Self-assembly amphipathic peptides induce active enzyme aggregation that dramatically increases the operational stability of nitrilase
Self-assembly amphipathic peptides induce active enzyme aggregation that dramatically increases the operational stability of nitrilase
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DOI：
10.1039/c4ra11236a
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发表时间：
2014-11
期刊：
影响因子：
3.9
通讯作者：
Xiaofeng Yang;A. Huang;Jizong Peng;Jufang Wang;Xiaoning Wang;Zhanglin Lin;Shuang Li
中科院分区：
文献类型：
--
作者：
Xiaofeng Yang;A. Huang;Jizong Peng;Jufang Wang;Xiaoning Wang;Zhanglin Lin;Shuang Li
寡聚腈水解酶与两亲性自组装肽18 A在C-末端融合，并在大肠杆菌中表达。融合酶自发组装成具有>90%天然腈水解酶活性的活性聚集体。在较高温度下，融合腈水解酶（Nit-SEA）的比活性比天然腈水解酶（Nit）高得多。通过细胞裂解和离心纯化酶聚集体，从而容易制备固定化颗粒（Nit-iSEA）。大约86%的初始腈水解酶活性被掺入到海藻酸钙包埋珠中。四种不同腈水解酶变体的热稳定性显示，Nit-SEA和Nit-iSEA在45 °C下比天然腈水解酶稳定约6.7倍和10倍。腈耐受性也显著改善，在30至120 mM扁桃腈的范围内，未观察到Nit-iSEA的明显底物抑制。此外，Nit-iSEA可循环使用20次，活性损失约5%。
Oligomeric nitrilase was fused with an amphipathic self-assembly peptide 18A at the C-terminus and expressed in Escherichia coli. The fusion enzyme spontaneously assembled into active aggregates with >90% native nitrilase activity. Much higher specific activities than the native nitrilase (Nit) were recorded for the fusion nitrilase (Nit-SEA) at higher temperatures. The enzyme aggregates were purified through cell lysis and centrifugation led to the facile preparation of immobilized particles (Nit-iSEA). Approximately 86% of the initial nitrilase activity was incorporated into the Ca-alginate entrapment beads. The thermostability of the four kinds of different nitrilase variants showed that the Nit-SEA and Nit-iSEA at 45 °C were about 6.7- and 10-fold more stable than the native nitrilase. The nitrile tolerance was also dramatically improved, no apparent substrate inhibition was observed for Nit-iSEA over the range of 30 to 120 mM mandelonitrile. Additionally, the Nit-iSEA could be recycled 20 times with ∼5% loss in activity.