Optimization of Borassus flabellifer Amylase Extraction Procedure Using Box-Behnken Design and Development of Simple Affinity Chromatographic Technique for Purification of Amylases
Ranjith Kumar Bakku
Department of Biotechnology, University of Hyderabad, Gachibowli, Hyderabad, India.
Ramana Terli
Department of Biotechnology, Andhra University, Visakhapatnam, India.
Subba Rao Somalanka
Department of Chemical Engineering, Andhra University, Visakhapatnam, India.
Mary Anupama Palukurty *
Department of Biotechnology, ANITS, Sangivalasa, Visakhapatnam, India.
*Author to whom correspondence should be addressed.
Abstract
This work is aimed to study the optimal conditions of extraction and development of novel purification process for α-Amylase from pericarp of Borassus flabellifer fruit. Optimization was done by Response Surface Methodology (RSM) using Box-Behnken design. The variables chosen for this purpose are pH (X1), temperature (X2, °C) and CaCl2 concentration (X3, ppm). A total of 9 runs were conducted, varying each factor while others were kept constant. The contribution of each factor was established giving raise to significant t-values and p-values. This revealed that the three variables are essential for activity of the enzyme. Based on the obtained optimal conditions from RSM studies, enzyme formation was successfully achieved with an activity of 28.8 U and concentration of 1.4 mg/ml for the 90% ammonium sulfate precipitated dialyzed enzyme sample. A novel affinity chromatographic process was developed for purification process and by this process the enzyme concentration was found to be 0.032mg/ml and activity was determined as 4.76 U.
Keywords: Amylase, Borassus, partial purification, RSM, Box-Behnken, affinity chromatography