Process Biochemistry 47 (2012) 2234–2239 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u rn al hom epa ge: www .elsevier.com/locate/procbio Extraction of lipase from Aspergillus niger by insoluble complex formation with anionic and cationic polyelectrolytes Analía Marini, Natalia Imelio, Sebastián Marini, Diana Romanini, Beatriz Farruggia ∗ Laboratory of Chemical-Physical Applied to Bioseparation, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario – CIUNR – CONICET, Suipacha 570, 2000 Rosario, Argentina a r t i c l e i n f o Article history: Received 27 June 2012 Received in revised form 2 August 2012 Accepted 27 August 2012 Available online 2 September 2012 Keywords: Lipase Anionic polyelectrolytes Cationic polyelectrolytes Precipitation Bioseparation a b s t r a c t The insoluble complex formation between lipase from Aspergillus niger and the electrically charged polymers, polyacrylic acid (PAA), poly-vinil sulfonate (PVS) and chitosan (CHI), was studied by using turbidimetric and enzymatic methods on a commercial lyophilized (Ly) and a filtrate of solid culture medium (SCM). It could be shown that both electrostatic interactions as hydrophobic are involved in the formation of insoluble complexes. The kinetics of the complex formation were determined. Lipase enzymatic activity is maintained through time in the presence of polyelectrolytes. On the Ly the three polymers produced insoluble complex, with a stoichiometric ratio (polymer mass per mass of Ly from Aspergillus niger) of PAA/Ly: 0.035, PVS/Ly: 0.099 and CHI/Ly: 0.071 mg/mg Ly. For the anionic polyelectrolytes, the PAA presents slightly better results than PVS to be used when the protein concentration is similar to the lyophilized. The filtrate of the SCM has a total protein concentration much lower than commercial lyophilized. Working with CHI as cationic polymer a recovery of the activity in the re-dissolved precipitate higher than 80%, with purification factors greater than 3 were achieved, both at 8 and 20 ◦ C. Therefore, this methodology could be used as a first step of purification. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction From Morawetz and Hugues work [1], it is now recognized that globular proteins can form tight complexes with polyelectrolytes. These associations may result in soluble species [2], complex coac- ervation [3], precipitation [4], or gelation [5]. The formation of complexes affects both the state of aggregation of the species and the environment immediately surrounding the bound moieties of the partners. Therefore, their detection reflects either the increase of the average size and the molecular weight of the species or a perturbation at a molecular level. Either an estimate of the light scattered by the samples using a spectrophotometer to measure the turbidity or a rapid measurement of the viscosity appears to be the simplest and most sensitive technique to be used for any kind of aggregation [6]. All the interactions (electrostatic, hydrophobic, hydrogen bonds, steric effects) can take place simultaneously among the Abbreviations: PVS, poly (vinyl sulfonic acid, sodiumsalt); PAA, poly (acrylic acid) partial sodium salt; CHI, chitosan; Ly, lyophilized; pNPL, 4-nitrophenyl laurate; pNP, p-nitrophenol; SCM, solid culture medium. ∗ Corresponding author. E-mail addresses: bfarrug@fbioyf.unr.edu.ar, bfarrug@gmail.com (B. Farruggia). polypeptide chains and parts of another polymer, directing both protein folding and complexation [6]. The practical approach of the protein–polyelectrolyte complex formation includes protein sepa- ration and protein recovery [7], immobilization or stabilization of enzymes [8], modification of protein–substrate affinity [9]. Today, technological development requires the production of biological catalysts for many industrial processes. This has led to the devel- opment of techniques to obtain large-scale enzyme. The need to generate enzymes for industrial use along with the synthesis of new polymers capable of interacting with them has allowed the devel- opment of rapid and simple methods of purification thus allowing the production of these macromolecules from natural sources or recombinant organisms where they are expressed. Lipases (EC 3.1.1.3) are a group of enzymes which catalyze the hydrolysis and synthesis of triglycerides in vivo, producing or con- suming fatty acid esters and even the synthesis of these ones in low water content environments. This feature has increased the biotechnological interest in these enzymes for a number of industri- ally significant biotransformations. Potential applications include modification of sugars, synthesis of flavor esters for the food indus- try, the resolution of racemic mixtures and biofuel production [10]. From an industrial standpoint, fungi are more interesting sources of these enzymes than animals or plants for their potential use in biotechnology due to their availability and high stability. Lipase 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.08.020