Biotechnology Information (NCBI) using the Proteome Discoverer (1.3) and SEQUEST algorithm
Assay of α-amylase in the Intracellular and extracellular Proteomes by the modified method of Bernfeld (1955).
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ACKNOLEDGEMENT
MMA thanks the Federal Government of Nigeria under the Tertiary Education Trust Fund (TETFUND) for the funding of this work.