Cloning, expression, purification and characterisation of serine alkaline protease from Bacillus subtilis RD7
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Date
2019-07-24
Authors
Akande, Idowu
Samuel, Titilola
Lawal, Adekunle
Olaniran, Ademola
Journal Title
Journal ISSN
Volume Title
Publisher
Elsevier
Abstract
Proteases (E.C 3.4) are enzymes that break peptide bonds between amino acid groups of proteins. Bacillus subtilis
RD7, an extracellular protease producer was genetically characterised, B. subtilis genomic DNA was isolated,
oligonucleotide primers specific to serine alkaline protease gene of B. subtilis were designed and its PCR amplification
was done. The purified PCR product and pET15b vector were subjected to restriction digestion with
Nde1 and BamH1 and transformed into Escherichia coli DH5α competent cells. The recombinant expression of
serine alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the
serine alkaline protease protein had an estimated molecular size of 43 kDa. Gene sequencing of the insert from
selected recombinant clone showed it to be a 1203bp gene encoding a protein of 400 amino acids. The sequence
was blasted and aligned with known serine alkaline protease genes for comparison with their nucleotide and
amino acid sequences. This identified major matches with closely related subsp. of B. subtilis. The insert also
showed many substitutions (mutations with other sp. of Bacillus which established that serine alkaline protease
of B. subtilis RD7 is a novel gene. The phylogenetic analysis of serine alkaline protease gene and its predicted
amino acid sequences also validated that serine alkaline protease gene is a novel gene and has been accessioned
in GenBank with accession number (MN097797). When expressed in E. coli, the recombinant enzyme was over
expressed in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by PMSF.
The enzyme showed maximum activity at pH 10 and 40 °C. It was stable at pH from 6 to 11 and below 70 °C. The
aim of this study is to clone serine alkaline protease gene from B. subtilis RD7 (MG255317), its expression in
mesophilic E. coli strain BL21, Purification and characterisation of the expressed protein.
Description
Keywords
Research Subject Categories::NATURAL SCIENCES::Chemistry::Biochemistry , Cloning , Bacillus , Serine alkaline protease , Phylogenetic analysis , Sequencing
Citation
Suberu Yewande, Akande Idowu, Samuel Titilola, Lawal Adekunle, Olaniran Ademola. Biocatalysis and Agricultural Biotechnology 20 (2019) 101264