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Cloning, microbial expression and structure–activity relationship of polyphenol oxidases from Camellia sinensis

Paper ID Volume ID Publish Year Pages File Format Full-Text
24575 43527 2010 7 PDF Available
Title
Cloning, microbial expression and structure–activity relationship of polyphenol oxidases from Camellia sinensis
Abstract

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.

Keywords
Polyphenol oxidase; Camellia sinensis; Prokaryotic expression; Yeast expression; Pichia pastoris; Structure–activity relationship
First Page Preview
Cloning, microbial expression and structure–activity relationship of polyphenol oxidases from Camellia sinensis
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 145, Issue 1, 1 January 2010, Pages 66–72
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering