Construction Of Supermolecular Artificial Hydrolase Based On Amphiphilic Short Peptide Self- Or Co-assembled Nanofiber

Inspired by the catalytic triad(serine/histidine/aspartate) of native hydolase, supermolecular artificial hydrolase was prepared based on the self- or co-assembly of Fmoc-FFH, Fmoc-FFD, and Fmoc-FFS. The structure and morphology of artificial hydrolase were characterized by SEM, TEM, CD, FTIR, and so on. P-nitrophenyl acetate(PNPA) was used as hydrolysis substrate to test catalytic activity, enzymatic properties, and dynamic behavior of the artificial hydrolase. Futhermore, in order to enhance the catalytic activity, some improvements of preparation process were made and molecular imprinting technique was introduced to optimize the micro-structure of catalytic center of the artificial hydrolase. The detailed conclusions can be summarized as follows:(1) Supermolecular self-assembled artificial hydrolase: Self-assembled artificial hydrolase(SA-H, SA-D, and SA-S) was prepared based on self-assembling of Fmoc-tripeptide. Studies showed that artificial hydrolase was composed of nanofibers, which self-assembled in an anti β-sheet manner. Only SA-H exhibited catalytic activity, and its catalytic rate was 2.41×10-4mM·L-1·s-1. The optimum temperature and pH for SA-H is 35 ℃ and 8.0. The kinetics behavior studies showed the typical enzymatic characteristics of SA-H as that of natural enzyme. In addition, metal ions Ca2+, Ba2+ can activate the artificial hydrolase, whereas Mg2+, Ni2+, Co2+, Cu2+, Zn2+ inhibit the activity.(2) Supermolecular co-assembled artificial hydrolase: Co-assembled artificial hydrolase(CoA-HS, CoA-HD, and CoA-HSD) was prepared using co-assembly of Fmoc-tripeptide. Multiple composition co-assembling led to the disorder of nanofiber, which inhibited the activity of co-assembled artificial hydrolase. Highly regular nanofiber was prepared through the modulation of the ratio of each peptide, and the catalytic activity of CoA-HSD maximized when the ratio of Fmoc-FFH, Fmoc-FFS, and Fmoc-FFD was 40:1:1. The kinetics behavior studies showed CoA-HSD has better catalytic efficiency and substrate binding ability than native PPL. Besides, metal ions Cr3+ and Fe2+ can activate the artificial hydrolase, whereas Ni2+, Co2+, Cu2+, and Zn2+ inhibit the activity. In addition, CoA-HSD also showed an activity for transesterification reaction.(3) Molecular imprinting-supermolecular artificial hydrolase: To enhance the catalytic activity, three molecular imprinting-supermolecular artificial hydrolase was prepared, including MI-SA-H, MI-CoA-HSD, and MI-CoA-HR. Molecular imprinting technique was used to improve the micro structure of catalytic center of artificial hydrolase. Study showed that catalytic efficiency of MI-SA-H, MI-CoA-HSD, and MI-CoA-HR has been greatly improved compared to SA-H, CoA-HSD, and CoA-HR, and MI-CoA-HSD exhibited the highest activity, which is 2.59×10-3mM·L-1·s-1. The ideal template was PNPA with concentration of 2 mg/m L.