(-)--Lactam ((-)-2-azabicyclo[2.2.1]hept-5-en-3-one) has attracted increasing attention as the chiral intermediate of carbocyclic nucleosides most of which serve as pharmaceutical agents such as anti-HIV/HBV drugs abacavir and carbovir. So far, developing in vitro (+)--lactamase-mediated biotransformation has been one of the most efficient approaches for the production of (-)--lactam. In this study, the catalytic activity of the (+)--lactamase from Sulfolobus solfataricus P2 was engineered by semi-rational design. Molecular docking and molecular dynamics simulation were carried out to target the key positions relevant to catalytic activity. Nine amino acid residues were selected for site saturation mutagenesis. To expedite the screening process, a sensitive colorimetric high-throughput screening method was established based on the Rimini test which was originally applied to distinguish primary amines from secondary amines. The screening process resulted in the achievement of several efficient mutants: V203N, V203Q, I336H, I336R, and Y388H. Synergy effects led to four final mutants (V203N/I336R, V203N/Y388H, I336R/Y388H, and V203N/I336R/Y388H) with enhanced enzyme activity after the combination of positive single mutants. The best mutant V203N/Y388H/I336R displayed a 21-fold higher enzyme efficiency (k(cat)/K-M) compared to the wild-type enzyme. The result demonstrated that the biotransformation using the triple mutant as the catalyst reached >49% conversion and >99% enantiomeric excess at 80 degrees C after 2h, which made it a good catalyst candidate to produce (-)--lactam. The possible mechanism responsible for the improvement in the catalytic activity was explicated by analyzing the protein-ligand binding modes and interaction between the protein and the ligand.