Abstract
Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.
Highlights
Aqueous Two-Phase Systems (ATPS) (PEG4000/ Potassium sodium tartrate) and Three Phase Partitioning (TPP) (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme
The use of PEG 4000/ Potassium sodium tartrate ATPS was investigated for isolation of lipase produced in solid-state cultivation of Rhizopus arrhizus
The results showed that all ATPSs tested were characterized by the partition coefficient of lipase Ke > 1, indicating that the enzyme was predominantly distributed in the top PEG-rich phase
Summary
Various conventional purification methods are applied for enzymes’ isolation. They include ammonium sulfate precipitation followed by size-exclusion and ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, or some combination of these methods [3]. The use of chromatographic techniques in some cases may lead to low yields of enzyme activity, as was observed for the process of lipase purification. Bhosale et al isolated lipase from Bacillus sonorensis 4R yielding 1.98% by sequential ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography [5]. Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree
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