One-step purification of L-asparaginase from cell extracts using carbon xerogels

Abstract

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme with wide applications in the pharmaceutical sector and food processing industries. It is mainly used as a biotherapeutic for treating Acute Lymphoblastic Leukemia (ALL) and to reduce acrylamide formation in starchy compounds. Despite its relevance, current purification methods for microbial enzymes involve complex and expensive techniques. To overcome this drawback, Carbon Xerogels (CXs) were here investigated as novel adsorbents to be applied in an one-step ASNase purification process, using a flow-through-like setup, from a cell extract of genetically engineered Bacillus subtilis. Different operating conditions were studied for optimizing the adsorption onto CXs, including total protein concentration (3–15 mg/mL), CXs amount (12, 18 and 24 mg), and adsorption volume of cell extract (1.5, 2.0 and 15 mL). Ultimately, CXs were packed into a column to evaluate the feasibility of semi-continuous ASNase purification. CXs have high affinity for other proteins present in the cell extract, while leaving ASNase in the supernatant or eluted sample. Purification folds of 2.5 and 3.8 for ASNase were obtained in batch and semi-continuous experiments, respectively, revealing the potential of CXs as novel adsorbent materials for ASNase purification directly from a complex matrix.