基于短肽自组装与共组装的纳米纤维人工水解酶

Artificial Hydrolase Based on Short Peptides Self- and Co-assembly Nanofiber

将水解酶活性中心催化三联体氨基酸(His/Ser/Asp)引入9-芴亚甲氧羰基苯丙氨酸二肽(Fmoc-FF)双亲短肽序列中,利用短肽的自组装性能,构建了具有对硝基苯酚乙酸酯水解活性的超分子纳米纤维人工水解酶.研究结果表明,形成规则的纳米纤维结构是获得催化活性的必要条件.9-芴亚甲氧羰基(Fmoc)基团间的弱相互作用促使β-折叠二级结构的形成.通过对比天然水解酶的米氏动力学方程、最适催化温度及p H结果可知,所制备的超分子纳米纤维人工水解酶具有与天然酶相似的酶学性质.金属离子Ca2+和Ba2+对人工水解酶活性具有激活作用,而Mg2+,Ni2+,Co2+,Cu2+和Zn2+则抑制酶活性.

The catalytic triad（Ser/His/Asp） of natural hydrolase were introduced into the pepticte segment of 9-fluorenylmethoxycarbonyl-diphenylalanine （ Fmoc-FF）, 9-fluorenylmethoxycarbonyl-phenylalanine-phenylalanine-histidine （ Fmoc-FFH）, 9-fluorenylmethoxycarbonyl-phenylalanine-phenylalanine-serine （ Fmoc-FFS ） and 9-fluorenylmethoxycarbonyl-phenylalanine-phenylalanine-aspartate（Fmoc-FFD） were designed to construct the artificial hydrolase. Based on the self-assembly and co-assembly of Fmoc-peptides, a series of supramolecular nanofibers built from the self-assembly of Fmoc-FFH（SA-H） , self-assembly of Fmoc-FFS（SA-S） , self- assembly of Fmoc-FFD （ SA-D）, co-assembly of Fmoc-FFH and Fmoc-FFS （CoA-HS） , co-assembly of FmocFFH and Fmoc-FFD （CoA-HD） , co-assembly of Fmoc-FFD and Fmoc-FFS （CoA-DS）, co-assembly of FmocFFH, Fmoc-FFD and Fmoc-FFS （CoA-HDS） were obtained as artificial hydrolases. SA-H exhibited the highest hydrolysis activity for p-nitrophenyl acetate （PNPA）. It was found that the well-ordered structure of nanofiber is important for catalytic activity, and the fl-sheet structure come from the week interactions between Fmoc groups. The kinetics behavior, optimum temperature and pH studies showed the typical enzymatic cha- racteristics of artificial hydrolase 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.