EVALUATION OF ACTIVITY AND MASS SPECTROMETRIC CHARACTERIZATION OF TWO PARTIALLY PURIFIED THERMO-TOLERANT LIPASE FRACTIONS FROM THE FUNGAL CULTURE OF Aspergillus niger GN1
KOTA SOBHA *
Department of Chemical Engineering, RVR and JC College of Engineering, Chowdavaram, Guntur 522 019, Andhra Pradesh, India.
DUMPALA PRADEEP
Department of Chemical Engineering, RVR and JC College of Engineering, Chowdavaram, Guntur 522 019, Andhra Pradesh, India.
GOLLAPUDI GEETHANJALI
Department of Chemical Engineering, RVR and JC College of Engineering, Chowdavaram, Guntur 522 019, Andhra Pradesh, India.
DEVARAI SANTHOSH KUMAR
Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India.
K. A. ABDUL JALEEL
Proteomics Core Facility, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014, Kerala, India.
*Author to whom correspondence should be addressed.
Abstract
Fungal infestations of groundnut seeds were screened for thermo-tolerant lipases with stability over a wide pH range and temperature, desirable for industrial applications. DNA sequencing of ITS 1, 5.8S and ITS 2 regions indicated the isolate closer to Aspergillus niger with 99% identity and therefore designated as A. niger GN1 in this study. The pellet obtained from culture extract subjected to 65% ammonium sulphate precipitation was suspended in Tris-buffer and assayed for lipase activity. Two fractions of lipase (1 and 2) could be extracted at pH 4 and pH 9 respectively. Both lipase 1 and 2 fractions were characterized by LC-MS/MS spectroscopic analysis. The relative and residual activities of the enzyme fractions were high in the temperature range of 60 – 80°C and over a pH of 4 & 8 for lipase 1 and 2-6 for lipase 2 fractions. LC-MS/MS confirmed the presence of Lipase 1 (~32 kDa) and Lipase 2 (~30 kDa) fractions with 4 and 2 unique peptides respectively.
Keywords: Lipase, A. niger, thermo-tolerance, pH stability, protein characterization.