The Alzheimer’s disease (AD) is characterized by a progressive cerebral deposition of amyloid b-peptide (Ab) in the brain. We have recently shown that the Ab1-40 is cleaved by E. coli pitrilysin, a homologue of the human insulysin at identical sites which provides an attractive therapeutic avenue towards this neurodegenerative disorder. To facilitate studies on the mechanism of recognition of Ab by pitrilysin, an overproduction system of Ab1-40 was constructed as a fusion protein with E. coli RNase HI. The Ab1-40 peptide derivative, Ab1-40*, although more amyloidogenic was conformationally similar to the wild-type Ab1-40 and was cleaved by pitrilysin at identical sites. The N-terminal truncated Ab15-40* peptide formed reduced amyloid fibrils suggesting that cleavage of this enzyme predominantly at the His14-Gln15 position decreases the amyloidogenecity of Ab1-40*. The well preserved hydrophobic core (Leu17, Val18, Phe19) was demonstrated to be vital for amyloid formation because series of Ab1-40* derivatives with collective or individual substitution of these amino acid residues to hydrophilic serine markedly inhibited amyloid formation as determined by the ThT binding assay. Meanwhile, substitutions to these amino acid residues to alanine slightlyincreased its ability to form amyloid fibrils in vitro. However, both the Km and kcat for cleavage of pitrilysin were not greatly affected for these mutant peptides irregardless of solubilities suggesting that these amino acids in the hydrophobic core although involved in amyloid formation may not be necessarily be required for binding of Ab to pitrilysin.