There is a constant need to identify novel inhibitors to combat 
β-lactamase-mediated antibiotic resistance. In this study, we identify three 
penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 
(SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) 
is utilized for quantitative determination and comparison of the binding 
specificity of selected peptides to penicillinase. An SPR biosensor 
functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of 
penicillinase with excellent sensitivity (15.8 RU nM(-1)) and binding affinity 
(KD = 0.56 nM). To determine if peptides can be good inhibitors for 
penicillinase, these peptides are mixed with penicillinase and their inhibition 
efficiency is determined by measuring the hydrolysis of substrate penicillin G 
using UV-vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a 
promising penicillinase inhibitor with a Ki of 9.22 μM and a Ki' of 33.12 μM, 
suggesting that the inhibition mechanism is a mixed pattern. This peptide 
inhibitor (P2) can be used as a lead compound to identify more potent small 
molecule inhibitors for penicillinase. This study offers a potential approach to 
both detection of β-lactamases and development of novel inhibitors of 
β-lactamases.