Cloning, microbial expression and structure-activity relationship of polyphenol oxidases from Camellia sinensis.
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.
Wu
Yi-Liang
YL
South China Normal University, Shipai, Guangzhou, Guangdong, China.
Pan
Li-Ping
LP
Yu
Si-Li
SL
Li
Hai-Hang
HH
D000595:Amino Acid Sequence; D028241:Camellia sinensis / Q000235:genetics*; D004156:Catechol Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D010940:Plant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004156:Catechol Oxidase