Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum.
An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis. The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH8.0 and 60°C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry.
Department of Microbiology, DAV University, Jalandhar, Punjab144012, India. Electronic address: shg1988@gmail.com.
Shiwani
S
Guleria
Department of Microbiology, DAV University, Jalandhar, Punjab144012, India. Electronic address: sunny_0999walia@yahoo.co.in.
Abhishek
A
Walia
Department of Basic Sciences (Microbiology Section), Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan 173230 (H.P.), India. Electronic address: anjali_chauhan22@yahoo.co.in.
Anjali
A
Chauhan
Department of Basic Sciences (Microbiology Section), Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan 173230 (H.P.), India. Electronic address: profckshirkot@gmail.com.
C K
CK
Shirkot
D000595:Amino Acid Sequence; D000069977:Bacillus amyloliquefaciens / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D061046:Biological Control Agents / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000254:growth & development*; D011480:Protease Inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D013381:Subtilisins / Q000235:genetics
D001426:Bacterial Proteins; D061046:Biological Control Agents; D004269:DNA, Bacterial; D011480:Protease Inhibitors; D010450:Endopeptidases; D013381:Subtilisins; C087822:alkaline protease
Bacillus amyloliquefaciens SP1; Biocontrol; Cloning; Fusarium oxysporum; Protease; Structure Prediction