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Jeifu Shaw - One of the best experts on this subject based on the ideXlab platform.
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a novel recombinant chlorophyllase1 from chlamydomonas reinhardtii for the production of Chlorophyllide derivatives
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Yili Chou, Chihchung Yen, Longfang O Chen, Jeifu ShawAbstract:Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce Chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce Chlorophyllide derivatives.
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purification and immobilization of the recombinant brassica oleracea chlorophyllase 1 boclh1 on diaion cr11 as potential biocatalyst for the production of Chlorophyllide and phytol
Molecules, 2015Co-Authors: Yili Chou, Chihchung Yen, Longfang O Chen, Jeifu ShawAbstract:Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to Chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of Chlorophyllide and phytol.
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Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol
MDPI AG, 2015Co-Authors: Yili Chou, Chihchung Yen, Longfang O Chen, Jeifu ShawAbstract:Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to Chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of Chlorophyllide and phytol
Hitoshi Tamiaki - One of the best experts on this subject based on the ideXlab platform.
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in vitro hydrolysis of zinc Chlorophyllide a homologues by a bcic enzyme
Biochemistry, 2020Co-Authors: Mitsuaki Hirose, Jiro Harada, Hitoshi TamiakiAbstract:Chlorosomes in green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, or e molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of Chlorophyllide a. In this study, the in vitro enzymatic reactions of Chlorophyllide a analogues, C132-methylene- and ethylene-inserted zinc complexes, were examined using a BciC protein from Chlorobaculum tepidum. As the products, their hydrolyzed free carboxylic acids were observed without the corresponding demethoxycarbonylated compounds. The results showed that the in vivo demethoxycarbonylation of Chlorophyllide a by an action of the BciC enzyme would occur via two steps: (1) an enzymatic hydrolysis of a methyl ester at the C132-position, followed by (2) a spontaneous (nonenzymatic) decarboxylation in the resulting carboxylic acid.
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Chlorophyllide a oxidoreductase preferentially catalyzes 8 vinyl reduction over b ring reduction of 8 vinyl Chlorophyllide a in the late steps of bacteriochlorophyll biosynthesis
ChemBioChem, 2020Co-Authors: Haruki Yamamoto, Yusuke Tsukatani, Hitoshi Tamiaki, Tadashi Mizoguchi, Genji Kurisu, Yuichi FujitaAbstract:Bacteriochlorophyll a (BChl) is an essential pigment for anoxygenic photosynthesis. In late steps of the BChl biosynthesis of Rhodobacter capsulatus, the C8 vinyl group and C7=C8 double bond of 8-vinyl Chlorophyllide a (8 V-Chlide) are reduced by a C8 vinyl reductase (8VR), BciA, and a nitrogenase-like enzyme, Chlorophyllide a oxidoreductase (COR), respectively, to produce 3-vinyl-bacteriochlorphyllide a. Recently, we discovered 8VR activity in COR. However, the kinetic parameters of the COR 8VR activity remain unknown, while those of the COR C7=C8 reductase activity and BciA have been reported. Here, we determined the kinetic parameters of COR 8VR activity by using 8 V-Chlide. The Km value for 8 V-Chlide was 1.4 μM, which is much lower than the 6.2 μM determined for the C7=C8 reduction of Chlide. The kinetic parameters of the dual activities of COR suggest that COR catalyzes the reduction of the C8 vinyl group of 8 V-Chlide preferentially over C7=C8 reduction when both substrates are supplied during BChl biosynthesis.
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bcic catalyzed c132 demethoxycarbonylation of metal pheophorbide a alkyl esters
ChemBioChem, 2020Co-Authors: Mitsuaki Hirose, Jiro Harada, Misato Teramura, Hitoshi TamiakiAbstract:Bacteriochlorophyll c molecules self-aggregate to form large oligomers in the core part of chlorosomes, which are the main light-harvesting antenna systems of green photosynthetic bacteria. In the biosynthetic pathway of bacteriochlorophyll c, a BciC enzyme catalyzes the removal of the C132 -methoxycarbonyl group of Chlorophyllide a, which possesses a free propionate residue at the C17-position and a magnesium ion as the central metal. The in vitro C132 -demethoxycarbonylations of chlorophyll a derivatives with various alkyl propionate residues and central metals were examined by using the BciC enzyme derived from one green sulfur bacteria species, Chlorobaculum tepidum. The BciC enzymatic reactions of zinc pheophorbide a alkyl esters were gradually suppressed with an increase of the alkyl chain length in the C17-propionate residue (from methyl to pentyl esters) and finally the hexyl ester became inactive for the BciC reaction. Although not only the zinc but also nickel and copper complexes were demethoxycarbonylated by the BciC enzyme, the reactions were largely dependent on the coordination ability of the central metals: Zn>Ni>Cu. The above substrate specificity indicates that the BciC enzyme would not bind directly to the carboxy group of Chlorophyllide a, but would bind to its central magnesium to form the stereospecific complex of BciC with Chlorophyllide a, giving pyroChlorophyllide a, which lacks the (132 R)-methoxycarbonyl group.
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rapid c8 vinyl reduction of divinyl Chlorophyllide a by bcia from rhodobacter capsulatus
Journal of Photochemistry and Photobiology A-chemistry, 2018Co-Authors: Chihiro Azai, Hitoshi Tamiaki, Tadashi Mizoguchi, Manami Kobayashi, Kazuki Terauchi, Yusuke TsukataniAbstract:Abstract Divinyl-Chlorophyllide a (DV-Chlide a ) is a universal precursor for chlorophyll or bacteriochlorophyll biosynthesis in all photosynthetic organisms. Previous mutational analyses revealed that BciA works for reduction of the C8-vinyl group of DV-Chlide a. Chlorophyllide oxidoreductase (COR) reduces the C7=C8 double bond of Chlide a bearing the C8-ethyl group, but also potentially catalyzes the reduction of the C8-vinyl group of DV-Chlide a. In this study, we prepared a recombinant BciA protein from the purple photosynthetic bacterium Rhodobacter capsulatus and analyzed its C8-vinyl reduction activity towards DV-Chlide a . BciA formed a functional oligomeric complex consisting of at least three rigid dimers. BciA required NADPH as an electron donor for its C8-vinyl reduction activity. The enzymatic activity of BciA towards the substrate DV-Chlide a was much higher than that of COR towards Chlide a . Phylogenetic distribution and the enzymatic parameters of BciA and COR suggest that BciA is a more recently acquired auxiliary C8-vinyl reductase, attained to meet the high demand for bacteriochlorophylls used to produce large amounts of light-harvesting complexes.
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Rhodobacter sphaeroides mutants overexpressing Chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways
Scientific reports, 2015Co-Authors: Yusuke Tsukatani, Jiro Harada, Jiro Nomata, Haruki Yamamoto, Yuichi Fujita, Tadashi Mizoguchi, Hitoshi TamiakiAbstract:Rhodobacter sphaeroides mutants overexpressing Chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways
Constantin A Rebeiz - One of the best experts on this subject based on the ideXlab platform.
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chloroplast biogenesis 84 solubilization and partial purification of membrane bound 4 vinyl Chlorophyllide a reductase from etiolated barley leaves
Analytical Biochemistry, 2001Co-Authors: Vladimir L Kolossov, Constantin A RebeizAbstract:Abstract [4-Vinyl] Chlorophyllide a reductase (4VCR) is a key enzyme of the chlorophyll (Chl) biosynthetic pathway. It catalyzes the conversion of divinyl Chlorophyllide (Chlide) a to monovinyl Chlide a by reduction of the vinyl group at position 4 of the macrocycle to ethyl. 4VCR is a membrane-bound enzyme, embedded in etioplast and etiochloroplast membranes. A study of the regulation and properties of this enzyme is mandatory for a comprehensive understanding of the biosynthetic heterogeneity of Chl biosynthesis. Solubilization and partial purification of 4VCR are described for the first time. The enzyme was solubilized with 5 mM Chaps and was partially purified by chromatography on DEAE-Sephacel and Cibacron Blue 3GA-1000 agarose. An overall 20-fold purification was achieved. The partially purified enzyme was stable for several months at −80°C.
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chloroplast biogenesis 72 a 4 vinyl Chlorophyllide a reductase assay using divinyl Chlorophyllide a as an exogenous substrate
Analytical Biochemistry, 1995Co-Authors: Ramin Parham, Constantin A RebeizAbstract:Abstract [4-Vinyl] Chlorophyllide a reductase (4VCR) catalyzes the the conversion of 2,4-divinyl Chlorophyllide a (DV Chlide a ) to 2-vinyl,4-ethyl Chlorophyllide a (MV Chlide a ) via an NADPH-dependent reaction. MV Chlide a is the immediate precursor of monovinyl chlorophyll a in plants. In etiolated cucumber ( Cucumis sativus L.) cotyledons, 4VCR is a plastidic membrane-bound enzyme. Further research on this enzyme required the development of an assay that utilizes DV Chlide a as an exogenous substrate. Such an assay is now described. It involves conversion of exogenous DV Chlide a to MV Chlide a at high rates by etioplast membranes of cucumber, corn ( Zea mays L.), and barley ( Hordeum vulgare L.). 4VCR exhibits high activity between 30 and 40°C and in the pH range of 6.3 to 7.0. Activity is quasilinear for the first 60 s of incubation.
