South China Normal University, Shipai, Guangzhou, Guangdong, China.
J Biotechnol. 2010 Jan 1;145(1):66-72. doi: 10.1016/j.jbiotec.2009.10.008.
Polyphenol oxidase (PPO) can be used for organic synthesis and degradation of wastes and dyes in industries. Lack of enzyme sources is a major barrier for its application. A PPO gene, with a full length of 1.8kb without introns, was cloned by PCR from genomic DNA of five common cultivars of Camellia sinensis. They had a 98.2-99.9% degree of identity in nucleotides and 94.7-96.1% in amino acids and encoded a polypeptide of 599 amino acids with a signal peptide targeting the chloroplast and three Cu-binding domains. The mature PPO showed high expression and enzyme activity after refolding the inclusion bodies in Escherichia coli BL21 (DE3) using pET30c expression vector, but low expression in Pichia pastoris GS115 using both the secretory and non-secretory vectors pPICZalphaA and pPICZA. The expression of PPO mutants demonstrated that the signal sequences prevented recombinant gene expression in E. coli. PPO activity was not affected by the C-terminus and was slightly inhibited by the CuC domain. Other domains were important for its activity. A 3.1-fold increase in PPO activity over non-recombinant controls was obtained by expressing the PPO fragment without signal sequences and the CuC domain in E. coli BL21 (DE3) using the pET30c vector.
多酚氧化酶（PPO）可用于有机合成和工业废物及染料的降解。缺乏酶源是其应用的主要障碍。本研究通过 PCR 从 5 个常见茶树品种的基因组 DNA 中克隆到一个全长 1.8kb、无内含子的 PPO 基因，其核苷酸序列的同源性为 98.2-99.9%，氨基酸序列的同源性为 94.7-96.1%，编码 599 个氨基酸的多肽，具有靶向叶绿体的信号肽和 3 个 Cu 结合结构域。利用 pET30c 表达载体在大肠杆菌 BL21（DE3）中复性包涵体后，成熟的 PPO 表达量高，酶活性也高，但在毕赤酵母 GS115 中利用分泌型和非分泌型载体 pPICZalphaA 和 pPICZA 表达时，表达量较低。PPO 突变体的表达表明，信号序列阻止了重组基因在大肠杆菌中的表达。PPO 活性不受 C 末端影响，CuC 结构域略有抑制，其他结构域对其活性很重要。通过在大肠杆菌 BL21（DE3）中利用 pET30c 载体表达无信号序列和 CuC 结构域的 PPO 片段，使 PPO 活性比非重组对照提高了 3.1 倍。
