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

Catalysts

Maria Romero-Fernández¹  Alejandro H. Orrego¹  JoséM. Guisán¹  Javier Rocha-Martin¹  Maríadel Mar Yust²  Maríadel Carmen Millán-Linares² 

[1] Department of Biocatalysis, Institute of Catalysis and Petrochemistry (ICP) CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain;Group of Plant Proteins, Instituto de la Grasa CSIC, Carretera Utrera Km 1, 41012 Seville, Spain;

关键词: enzyme immobilization;    2-picoline borane;    biocatalysis;    glyoxyl agarose;    Schiff base;    enzyme stabilization;   

DOI  :  10.3390/catal8080333

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown