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A potentially non\u2010inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze A\u03b2 at its \u03b1\u2010secretase site. We have previously identified a light chain fragment, mk18, with \u03b1\u2010secretase\u2010like catalytic activity, producing the 1\u201316 and 17\u201340 amino acid fragments of A\u03b240 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 \u03b1\u2010secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for A\u03b2. 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 \u03b1\u2010secretase site of A\u03b2. 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\u2010 and 6\u2010fold increase in catalytic activity (<jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/<jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub>) toward the synthetic A\u03b2 substrate compared to the original scFv primarily due to an expected decrease in <jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub> rather than an increase in <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for A\u03b2. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble A\u03b2 levels in vivo. \u00a9 2009 American Institute of Chemical Engineers. Biotechnol. 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