Care Andrew, Nevalainen Helena, Bergquist Peter L, Sunna Anwar
Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, North Ryde, Sydney, NSW 2109, Australia.
Appl Biochem Biotechnol. 2014 Aug;173(8):2225-40. doi: 10.1007/s12010-014-1027-7. Epub 2014 Jun 27.
An inorganic-binding peptide sequence with high affinity to silica-containing materials was fused to a glycoside hydrolase GH26 mannanase, ManA, from the extremely thermophilic bacterium Dictyoglomus thermophilum. The resulting recombinant enzyme produced in Escherichia coli, ManA-Linker, displayed high binding affinity towards synthetic zeolite while retaining its catalytic activity at 80 °C. ManA-Linker was able to bind to the zeolite at different pH levels, indicating a true pH-independent binding. However, complete degradation of the peptide linker was observed when the recombinant ManA-Linker was exposed to the supernatant from the filamentous fungus Trichoderma reesei. This degradation was caused by extracellular proteinases produced by T. reesei during its growth phase. Several derivatives of ManA-Linker were designed and expressed in E. coli. All the derivatives carrying a single sequence of the linker were still susceptible to T. reesei proteinase degradation. Complete substitution of the linker sequence by (GGGGS)16 resulted in a proteinase-resistant ManA derivative, ManA-Linker-(GGGGS)16, which was able to bind to zeolite in a pH-dependent manner.
将对含硅材料具有高亲和力的无机结合肽序列与来自极端嗜热细菌嗜热栖热袍菌的糖苷水解酶GH26甘露聚糖酶ManA融合。在大肠杆菌中产生的重组酶ManA-Linker对合成沸石显示出高结合亲和力，同时在80°C下保留其催化活性。ManA-Linker能够在不同pH水平下与沸石结合，表明其结合真正不依赖于pH。然而，当重组ManA-Linker暴露于丝状真菌里氏木霉的上清液时，观察到肽接头完全降解。这种降解是由里氏木霉在其生长阶段产生的细胞外蛋白酶引起的。设计了几种ManA-Linker的衍生物并在大肠杆菌中表达。所有携带单个接头序列的衍生物仍然易受里氏木霉蛋白酶降解。用(GGGGS)16完全取代接头序列产生了一种抗蛋白酶的ManA衍生物ManA-Linker-(GGGGS)16，它能够以pH依赖的方式与沸石结合。
