Partitioning of thermostable glucoamylase in polyethyleneglycol/salt aqueous two-phase system

Abstract

A major challenge in downstream processing is the separation and purification of a target biomolecule from the fermentation broth which is a cocktail of various biomolecules as impurities. Aqueous two phase system (ATPS) can address this issue to a great extent so that the separation and partial purification of a target biomolecule can be integrated into a single step. In the food industry, starch production is carried out using thermostable glucoamylase. Humicola grisea serves as an attractive source for extracellular production of glucoamylase. In the present investigation, the possibility of using polyethylene glycol (PEG)/salt-based ATPS for the partitioning of glucoamylase from H. grisea was investigated for the first time. Experiments were conducted based on one variable at a time approach in which independent parameters like PEG molecular weight, type of phase-forming salt, tie line length, phase volume ratio, and neutral salt concentration were optimized. It has been found that the PEG 4000/potassium phosphate system was suitable for the extraction of glucoamylase from the fermentation broth. From the results, it was observed that, at a phase composition of 22 % w/w PEG 4000 and 12 % w/w phosphate in the presence of 2 % w/w NaCl and at pH 8, glucoamylase was partitioned into the salt-rich phase with a maximum yield of 85.81 %. A range of parameters had a significant influence on aqueous two-phase extraction of glucoamylase from H. grisea. The feasibility of using aqueous two-phase extraction (ATPE) as a preliminary step for the partial purification of glucoamylase was clearly proven.