Kinetic and microstructural characterization of immobilized penicillin acylase fromStreptomyces lavendulaeon Sepabeads EC-EP

Abstract

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303 † . Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2 IU g wet carrier (1 in the hydrolysis of penicillin V at pH 8.0 and 408C. This biocatalyst showed the highest activity at pH 8.5 and 658C, 1.5 pH units lower and 58C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 408C and pH 8.0 (t 1/2 0625 h vs. t 1/2 0397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…Soaking the support with liquid of comparable refractive index is a possible solution to enable direct measurements also in such cases [12,62,63]. Evidence from CLSM studies on the interaction of proteins with chromatographic supports can be translated almost directly to enzyme immobilization [50,62,[71][72][73][74][75][76][77][78][79][80][81][82][83]. Relationships between the dynamics of protein adsorption and the external and internal geometrical features of the support were established [84][85][86].…”

Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

“…PVA from Streptomyces lavendulae ATCC 13664 (SlPVA) is an extracellular enzyme which has been exhaustively characterized (7)(8)(9)(10) and immobilized (11,12) due to its ability to hydrolyze very efficiently PV and other natural aliphatic penicillins that contaminate PV and usually reduce 6-APA yield at the end of the process. The broad substrate specificity of SlPVA allows this enzyme to hydrolyze several penicillins with aliphatic acyl chains, e.g., 3-hexenoyl-penicillin (penicillin F [PF]), hexanoyl-penicillin (penicillin dihydro-F [PdF]), and octanoyl-penicillin (penicillin K [PK]), as the catalytic constant for PK was even higher than that for PV (13).…”

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from Pseudomonas putida KT2442 isolated from inclusion bodies

“…-NTA agarose beads. Unfortunately, hydrolysis of polyhydroxyoctanoate (PHO) catalyzed by immobilized enzyme failed, likely due to the substrate hindrance to enter into the active site of the immobilized depolymerase, which is linked inside the support and/or near to the agarose surface as observed by confocal scanning microscopy of immobilized enzyme, which was labeled with Alexa Fluor-488 dye antibody conjugates, a very useful technique for the determination of enzyme distribution in activated carriers [8,9]. Such catalytic behavior was not observed in PHO depolymerase from Pseudomonas fluorescens GK13 adsorbed onto Accurel MP-1000, which was successfully used in the production of (R)-3-hydroxyoctanoic acid from PHO [6].…”