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Cloning, microbial expression and structure–activity relationship of polyphenol oxidases from Camellia sinensis
Ist Teil von
Journal of biotechnology, 2010, Vol.145 (1), p.66-72
Ort / Verlag
Amsterdam: Elsevier B.V
Erscheinungsjahr
2010
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
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 1.8
kb 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 pPICZαA 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.