Sitian Gu1,2, Xiaojun Dai3, Zhengjun Xu3, Qiwen Niu3, Jiang Jiang4, Yuanfa Liu5. 1. State Key Laboratory of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, National Engineering Research Center for Functional Food, National Engineering Laboratory for Cereal Fermentation Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Jiangsu, 214122, Wuxi, People's Republic of China. gusitian1983@163.com. 2. Wilmar Biotechnology Research & Development Center Co., Ltd, 200137, Shanghai, People's Republic of China. gusitian1983@163.com. 3. Wilmar Biotechnology Research & Development Center Co., Ltd, 200137, Shanghai, People's Republic of China. 4. State Key Laboratory of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, National Engineering Research Center for Functional Food, National Engineering Laboratory for Cereal Fermentation Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Jiangsu, 214122, Wuxi, People's Republic of China. 5. State Key Laboratory of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, National Engineering Research Center for Functional Food, National Engineering Laboratory for Cereal Fermentation Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Jiangsu, 214122, Wuxi, People's Republic of China. yfliu@jiangnan.edu.cn.
Abstract
BACKGROUND: Chlorophyllase catalyzes the hydrolysis of chlorophyll and produces chlorophyllide and phytol. Cyanobacterial chlorophyllases are likely to be more highly heterologously expressed than plant chlorophyllases. A novel recombinant chlorophyllase from the cyanobacterium Oscillatoria acuminata PCC 6304 was successfully expressed in Escherichia coli BL21(DE3). RESULTS: The putative N-terminal 28-amino-acid signal peptide sequence of O. acuminata chlorophyllase (OaCLH) is essential for its activity, but may confer poor solubility on OaCLH. The C-terminal fusion of a 6 × His tag caused a partial loss of activity in recombinant OaCLH, but an N-terminal 6 × His tag did not destroy its activity. The optimal pH and temperature for recombinant OaCLH activity are 7.0 and 40 °C, respectively. Recombinant OaCLH has hydrolysis activities against chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, but prefers chlorophyll b and chlorophyll a as substrates. The results of site-directed mutagenesis experiments indicated that the catalytic triad of OaCLH consists of Ser159, Asp226, and His258. CONCLUSIONS: The high-level expression and broad substrate specificity of recombinant OaCLH make it suitable for genetically engineering and a promising biocatalyst for industrial production, with applications in vegetable oil refining and laundry detergents.
BACKGROUND: Chlorophyllase catalyzes the hydrolysis of chlorophyll and produces chlorophyllide and phytol. Cyanobacterial chlorophyllases are likely to be more highly heterologously expressed than plant chlorophyllases. A novel recombinant chlorophyllase from the cyanobacteriumOscillatoria acuminata PCC 6304 was successfully expressed in Escherichia coli BL21(DE3). RESULTS: The putative N-terminal 28-amino-acid signal peptide sequence of O. acuminata chlorophyllase (OaCLH) is essential for its activity, but may confer poor solubility on OaCLH. The C-terminal fusion of a 6 × His tag caused a partial loss of activity in recombinant OaCLH, but an N-terminal 6 × His tag did not destroy its activity. The optimal pH and temperature for recombinant OaCLH activity are 7.0 and 40 °C, respectively. Recombinant OaCLH has hydrolysis activities against chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, but prefers chlorophyll b and chlorophyll a as substrates. The results of site-directed mutagenesis experiments indicated that the catalytic triad of OaCLH consists of Ser159, Asp226, and His258. CONCLUSIONS: The high-level expression and broad substrate specificity of recombinant OaCLH make it suitable for genetically engineering and a promising biocatalyst for industrial production, with applications in vegetable oil refining and laundry detergents.
Authors: Elisabeth Gasteiger; Alexandre Gattiker; Christine Hoogland; Ivan Ivanyi; Ron D Appel; Amos Bairoch Journal: Nucleic Acids Res Date: 2003-07-01 Impact factor: 16.971
Authors: T Tsuchiya; H Ohta; K Okawa; A Iwamatsu; H Shimada; T Masuda; K Takamiya Journal: Proc Natl Acad Sci U S A Date: 1999-12-21 Impact factor: 11.205