Abstract
Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30–60 °C and pH range of 6–11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na+, Mg2+, K+ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.
Highlights
The demand for enzymes to be used in different related industries is growing, the high cost of enzymes is still a major concern for the most of them
Mehrnoush et al [5,7] have reported that the partitioning of enzymes in the presence of high concentrations of Triton X-100 was reduced in aqueous two-phase system (ATPS) as the stability of the targeted enzymes significantly decreased in the presence of the high concentrations of surfactants
To the best of author’s knowledge, this work is the first study to investigate and develop the purification method using ATPS composed of Triton X-100/Maltitol
Summary
The demand for enzymes to be used in different related industries is growing, the high cost of enzymes is still a major concern for the most of them. The purification methods of enzymes traditionally used in the industries include several steps such as ultrafiltration, precipitation, and chromatography, which might result in the loss of product in each step [2]. These conventional methods of purification, which includes the preparation of lipases for further purification, chromatographic steps, and membrane processes, are expensive, multisteps, and discontinuous as well as they are labor and time extensive. ATPS based on polymer/polymer and polymer/salt are the most common systems used in the purification of biomolecules These two traditional systems have some drawbacks such as the use of salt in the system might lead to the denaturation or certain target proteins may precipitate due to the high ionic-strength environment [4]
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