Gao Yu, Li Jian-Jun, Zheng Lanyan, Du Yuguang
Department of Microbiology and Parasitology, China Medical University, No. 77 Puhe Road, Shenyang 110122, Liaoning Province, China.
National Key Laboratory of Biochemical Engineering, National Engineering Research Center for Biotechnology (Beijing), Key Laboratory of Biopharmaceutical Production & Formulation Engineering, PLA, Institute of Process Engineering, Chinese Academy of Sciences, No. 1 North 2nd Street, Beijing 100190, China.
Protein Eng Des Sel. 2017 Nov 1;30(11):743-751. doi: 10.1093/protein/gzx055.
Versatile peroxidase (VP) from Pleurotus eryngii is a high redox potential peroxidase. It has aroused great biotechnological interest due to its ability to oxidize a wide range of substrates, but its application is still limited due to low pH and thermal stability. Since CiP (Coprinopsis cinerea peroxidase) and PNP (peanut peroxidase) exhibited higher pH and thermal stability than VP, several motifs, which might contribute to their pH and thermal stability, were identified through structure and sequence alignment. Six VP variants incorporating the beneficial motifs were designed and constructed. Most variants were nearly completely inactivated except V1 (Variant 1) and V4. V1 showed comparable activity to WT VP against ABTS, while V4 exhibited reduced activity. V1 displayed improved pH stability than WT VP, at pH 3.0 in particular, whereas the pH stability of V4 did not change a lot. The thermal stabilities of V1 and V4 were enhanced with T50 raised by 3°C. The results demonstrated that variants containing the beneficial motifs of CiP and PNP conferred VP with improved pH and thermal stability.
杏鲍菇中的多功能过氧化物酶（VP）是一种高氧化还原电位过氧化物酶。由于其能够氧化多种底物，它引起了极大的生物技术兴趣，但由于其低pH值和热稳定性，其应用仍然有限。由于灰盖鬼伞过氧化物酶（CiP）和花生过氧化物酶（PNP）表现出比VP更高的pH值和热稳定性，通过结构和序列比对确定了几个可能有助于其pH值和热稳定性的基序。设计并构建了六个包含有益基序的VP变体。除了V1（变体1）和V4外，大多数变体几乎完全失活。V1对ABTS的活性与野生型VP相当，而V4的活性降低。V1在pH 3.0时表现出比野生型VP更好的pH稳定性，特别是在pH 3.0时，而V4的pH稳定性变化不大。V1和V4的热稳定性得到增强，T50提高了3°C。结果表明，含有CiP和PNP有益基序的变体赋予了VP更好的pH值和热稳定性。
