Key Laboratory of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore 599489, Singapore.
Int J Mol Sci. 2018 Sep 30;19(10):2989. doi: 10.3390/ijms19102989.
Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in . Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.
漆酶由于其绿色催化特性和广泛的底物特异性，在工业应用中具有巨大的潜力，因此许多研究都试图提高这些酶的催化性能。在这里，据我们所知，我们首次报道了通过随机诱变和体内组装，对融合有α-因子前导肽的同源二聚体漆酶 BBP6 的定向进化。从约 3500 个克隆中筛选出的 3 个进化融合变体，其总漆酶活性提高了 31-37 倍，具有更好的热稳定性和更宽的 pH 谱。经过进化的α-因子前导肽可将漆酶表达水平提高高达 2.4 倍。对这些变体的蛋白模型分析表明，有利的突变会影响蛋白 pKa 偏移、亚基相互作用、底物进入和 C 末端功能。
