Mass Spectrometric Determination of the Primary Structure of β Amylase Isolated from Amylolytic Nigerian Maize Cultivar
O. A. Awoyinka *
Department of Medical Biochemistry, College of Medicine, Ekiti State University Ado Ekiti, Nigeria
O. Daini
Kampala International University, Western Campus Ishaka Kampala, Uganda
K. Satisha
School of Molecular and Biomedical Sciences, The University of Adelaide, Australia
Dipankar Chatterji
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 56000-12, India
*Author to whom correspondence should be addressed.
Abstract
Mass Spectrometric Determination of the Primary Structure of β Amylase Isolated from Amylolytic Nigerian Maize Cultivar
This study was carried out after a five day germination period on TZEE*TZEE-W*DEMARSCUS*TZEE-W one of the most recommended high amylolytic Nigerian maize cultivar. Purification steps comprising of fractional precipitation by ammonium sulphate, gel filtration and anion exchange chromatography, was used respectively to purify β amylase (EC 3.2.1.2) from the malt. The sensitivity of the beta amylase indicated that it has serine at its active site. An apparent 60KDa monomeric protein was detected by one dimensional sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). Identity assigned to the purified protein by Matrix-assisted laser desorption ionization time –of- flight mass spectrometry ( MALDI-ToF) reference to electronic protein data base is a 58542Da high putative beta amylase – Q9AV88-ORSA from Oryza sativa. Complete primary structure thereafter deduced with the aid of MS/MS MALDI- ToF showed a signature of a highly conserved ubiquitous not yet reported beta amylase. This study paved an insight to the gene encoding the β amylase in TZEE*TZEE-W*DEMARSCUS*TZEE-W and better understanding of the activity of the enzyme at molecular level.
Keywords: β-amylase, maize, barley, MALDI-TOF-MS, SDS-PAGE