Immobilization and Characterization of Enzymes

Agarose 4 BCL was purchased from Agarose Bead Technologies (Madrid, Spain). Epichlorohydrin, iminodiacetic acid, triethylamine, sodium borohydride, sodium periodate, 1-octanothiol, bovine serum albumin, O-nitro-phenyl-β-d-galactopyranoside (O-NPG), p-nitrophenyl proprionate (p-NPP), 4-nitrophenyl-β-d-glucopyranoside (p-NPG), and high molecular weight protein (Sigma Marker^TM, St. Louis, MO, USA) were purchased from Sigma (Sigma-Aldrich^®, St. Louis, MO, USA). Glutaraldehyde solution (25%, v/v) and ethylenediamine were purchased from Alfa Aesar (Thermo Fisher Scientific^®, Waltham, MA, USA). Octyl-Sepharose CL-4B was purchased from GE Healthcare Bio-Sciences (Uppsala, Sweden). The β-galactosidase from Kluyveromyces lactis (Lactozym pure 6500 L) (KlBgal) was kindly supplied by Novozymes A/S (Copenhagen, Denmark). The lipase from Candida rugosa (CRL) was purchased from Sigma (Sigma-Aldrich^®, St. Louis, MO, USA). Overexpression and purification of lipase (LipC12) and β-glucosidase (EaBglA) were performed as previously described by Glogauer et al. [40 (link)] and Crespim et al. [47 (link)], respectively. All other chemicals used were of analytical grade.