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

Abstract

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.

Parameters necessary to define an immobilized enzyme preparation

“…If used at a high concentration, sodium borohydride can even break peptide bonds (66). Some alternative reagents have been proposed, but are rarely used (67). The case of glutaraldehyde is special, because it loses chemical reactivity after several days and gives very stable enzyme-support bonds (68).…”

Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System

“…The usual way to stabilize the labile azomethine group is to reduce it to a secondary amine bond, so that the overall process (condensation and reduction) is called reductive amination [135]. There are different reducing agents that can be used, such as sodium cyanoborohydride (ideally, for pH values between 6 and 8) and different borane complexes [136], specially α-picoline borane [137][138][139][140][141]. Sodium borohydride could be also used for reducing the imine, but, as it is 5-fold stronger than sodium cyanoborohydride, it could directly reduce the aldehyde groups (avoiding the condensation with the amine) or even the disulfide bridges, leading to enzyme denaturation [134].…”

Stable immobilization of aldehyde ketone reductase mutants containing nonstandard amino acids on an epoxy resin via strain-promoted alkyne–azide cycloaddition

“…Although multi-point covalent immobilization can enhance the rigidity of the enzyme, consequently improving the thermal stability of the immobilized enzyme, this promiscuous multi-point immobilization also reduced enzyme activity 5,6 due to the decrease in the motion of the enzyme. 7,8 Thus, uncertainties still exist 9,10 as to how to precisely control the exact number of covalent bonds between enzyme and support and sites where the linkages occurs on the enzyme surface with regard to multi-point covalent immobilization. Site-directed mutagenesis seems to be an elegant and versatile way to control the direction of a protein.…”