title: A novel recombinant chlorophyllase from cyanobacterium Cyanothece sp. ATCC 51142 for the production of bacteriochlorophyllide a.
abstract: Bacteriopheophorbide a (BPheid a) is used as a precursor for bacteriochlorin a (BCA), which can be used for photodynamic therapy in both in vitro and in vivo biochemical applications. This study successfully isolated and expressed a photosynthetic bacterium (Cyanothece sp. ATCC 51142) chlorophyllase called CyanoCLH, which can be used as a biocatalyst for the production of a BCA precursor by degrading bacteriochlorophyll a (BChl a). Substrate specificity and enzyme kinetic analyses were performed and the results verified that the recombinant CyanoCLH preferred hydrolyzing BChl a to produce bacteriochlorophyllide a (BChlide a), which can be converted to BPheid a by removing magnesium ion. The recombinant CyanoCLH was cloned and expressed in Escherichia coli BL-21 (DE3), and its molecular weight was 54.7 kDa. The deduced amino acid sequence of the recombinant CyanoCLH comprised a unique lipase-motif GHSLG, which differs from the GHSRG sequence of other plants and lacks a histidine of the typical and conserved catalytic triad Ser-Asp-His. The recombinant CyanoCLH was subjected to biochemical analyses, and the results indicated that its optimal pH and temperature were 7.0 and 60 °C, respectively.
authors.affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan.|Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Yi-Li
authors.initials: YL
authors.lastname: Chou
authors.affiliation: Biotechnology Division, Taiwan Agricultural Research Institute, Taichung, Taiwan.
authors.forename: Ya-Lin
authors.initials: YL
authors.lastname: Lee
authors.affiliation: Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan.
authors.forename: Chih-Chung
authors.initials: CC
authors.lastname: Yen
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Long-Fang O
authors.initials: LF
authors.lastname: Chen
authors.affiliation: Department of Nutrition, I-Shou University, Kaohsiung, Taiwan.
authors.forename: Li-Chiun
authors.initials: LC
authors.lastname: Lee
authors.affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan.|Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D025541:Bacteriochlorophyll A / Q000096:biosynthesis*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D046889:Cyanothece / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D025541:Bacteriochlorophyll A; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase
keywords: Cyanothece sp. ATCC 51142; bacteriochlorin a; biocatalyst; biochemical analyses; chlorophyllase; kinetic analyses