Co-Immobilization and Co-Localization of Oxidases and Catalases: Catalase from Bordetella Pertussis Fused with the Zbasic Domain

Oxidases catalyze selective oxidations by using molecular oxygen as an oxidizing agent. This process promotes the release of hydrogen peroxide, an undesirable byproduct. The instantaneous elimination of hydrogen peroxide can be achieved by co-immobilization and co-localization of the oxidase and an auxiliary catalase inside the porous structure of solid support. In this paper, we proposed that catalase from Bordetella pertussis fused with a small domain (Zbasic) as an excellent auxiliary enzyme. The enzyme had a specific activity of 23 U/mg, and this was almost six-fold higher than the one of the commercially available catalases from bovine liver. The Zbasic domain was fused to the four amino termini of this tetrameric enzyme. Two domains were close in one hemisphere of the enzyme molecule, and the other two were close in the opposite hemisphere. In this way, each hemisphere contained 24 residues with a positive charge that was very useful for the purification of the enzyme via cationic exchange chromatography. In addition to this, each hemisphere contained 10 Lys residues that were very useful for a rapid and intense multipoint covalent attachment on highly activated glyoxyl supports. In fact, 190 mg of the enzyme was immobilized on one gram of glyoxyl-10% agarose gel. The ratio catalase/oxidase able to instantaneously remove more than 93% of the released hydrogen peroxide was around 5–6 mg of catalase per mg of oxidase. Thirty milligrams of amine oxidase and 160 mg of catalase were co-immobilized and co-localized per gram of glyoxyl-agarose 10BCL (10% beads cross-linked) support. This biocatalyst eliminated biogenic amines (putrescine) 80-fold faster than a biocatalyst of the same oxidase co-localized with the commercial catalase from bovine liver.

Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.

Stabilization of β-Gal-3 ATCC 31382 on agarose gels: synthesis of β-(1→3) galactosides under sustainable conditions

β-Gal-3 was immobilized by multipoint covalent attachment on an agarose support. The derivative was characterized and usefully applied as a recoverable and reusable biocatalyst for the synthesis of β-(1 → 3) galactosides under sustainable conditions.

Characterization of supports activated with divinyl sulfone as a tool to immobilize and stabilize enzymes via multipoint covalent attachment. Application to chymotrypsin

DVS supports are very suitable to stabilize enzymes via multipoint covalent attachment.