Extraction of lipase fromRhizopus microsporusfermentation culture by aqueous two-phase partitioning

Abstract

The purification of a desired protein from the complex mixture of biomolecules in a fermentation broth is one of the main challenges in industrial-scale biotechnological processes. For partial purification of industrial enzymes, one possible approach is to use aqueous two-phase extraction (APTE) as a primary downstream processing step. In the present study, the feasibility of utilizing ATPE for the purification of lipase from Rhizopus microsporus fermentation culture was investigated. The effects of the phase composition and salt type, molecular weight of the polyethylene glycol (PEG), pH of the system, sample loading and addition of neutral salts on lipase partitioning were investigated at 24 C. In most of the examined aqueous two-phase systems (ATPSs), lipase showed affinity to the top phase. Optimum conditions for lipase purification were obtained in 20% PEG 2000/12% (NH 4 ) 2 SO 4 , with 5% Na 2 CO 3 addition at pH D 8 for 30% crude load. For the optimized ATPS, the recovery yield and purification factor in the top phase were determined to be 92.3% and 19.8, respectively.

Partitioning of Geotrichum candidum Lipase from fermentative crude extract by aqueous two-phase system of polyethylene glycol and sodium citrate

“…Gum Arabic and anhydrous ethanol (purity > 99.5% m/m) were purchased from Synth ® (São Paulo, Brazil). All other chemical reagents and solvents were supplied by Synth ® and Vetec Química Ltd. (São Paulo, SP, Brazil).The reagents used in the purification step were poly (ethylene glycol) with average molar mass of 1500, 4000 and 6000 g mol -1 (Aldrich, USA); the triblock copolymer L35, (EO) 11 (PO) 16 …”

“…While there previously published works about using ATPS in the study of extraction/purification processes of lipases is important to note that these lipases are different in size, shape, and other characteristics and are obtained by fermentation processes involving other microorganisms [16][17][18]. In this sense, so far, there has been no report in the literature on the use of ATPS for the purification of the extract fermentation of extracellular lipase of Geotrichum candidum.…”

Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

“…Similar results were obtained for lipase purification with PEG/ammonium sulfate ATPS by Anvari, with an increase in the PEG concentration of 10% to 20%. Moreover, the partitioning yield of lipase activity increased from 18.25% to 76.58% in PEG2000/ammonium sulfate ATPS and from 29.73% to 42.15% in PEG4000/ ammonium sulfate ATPS [9].…”

“…A purification rate of 5.84 folds and an enzyme yield of 80.4% were obtained [8]. Anvari [9] extracted lipase from Rhizopus microsporus during fermentation process by ATPS of 20% PEG 2000/12% (NH 4 ) 2 SO 4 and 5% Na 2 CO 3 at pH 8.0. The yield was 92.3% and the purification degree was 19.8 folds [9].…”

Extractive purification of recombinant thermostable lipase from fermentation broth of Escherichia coli using an aqueous polyethylene glycol impregnated resin system

“…Aqueous two-phase extraction (ATPE) is commonly used to separate the biomolecule from the cell culture. It has been used for the purification of lipase from different microorganisms such as Rhizopus microspores and Burkholderia pseudomallei (Anvari 2015;Ooi et al 2009). However, this method of extraction has no applications in industry (Iqbal et al 2016), which is substantially based on very long equilibration time needed for separation of phase and partitioning of protein, causes an increased footprint of the required settler devices or the application of additional equipment such as energy consuming centrifuges (van Winssen et al 2014b).…”