Journal of Molecular Catalysis B: Enzymatic 133 (2016) 107–116 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb Diffusion effects of bovine serum albumin on cross-linked aggregates of catalase Agnes Cristina Oliveira Mafra a , Willian Kopp a , Maisa Bontorin Beltrame b , Raquel de Lima Camargo Giordano a,b , Marcelo Perencin de Arruda Ribeiro b , Paulo Waldir Tardioli a,b,∗ a Postgraduate Program in Chemical Engineering of the Federal University of São Carlos (PPG-EQ/UFSCar), Rodovia Washington Luiz, km. 235, 13565-905 São Carlos, SP, Brazil b Department of Chemical Engineering, Federal University of São Carlos, Rodovia Washington Luiz, km. 235, 13565-905 São Carlos, SP, Brazil a r t i c l e i n f o Article history: Received 5 May 2016 Received in revised form 20 July 2016 Accepted 5 August 2016 Available online 6 August 2016 Keywords: Catalase CLEA BSA Kinetic model Diffusional delay a b s t r a c t Stabilization of multimeric enzymes is one of the major challenges in biocatalysis since dissociation of subunits can inactivate the enzyme. Particularly, catalase that breaks down hydrogen peroxide in water and molecular oxygen is an enzyme difficult to stabilize by conventional immobilization techniques, because it is a tetrameric structure containing Fe-protoporphyrin IX in its active site. Cross-linking of enzyme aggregates is a methodology that can overcome this bottleneck, but diffusional delay of mass transport within the particles is a recurrent drawback. In this work, cross-linked aggregates of catalase from bovine liver were prepared, evaluating the influence of precipitant and cross-linking agents, as well as bovine serum albumin (BSA) as feeder protein on the catalytic properties, thermal stability, and mass transport resistance of the derivatives. The most active derivatives were prepared using ammonium sulfate as precipitant agent, 50 mM glutaraldehyde as cross-linker, and mass ratio BSA/catalase of 3.0. These derivatives in the absence of diffusive effects showed recovered activity of 98 ± 1.7% and high stability at 40 ◦ C and pH 7.0 (∼80% of the initial activity was recovery after 200 h under these conditions). The co-precipitation of BSA together with catalase reduced the size of clusters suggesting a decrease of diffusive effects within the biocatalyst. Empirical kinetic model was fitted to the experimental data of initial rate vs. substrate concentration and used to make a comparative analysis of mass transfer into derivatives with and without BSA. Results suggested that the main effect that differentiates the free enzyme and the two derivatives analyzed was of diffusive nature. In fact, the effectiveness factor of the cross-linked aggregates of catalase with BSA increased around 4 times. Statistical design of experiments and the analysis of the response surface methodology showed that the immobilization did not alter the conditions of maximum activity of the catalase, which were found to be 30 ◦ C and pH ∼7.0 for all biocatalysts. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Catalase (CAT; EC 1.11.16) is a homotetramer containing Fe- protoporphyrin IX in its active site [1], which is normally obtained from bovine liver or from microbial sources. CAT from bovine liver has a molecular mass of 250 kDa and each subunit has a molecular weight above 65 kDa [2]. ∗ Corresponding author at: Postgraduate Program in Chemical Engineering of the Federal University of São Carlos (PPG-EQ/UFSCar), Rodovia Washington Luiz, km. 235, 13565-905 São Carlos, SP, Brazil. E-mail addresses: pwtardioli@ufscar.br, pwtardioli@hotmail.com (P.W. Tardioli). CAT catalyzes the hydrogen peroxide (H 2 O 2 ) decomposition through Bi–Bi Ping-Pong mechanism [3–5]. The reaction of CAT with H 2 O 2 follows two steps. In the first step of the reaction, a molecule of H 2 O 2 oxidizes the ion Fe 3+ in the prosthetic group, with the condensation of one molecule of water. In the second step, a second molecule of H 2 O 2 reduces the prosthetic group, which was oxidized in the first step (O–Fe 4+ ), generating Fe 3+ and releasing H 2 O and O 2 [3–5]. Commercially, CAT is used to remove H 2 O 2 from milk before cheese processes [6]. It can also be found in disinfectants and food containers to prevent oxidation [6], keeping the food fresh for longer periods of time, and as an oxygenator for skin rejuvenation [7]. Enzymes play an important role in industrial chemical reactions. However, low operational stability and high costs may limit their http://dx.doi.org/10.1016/j.molcatb.2016.08.002 1381-1177/© 2016 Elsevier B.V. All rights reserved.