Novel flow-through like mode platform for the purification of L-asparaginase

Abstract

L-Asparaginase, an amidohydrolase enzyme applied in the pharmaceutical and food industries, is widely distributed in nature. Microorganisms are the preferential source of L-asparaginase production since they easily grow on inexpensive substrates. However, high levels of enzyme purity are required for all industrial applications, highlighting the need of developing cost-effective L-asparaginase purification processes. The main focus of this work is the development of an alternative and simple semi-continuous flow-through downstream process for L-asparaginase purification through silica-based supported ionic liquid-like phase (SSILLP) materials. SSILLP materials comprise ionic liquids (ILs) that are covalently attached to the silica, enabling distinct interactions to be established among the target compounds and the support. SSILLP materials based on quaternary ammonium cations with different alkyl chain lengths and the Cl− anion were synthesized and chemically, surface and morphologically characterized. The most promising SSILLP material for L-asparaginase purification was selected through an initial screening. The L-asparaginase purification conditions, e.g., pH and solid/liquid ratio, were then optimized using Response Surface Methodology. The semi-continuous L-asparaginase purification was ultimately addressed using the most promising SSILLP material (silica functionalized with dimethylbutylpropylammonium chloride ([Si][N3114]Cl)) and optimized purification conditions (pH 3 and solid/liquid ratio of 15), reaching a purification factor of 5.15. Through this methodology, process costs could be significantly decreased, leading to the enzyme price reduction, enabling its widespread application.