Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA, and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7 +/- 1.4% and 3,594.0 +/- 675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1 +/- 1.5% and 1,652.0 +/- 34.1, respectively). Higher amount of HA1/L/AcmA (approximate to 2.9 x 10(4) molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (approximate to 1.1 x 10(4) molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:154-162, 2017