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Chloroplast biogenesis: [4-vinyl] Chlorophyllide a reductase is a divinyl Chlorophyllide a-specific, NADPH-dependent enzyme.
Biochemistry, 1992Co-Authors: Ramin Parham, Constantin A RebeizAbstract:Some properties of [4-vinyl] Chlorophyllide a reductase are described. This enzyme converts divinyl Chlorophyllide a to monovinyl Chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] Chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protoChlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] Chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl Chlorophyllide a, and it does not reduce divinyl protoChlorophyllide a to monovinyl protoChlorophyllide a. The conversion of divinyl protoChlorophyllide a to monovinyl protoChlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protoChlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protoChlorophyllide a and divinyl Chlorophyllide a to their respective 4-ethyl analogues.
Yusuke Tsukatani - One of the best experts on this subject based on the ideXlab platform.
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Chlorophyllide a oxidoreductase preferentially catalyzes 8 vinyl reduction over b ring reduction of 8 vinyl Chlorophyllide a in the late steps of bacteriochlorophyll biosynthesis
ChemBioChem, 2020Co-Authors: Haruki Yamamoto, Yusuke Tsukatani, Hitoshi Tamiaki, Tadashi Mizoguchi, Genji Kurisu, Yuichi FujitaAbstract:Bacteriochlorophyll a (BChl) is an essential pigment for anoxygenic photosynthesis. In late steps of the BChl biosynthesis of Rhodobacter capsulatus, the C8 vinyl group and C7=C8 double bond of 8-vinyl Chlorophyllide a (8 V-Chlide) are reduced by a C8 vinyl reductase (8VR), BciA, and a nitrogenase-like enzyme, Chlorophyllide a oxidoreductase (COR), respectively, to produce 3-vinyl-bacteriochlorphyllide a. Recently, we discovered 8VR activity in COR. However, the kinetic parameters of the COR 8VR activity remain unknown, while those of the COR C7=C8 reductase activity and BciA have been reported. Here, we determined the kinetic parameters of COR 8VR activity by using 8 V-Chlide. The Km value for 8 V-Chlide was 1.4 μM, which is much lower than the 6.2 μM determined for the C7=C8 reduction of Chlide. The kinetic parameters of the dual activities of COR suggest that COR catalyzes the reduction of the C8 vinyl group of 8 V-Chlide preferentially over C7=C8 reduction when both substrates are supplied during BChl biosynthesis.
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rapid c8 vinyl reduction of divinyl Chlorophyllide a by bcia from rhodobacter capsulatus
Journal of Photochemistry and Photobiology A-chemistry, 2018Co-Authors: Chihiro Azai, Hitoshi Tamiaki, Tadashi Mizoguchi, Manami Kobayashi, Kazuki Terauchi, Yusuke TsukataniAbstract:Abstract Divinyl-Chlorophyllide a (DV-Chlide a ) is a universal precursor for chlorophyll or bacteriochlorophyll biosynthesis in all photosynthetic organisms. Previous mutational analyses revealed that BciA works for reduction of the C8-vinyl group of DV-Chlide a. Chlorophyllide oxidoreductase (COR) reduces the C7=C8 double bond of Chlide a bearing the C8-ethyl group, but also potentially catalyzes the reduction of the C8-vinyl group of DV-Chlide a. In this study, we prepared a recombinant BciA protein from the purple photosynthetic bacterium Rhodobacter capsulatus and analyzed its C8-vinyl reduction activity towards DV-Chlide a . BciA formed a functional oligomeric complex consisting of at least three rigid dimers. BciA required NADPH as an electron donor for its C8-vinyl reduction activity. The enzymatic activity of BciA towards the substrate DV-Chlide a was much higher than that of COR towards Chlide a . Phylogenetic distribution and the enzymatic parameters of BciA and COR suggest that BciA is a more recently acquired auxiliary C8-vinyl reductase, attained to meet the high demand for bacteriochlorophylls used to produce large amounts of light-harvesting complexes.
