Deposition of beta-amyloid (A beta) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of A beta levels by various therapeutic approaches is actively being pursued. A potentially non-inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze A beta at its alpha-secretase site. We have previously identified a light chain fragment, mk18, with alpha-secretase-like catalytic activity, producing the 1-16 and 17-40 amino acid fragments of A beta 40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking alpha-secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for A beta. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the alpha-secretase site of A beta. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3- and 6-fold increase in catalytic activity (k(cat)/K-M) toward the synthetic A beta substrate compared to the original scFv primarily due to an expected decrease in K-M rather than an increase in k(cat). This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for A beta. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble A beta levels in vivo. (C) 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 25: 1054-1063, 2009