Diffusion effects of bovine serum albumin on cross-linked aggregates of catalase

作者：Oliveira Mafra Agnes Cristina; Kopp Willian; Beltrame Maisa Bontorin; Camargo Giordano Raquel de Lima; de Arruda Ribeiro Marcelo Perencin; Tardioli Paulo Waldir^*

来源：Journal of Molecular Catalysis B: Enzymatic , 2016, 133: 107-116.

DOI：10.1016/j.molcatb.2016.08.002

• Catalase
• CLEA
• BSA
• Kinetic model
• Diffusional delay

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 degrees C and pH 7.0 (similar to 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 degrees C and pH similar to 7.0 for all biocatalysts.

• 出版日期2016-11