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Rhodobacter sphaeroides mutants overexpressing Chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways
Scientific reports, 2015Co-Authors: Yusuke Tsukatani, Jiro Harada, Jiro Nomata, Haruki Yamamoto, Yuichi Fujita, Tadashi Mizoguchi, Hitoshi TamiakiAbstract:Rhodobacter sphaeroides mutants overexpressing Chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways
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Chlorophyllide a oxidoreductase works as one of the divinyl reductases specifically involved in bacteriochlorophyll a biosynthesis
Journal of Biological Chemistry, 2014Co-Authors: Jiro Harada, Ayumi Tanaka, Yusuke Tsukatani, Tadashi Mizoguchi, Makio Yokono, Hitoshi TamiakiAbstract:Bacteriochlorophyll a is widely distributed among anoxygenic photosynthetic bacteria. In bacteriochlorophyll a biosynthesis, the reduction of the C8 vinyl group in 8-vinyl-Chlorophyllide a is catalyzed to produce Chlorophyllide a by an 8-vinyl reductase called divinyl reductase (DVR), which has been classified into two types, BciA and BciB. However, previous studies demonstrated that mutants lacking the DVR still synthesize normal bacteriochlorophyll a with the C8 ethyl group and suggested the existence of an unknown "third" DVR. Meanwhile, we recently observed that Chlorophyllide a oxidoreductase (COR) of a purple bacterium happened to show the 8-vinyl reduction of 8-vinyl-Chlorophyllide a in vitro. In this study, we made a double mutant lacking BciA and COR of the purple bacterium Rhodobacter sphaeroides in order to investigate whether the mutant still produces pigments with the C8 ethyl group or if COR actually works as the third DVR. The single mutant deleting BciA or COR showed production of the C8 ethyl group pigments, whereas the double mutant accumulated 8-vinyl-Chlorophyllide, indicating that there was no enzyme other than BciA and COR functioning as the unknown third DVR in Rhodobacter sphaeroides (note that this bacterium has no bciB gene). Moreover, some COR genes derived from other groups of anoxygenic photosynthetic bacteria were introduced into the double mutant, and all of the complementary strains produced normal bacteriochlorophyll a. This observation indicated that COR of these bacteria performs two functions, reductions of the C8 vinyl group and the C7=C8 double bond, and that such an activity is probably conserved in the widely ranging groups.
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Completion of biosynthetic pathways for bacteriochlorophyll g in Heliobacterium modesticaldum: The C8-ethylidene group formation.
Biochimica et biophysica acta, 2013Co-Authors: Yusuke Tsukatani, Haruki Yamamoto, Yuichi Fujita, Tadashi Mizoguchi, Hitoshi TamiakiAbstract:Abstract Heliobacteria have the simplest photosynthetic apparatus, i.e., a type-I reaction center lacking a peripheral light-harvesting complex. Bacteriochlorophyll (BChl) g molecules are bound to the reaction center complex and work both as special-pair and antenna pigments. The C8-ethylidene group formation for BChl g is the last missing link in biosynthetic pathways for bacterial special-pair pigments, which include BChls a and b as well. Here, we report that Chlorophyllide a oxidoreductase (COR) of Heliobacterium modesticaldum catalyzes the C8-ethylidene formation from 8-vinyl-Chlorophyllide a , producing bacterioChlorophyllide g , the direct precursor for BChl g without the farnesyl tail. The finding led to plausible biosynthetic pathways for 8 1 -hydroxy-chlorophyll a , a primary electron acceptor from the special pair in heliobacterial reaction centers. Proposed catalytic mechanisms on hydrogenation reaction of the ethylidene synthase-type CORs are also discussed.
Yili Chou - One of the best experts on this subject based on the ideXlab platform.
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a novel recombinant chlorophyllase1 from chlamydomonas reinhardtii for the production of Chlorophyllide derivatives
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Yili Chou, Chihchung Yen, Longfang O Chen, Jeifu ShawAbstract:Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce Chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce Chlorophyllide derivatives.
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purification and immobilization of the recombinant brassica oleracea chlorophyllase 1 boclh1 on diaion cr11 as potential biocatalyst for the production of Chlorophyllide and phytol
Molecules, 2015Co-Authors: Yili Chou, Chihchung Yen, Longfang O Chen, Jeifu ShawAbstract:Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to Chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of Chlorophyllide and phytol.
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Purification and Immobilization of the Recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as Potential Biocatalyst for the Production of Chlorophyllide and Phytol
MDPI AG, 2015Co-Authors: Yili Chou, Chihchung Yen, Longfang O Chen, Jeifu ShawAbstract:Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to Chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of Chlorophyllide and phytol