Bioaffinity extraction of glucoamylase in aqueous two-phase systems using starch as free bioligand

• Published in: Journal of chromatography. B, Biomedical sciences and applications Biomedical sciences and applications, 2000-06, Vol.743 (1), p.241-246
• Main Authors: de Gouveia, Thomaz, Kilikian, Beatriz V
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Applied sciences; Bioligands, free; Biological and medical sciences; Biotechnology; Chemical engineering; Chromatography, Affinity - methods; Enzymes; Enzymes and enzyme inhibitors; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Glucan 1,4-alpha-Glucosidase - isolation & purification; Glucoamylase; Hydrogen-Ion Concentration; Hydrolases; Ligands; Liquid-liquid extraction; Methods. Procedures. Technologies; Others; Starch - chemistry; Various methods and equipments; Water - chemistry
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(00)00031-1

Aqueous two-phase systems with bioligands bound to the polymer, usually polyethylene glycol (PEG), have high selectivity. However, such kind of systems can be costly since some specific synthetic ligands are expensive, making them non-viable for bioaffinity extraction of low-cost proteins. The use of free bioligands is an alternative for decreasing the high cost of these systems. This work describes the use of starch as a free bioligand on the partition of glucoamylase in PEG 300–phosphate system. The results were separated into two parts. In the first part, analysis of the starch distribution between the phases showed one-sided distribution to the bottom phase. In the second part, filtered broth from submerged cultivation of Aspergillus awamori, containing glucoamylase and contaminants, was submitted to the extraction in the system with starch. In the system without starch, glucoamylase and contaminants are partitioned in the upper phase. Upon starch addition, the partition coefficient of glucoamylase was decreased nine-fold without altering the partition of contaminants. These results indicate the possibility of separation of enzymes with high-molecular-mass and hydrophilic substrates, like glucoamylase, cellulase and pullulanase, from their contaminants in a one-step extraction. Since systems made of low-molecular-mass PEG partitioned almost all of the proteins to the upper phase, the separation can be achieved by extraction of the target enzyme in the bottom phase, as in the case of the presented study.