Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/62056
標題: Genes and Biochemical Characterization of Three Novel Chlorophyllase Isozymes from Brassica oleracea
作者: Lee, G.C.
蕭介夫
Chepyshko, H.
Chen, H.H.
Chu, C.C.
Chou, Y.F.
Akoh, C.C.
Shaw, J.F.
關鍵字: Chlorophyllase
Isozyme
Broccoli (Brassica oleracea)
subcellular-localization
candida-rugosa
higher-plants
degradation
purification
breakdown
senescence
hydrolase
lipases
cloning
期刊/報告no:: Journal of Agricultural and Food Chemistry, Volume 58, Issue 15, Page(s) 8651-8657.
摘要: Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.
URI: http://hdl.handle.net/11455/62056
ISSN: 0021-8561
文章連結: http://dx.doi.org/10.1021/jf1016384
Appears in Collections:食品暨應用生物科技學系

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