Bidirectional cell-surface anchoring function of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403.
With the aim of constructing an efficient protein display system for lactic acid bacteria (LABs), the effect of fusion direction on the cell-surface binding activity of the C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 was studied. CPH fused to the alpha-amylase (AMY) of Streptococcus bovis 148 either at its C-terminus (CPH-AMY) or at its N-terminus (AMY-CPH) was expressed intracellularly in Escherichia coli. This domain was able to direct binding of AMY to the surface of L. lactis ATCC 19435 in both constructs. However, the number of bound molecules per cell and the specific activity for starch digestion in the case of CPH-AMY were 3 and 14 times greater than those in the case of AMY-CPH, respectively. Of the LABs tested, L. lactis ATCC 19435 showed the highest binding capability for CPH-AMY, up to 6 x 10(4) molecules per cell, with a dissociation rate constant of 5.00 x 10(-5) s(-1). The binding of CPH-AMY to the surface of Lactobacillus delbrueckii ATCC 9649 cells was very stable with a dissociation rate constant of 6.96 x 10(-6) s(-1). The production of CPH-AMY in the soluble form increased 3-fold as a result of coexpression with a molecular chaperone, trigger factor. The results of this study suggest the usefulness of CPH as a bidirectional anchor protein for the production of cell-surface adhesive enzymes in E. coli. Furthermore, the importance of the fusion direction of CPH in determining cell-surface binding and enzymatic activities was shown.
Tarahomjoo
Shirin
S
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Katakura
Yoshio
Y
Satoh
Eiichi
E
Shioya
Suteaki
S
D002448:Cell Adhesion / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D013294:Lactococcus lactis / Q000166:cytology* / Q000502:physiology*; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000502:physiology
D011993:Recombinant Fusion Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase