Article pubs.acs.org/JAFC
A Novel Recombinant Chlorophyllase1 from Chlamydomonas reinhardtii for the Production of Chlorophyllide Derivatives Yi-Li Chou,† Chia-Yun Ko,‡ Chih-Chung Yen,§,∥ Long-Fang O. Chen,*,‡ and Jei-Fu Shaw*,†,∥ †
Department of Biological Science and Technology, I-Shou University, Kaohsiung 82445, Taiwan Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan § Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung 40227, Taiwan ∥ Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan ‡
ABSTRACT: 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. KEYWORDS: Chlamydomonas reinhardtii, chlorophyllase, chlorophyllide derivatives, enzyme kinetics
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INTRODUCTION
Chl breakdown during leaf senescence in Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum.6−8 In the other Chl degradation pathway, Chl first loses the magnesium ion required to produce pheophytin (Phein), and PPH then catalyzes Phein, removing phytol to yield Pheide.6 Afterward, the free phytol can be utilized to form vitamin E through tocopherol biosynthesis.9 The deduced amino acid sequences of all cloned Chlases share a highly conserved lipase motif GXSXG,10 in which serine is conserved in the catalytic triad (Ser-Asp-His).11 The first published Chlase gene was isolated from Chenopodium album leaves and encodes 347 amino acids (CaCLH). CaCLH contains typical endoplasmic reticulum and vacuolar-sorting signal peptides and is considered a vacuolar enzyme.10 AtCLH1 and AtCLH2 isozymes were isolated from A. thaliana,10 and Cterminal green fluorescent protein (GFP), fused with AtCLH1 and AtCLH2, was located outside the chloroplast, being transiently expressed in the mesophyll protoplast.12,13 Chlase from Citrus sinensis (CsCLH) was localized in a chloroplast membrane fraction in tobacco protoplasts.14,15 BoCLH1 and BoCLH2 from Brassica oleracea were predicted to be expressed in it is the chloroplast.16,17 On the basis of subcellular fractionations, the recombinant GbCLH from Ginkgo biloba was identified in the thylakoid membranes of the chloroplast.18 According to an in situ immunofluorescence study, ethyleneinduced Citrus limon Chlase (ClCLH) was located in the plastid.19,20 However, the subcellular localization of some
Chlorophyll (Chl) is the most abundant pigment on Earth involved in the sunlight absorption required for photosynthesis. Chl biosynthesis and metabolism are regulated by plant development, stress, and environmental responses.1,2 Previous studies have indicated that land plant chlorophyllase (Chlase, EC 3.1.1.14) is an important enzyme involved in Chl degradation during fruit ripening and pathogen infection.1,3 The Chl degradation pathway begins with the removal of phytol by Chlase, producing chlorophyllide (Chlide),3,4 and Mg-dechelatase then removes the magnesium ion from the tetrapyrrole macrocycle to yield pheophorbide (Pheide) in vivo5 (Figure 1). Recently, studies have indicated that pheophytin pheophorbide hydrolase (PPH) is essential for
Figure 1. Possible models for an early step of Chl metabolism. In this model, Chlase catalyzes Chl to produce Chlide and phytol and Mgdechelatase then removes the magnesium ion from Chlide to yield Pheide. Chl a and Chl b have one tetrapyrrole macrocycle and one reduced pyrrole ring. BChl a has one tetrapyrrole macrocycle and two reduced pyrrole rings (bacteriochlorin). Black asterisks indicate the site at which the Chl a and Chl b lack the reduced pyrrole ring compared with BChl a. © 2015 American Chemical Society
Received: Revised: Accepted: Published: 9496
June 7, 2015 October 17, 2015 October 19, 2015 October 19, 2015 DOI: 10.1021/acs.jafc.5b02787 J. Agric. Food Chem. 2015, 63, 9496−9503
Article
Journal of Agricultural and Food Chemistry
of C. reinhardtii, CLH1N (5′-GAATTCCCTTCCACGCAGTTC-3′) and CLH1C, were used to amplify and generate the CrCLH1 cDNA fragment [National Center for Biotechnology Information (NCBI) accession number 5721184]. The PCR conditions are listed as follows: 95 °C for 5 min, 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min for 35 cycles and then 72 °C for 7 min. The 969 bp DNA fragment was cloned into a pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing. Overexpression of CrCLH1 in E. coli. For gene cloning, we used one CrCLH1 forward primer containing an EcoRI site upstream from the translation start site of the open reading frame (ORF) and one reverse primer containing an XhoI site downstream from the ORF. After restriction enzyme digestion, the EcoRI−XhoI CrCLH1 fragment was cloned into the pET-21a (+) expression vector (Novagen, Darmstadt, Germany). The recombinant vector was transformed into an E. coli BL21(DE3) strain. The recombinant CrCLH1 included an N-terminal T7-Tag amino acid sequence (MASMTGGQQMG) for enhanced protein purification. Protein Expression and Purification. CrCLH1 fused with an Nterminal T7-Tag was overexpressed in the E. coli BL21(DE3) strain that was induced by adding a final concentration of 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to a refreshed culture at 16 °C for more than 20 h. The cells were harvested by centrifugation and lysed using a French press (Thermo Scientific) in a T7-Tag binding buffer [4.29 mM Na2HPO4, 1.47 mM KH2PO4, 2.7 mM KCl, 13.7 mM NaCl, 1% Tween 20, 0.02% sodium azide, and 0.24% Triton X-100 (pH 7.3)]. After centrifugation (10000g for 15 min at 4 °C), the supernatant was applied to the T7-Tag affinity chromatography column (Novagen). The recombinant CrCLH1 was eluted using a T7Tag elution buffer. The recombinant CrCLH1 concentration was quantified using the Bradford assay (Bio-Rad, Hercules, CA). Phylogenetic Analysis. The Chlase amino acid sequences of different species were obtained from the NCBI database. The amino acid sequences of AtCLH1 and AtCLH2 (GenBank entries NP_564094.1 and NP_199199.1) from A. thaliana, BoCLH1 and BoCLH2 (GenBank entries AAN51933.1 and AAN51934.1) from B. oleracea, GbCLH (GenBank entry AAP44978.1) from G. biloba, PbCLH (GenBank entry AAP92160.2) from P. betle L., TaCLH (GenBank entry AHJ14565.1) from T. aestivum, ClCLH (GenBank entry ACI06105.1) from Ci. limon, CsCLH (GenBank entry AAF59834.1) from Ci. sinensis, CaCLH (GenBank entry BAA93635.1) from Ch. album, PpCLH (GenBank entry EDQ81786.1) from Physcomitrella patens, CrCLH1 (GenBank entry XP001695577.1) from C. reinhardtii, CvCLH1 and CvCLH2 (GenBank entries EFN59881.1 and EFN58050.1) from Chlorella variabilis, OaCLH (GenBank entry AFY81906.1) from Oscillatoria acuminata, and CmCLH (GenBank entry WP015159362) from Chamaesiphon minutus were aligned and analyzed using MEGA 6.38 A consensus tree was created through the Maximum Likelihood method by using a 1000 bootstrap data set. The numbers in the tree indicated the percentage of bootstrap replicates that supported each node. The amino acid sequences of CrCLH1, CvCLH1, CvCLH2, OaCLH, CmCLH, AtCLH1, and AtCLH2 were aligned using ClustalW.39 Transient Transformation of A. thaliana Protoplasts. For subcellular localization, plasmids were transformed into Arabidopsis protoplasts by using a polyethylene glycol method.40 The cDNA encoding full-length CrCLH1 was amplified using a forward primer (5′-TCTAGAATGCCTTCCACGCAGTTC-3′) and a reverse primer (5′-CCCGGGTTGCGGCGACTGCAT-3′), including the XbaI and XmaI sites on the forward primer and reverse primer, respectively. The gusA gene was replaced with the sGFP gene to form the pBI221G vector. After restriction enzyme digestion, the XbaI−XmaI CrCLH1 fragment was cloned into the pBI-221G vector to form the CaMV35SP::CrCLH1-sGFP construct. The pBI221G vector was used as a negative control. The protoplasts were isolated from 3−4-week-old well-expanded leaves yet to flower. The protoplasts (100 μL; approximately 2 × 104 protoplasts) were transfected with 20 μg of plasmid DNA. Transformed cells were incubated for 16−18 h in the dark at room
Chlases, such as PbCLH and TaCLH isolated from Piper betle L.21 and Triticum aestivum,22 respectively, is not clearly understood. Chlase has a phytyl-removing activity of Chl to yield Chlide;16,18,22 subsequently, Chlide loses the magnesium ion under acidic conditions to form Pheide in vitro.23,24 Chlide and Chlide derivatives (such as Pheide) have been demonstrated to have antiviral, antioxidant, antimutagenic, and anticarcinogenic activity in vitro;23−27 for instance, Chlide and Pheide effectively reduced the level of formation of the aflatoxin B1−DNA adduct caused by hepatitis B virus infection in murine hepatoma cells23 and acted as free radical scavengers and chelating agents in protecting human lymphocyte DNA from oxidative damage.24 Furthermore, Pheide a conjugated with cancer-targeting moieties might form an efficient photodynamic reagent for cancer therapy.26,27 In addition, when the magnesium ion of Chlide is replaced with a copper ion to form a Cu−Chlide derivative, it can act as a potential antiviral agent for preventing hepatitis B virus infections.25 Many recombinant Chlases in plants have been isolated, but recombinant BoCLH1, BoCLH2,16 and TaCLH22 exhibit low catalytic efficiency during Chl degradation. Our objective was to identify Chlases with high catalytic efficiency in Chl degradation to obtain Chlide and its derivatives. Although Chlases from various algal species have been partially purified and characterized,28−32 gene cloning and protein expression of algal Chlases have not been performed. Chlamydomonas reinhardtii is a single-cell green alga with multiple mitochondria and a cup-shaped chloroplast that is a particularly crucial model organism in photosynthesis and biofuel-related studies.33,34 The C. reinhardtii genome has been completely sequenced;35 therefore, the novel Chlase1 gene (CrCLH1) can be isolated and investigated using C. reinhardtii. In this study, recombinant CrCLH1 was expressed in an Escherichia coli system and exhibited excellent catalytic efficiency in Chl degradation. The data indicate that the recombinant CrCLH1 can be used as a biocatalyst for a dephytylation activity to produce Chlide and its derivatives. Naturally occurring Chl is broken down into more than 10 billion tons per year,36 facilitating convenient extraction of Chl from agricultural waste. As a raw material, Chl can be converted to high-value Chlide derivatives by using recombinant CrCLH1 and thus can generate opportunities for developing new green industries.
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MATERIALS AND METHODS
Sample Preparation. The C. reinhardtii CC-503 cw92 mt+ strain, a cell wall-deficient mutant, was obtained from the Chlamydomonas Center (http://www.chlamy.org/), and 100 mL liquid cultures in TAP medium were maintained in Erlenmeyer flasks under orbital shaking at 25 °C in white light.37 The cultures were harvested through centrifugation during the logarithmic growth phase (approximately 7 × 106 cells/mL) and then ground in liquid nitrogen. Isolation of RNA and Cloning of CrCLH1 cDNA. Total RNA was extracted using a PureLinkRNA mini kit (Thermo Fisher). The C. reinhardtii cells (100 mg) were ground into powder by using a mortar and pestle in liquid nitrogen. The powdered sample was added to 0.6 mL of a lysis buffer containing 1% 2-mercaptoethanol and then mixed vigorously. After centrifugation, a half volume of 100% ethanol was added to the supernatant and mixed through vortexing. The sample was transferred to a spin cartridge and centrifuged at 12000g for 15 s. Subsequently, the spin cartridge was washed using wash buffers I and II. Finally, the total RNA was eluted in 50 μL of RNase-free water. cDNA was synthesized using the OneStep reverse transcription polymerase chain reaction (PCR) system (Qiagen) and the CLH1C primer (5′-CTCGAGTTGCGGCGACTGCAT-3′). The primer pairs 9497
DOI: 10.1021/acs.jafc.5b02787 J. Agric. Food Chem. 2015, 63, 9496−9503
Article
Journal of Agricultural and Food Chemistry
calculated the kinetic parameters kcat and Km according to the Michaelis−Menten kinetics for nonlinear regression (Origin software, version 6.1) and generated a plot of reaction velocity versus substrate concentration. Data from three independent reactions were used to calculate the mean for each kinetic analysis.
temperature being subjected to laser scanning confocal microscopic analysis (Zeiss LSM 510 Meta Confocal Microscope, Carl Zeiss). Chlase Activity Assay. The Chlase activity was measured according to the method described by Lee et al.16 Three substrates [Chl a from Anacystis nidulans algae, Chl b from spinach, and bacteriochlorophyll a (BChl a) from Rhodopseudomonas sphaeroides] were purchased from Sigma Chemical Co. (St. Louis, MO). The reaction mixture contained 1 μg of enzyme protein, 65 μL of reaction buffer [100 mM sodium phosphate and 0.24% Triton X-100 (pH 7.0)], and 7.5 μL of acetone-dissolved substrates (at a final concentration of 500 μM). The reaction mixture was incubated in a shaking water bath at 40 °C for 30 min. The enzyme reaction was stopped by adding 1 mL of a stop buffer [4:6:1 (v/v) acetone/nhexane/10 mM KOH]. The reaction mixture was vigorously vortexed and centrifuged at 12000g for 3 min to obtain two separate layers (or phases): the upper layer contained the unreacted Chl a, Chl b, or BChl a in n-hexane, and the lower layer contained the product Chlide a, Chlide b, or BChlide a in an aqueous acetone solution. The absorbance of the aqueous acetone layer was measured at 667 nm for Chlide a, 650 nm for Chlide b, and 771 nm for BChlide a by using a spectrophotometer.41 Millimolar extinction coefficients of 42.0, 42.1, and 54.1 mM−1 cm−1 were employed for calculating the amount of Chlide a, Chlide b, and BChlide a produced, respectively.22,41 One unit of enzyme activity was defined as 1 μmol of the substrate hydrolyzed by an enzyme per minute at 40 °C. The enzyme specific activity (SA) was defined as the enzyme activity (units) per milligram of enzyme proteins. Effects of pH and Temperature on Chlase Activity and Stability. The effects of pH and temperature on the purified recombinant CrCLH1 were determined by measuring its Chl a hydrolytic activity. The optimal pH was investigated by preparing reaction buffers in the pH range of 3.0−5.0 containing 50 mM sodium acetate and in the pH range of 6.0−10.0 containing 50 mM bicine, CAPS, sodium acetate, and bis-trispropane. The pH was adjusted by adding HCl or NaOH. The enzyme reaction of the recombinant CrCLH1 was performed at 40 °C for 30 min and considered the standard. To analyze the pH stability of the recombinant CrCLH1, the enzyme solutions were incubated in reaction buffers [containing 0.24% Triton X-100 (pH 3.0−10.0)] at 40 °C for 30 min. Enzyme activity was subsequently measured using the aforementioned standard method. To determine the effect of temperature on the recombinant CrCLH1, reactions were performed at temperatures ranging from 20 to 80 °C for 30 min. To analyze thermal stability, the enzyme solutions were preincubated at temperatures ranging from 20 to 80 °C for 10 min and subsequently cooled before activity analysis. All reactions were performed in triplicate. The resulting average values and standard deviations are presented. The highest activity is presented as 100%, with the other activities presented as percentages relative to the highest activity. High-Performance Liquid Chromatography (HPLC) Analysis of Chl Catabolites. To analyze the Chl catabolites (Chl, Chlide, and Pheide), the enzyme reaction mixtures were incubated in two pH reaction buffers [100 mM sodium phosphate with 0.24% Triton X-100 (pH 6.0), and 50 mM sodium acetate with 0.24% Triton X-100 (pH 5.0)] at 40 °C for 30 min. After the addition of 1 mL of acetone and centrifugation, the supernatants were analyzed using HPLC as described previously.42 Each Chl catabolite was detected according to absorption at a wavelength of 665 nm and identified by their absorption spectra, peak ratios, and comigration with authentic standards.6 Enzyme Kinetic Assay. To study the enzyme kinetics of the purified recombinant CrCLH1, reactions were performed at 40 °C for 30 min by using spectrophotometric determination. Five concentrations (100, 200, 300, 400, and 500 μM) of substrates were prepared in a reaction buffer at pH 6.0. The initial reaction velocity (V0) was determined according to the production of Chlide a, Chlide b, and BChlide a per minute and was detected on the basis of the maximal absorbance of the product (667, 650, and 771 nm, respectively). We
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RESULTS AND DISCUSSION Amino Acid Sequence Analysis of Recombinant CrCLH1. The phylogenetic tree classified 16 Chlases from various species into three clades (Figure2). The first clade
Figure 2. Phylogenetic analysis of Chlases. The amino acid sequences of AtCLH1 and AtCLH2 from A. thaliana, BoCLH1 and BoCLH2 from B. oleracea, GbCLH from G. biloba, PbCLH from P. betle, TaCLH from T. aestivum, ClCLH from Ci. limon, CsCLH from Ci. sinensis, CaCLH from Ch. album, PpCLH from Phy. patens, CrCLH1 from C. reinhardtii, CvCLH1 and CvCLH2 from Chl. variabilis, OaCLH from O. acuminata, and CmCLH from Cha. minutus were aligned and analyzed using MEGA 6. A consensus tree was created through the Maximum Likelihood method by using a 1000 bootstrap data set. The numbers in the tree indicate the percentage of bootstrap replicates that support each node.
comprised Chlases belonging to land plants, namely, AtCLH1/ 2, BoCLH1/2, ClCLH, CsCLH, CaCLH, GbCLH, PbCLH, PpCLH, and TaCLH. The second clade comprised Chlases belonging to chlorophyta, namely, CrCLH1 and CvCLH1/2. The third clade consisted of Chlases belonging to cyanobacteria, namely, OaCLH and CmCLH. The Chlases of land plants are 35−86% homologous. The recombinant CrCLH1 shared a higher degree of homology with algal Chlases (28−33%) than land plant Chlases (19−24%). The seven novel Chlase protein sequences were aligned with those of the other reported Chlases by using ClustalW.39 All proteins shared the lipase motif GXSXG.10 Specifically, a highly conserved GHSRG motif was discovered in land plants and chlorophyta (Figure 3). Three-dimensional structures of the GXSXG motif of GDSL lipases10 showed that the conserved βSer-α folding structure belongs to the α/β hydrolase superfamily.43 The PredictProtein server44 predicted that recombinant CrCLH1 also has a β-Ser-α folding structure and belongs to the α/β hydrolase superfamily. In Figure 3, the putative SerAsp-His catalytic triad11 is represented by three black triangles, except for OaCLH and CmCLH, which contained the catalytic dyad Ser-His. Previous studies have indicated that the catalytic triad (Ser162, Asp191, and His262) of CaCLH is essential for Chlase activity.10 The catalytic residues (Ser141, Asp170, and His247) of BoCLH2 were replaced with alanine at all three 9498
DOI: 10.1021/acs.jafc.5b02787 J. Agric. Food Chem. 2015, 63, 9496−9503
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Journal of Agricultural and Food Chemistry
Figure 3. Alignment of amino acid sequences of various Chlases. The amino acid sequences of CrCLH1, CvCLH1, CvCLH2, OaCLH, CmCLH, AtCLH1, and AtCLH2 were aligned using ClustalW. The black line represents the conserved GHSRG lipase motif, and the three black inverted triangles denote the catalytic triad (Ser, Asp, and His residues). The black dotted line represents the signal peptide prediction of CrCLH1. Asterisks, colons, and periods represent identity, strong similarity, and weak similarity, respectively.
sites. BoCLH2 mutants had either decreased or no enzyme activity during Chl a degradation.16 The data indicated that Chlases and GDSL lipases have the same catalytic triad for biocatalysis. Although OaCLH and CmCLH have only the catalytic dyad Ser-His, a study indicated that the catalytic mechanisms and substrates of Chlases differed between plants and cyanobacteria.45 Expression and Subcellular Localization of Recombinant CrCLH1. Chlases from various green plants have been isolated and studied; however, they have a low catalytic efficiency in Chl a or Chl hydrolysis. Therefore, we isolated Chlase from other species to identify Chlases with high catalytic efficiency in Chl degradation and obtain Chlide and Pheide. Chlase was discovered in plants and algae. Some algal Chlases exhibited Chl degradation activity after being partially purified. For instance, Chlorella protothecoides Chlase30 and Phaeodactylum tricornutum Chlase28 were evaluated. Because those Chlases were only partially purified, it was unclear whether only one Pha. tricornutum Chlase enzyme or a variety of isozymes was involved in Chl degradation. We first isolated a novel and complete Chlase gene from C. reinhardtii (CrCLH1) and overexpressed it in an E. coli system. The cDNA length of CrCLH1 was 969 bp, and it encoded 322 amino acids. Purified recombinant CrCLH1 fused with an N-terminal T7-Tag formed a soluble protein that exhibited Chlase activity, and its molecular mass was 36.5 kDa (Figure 4). Most Chlases are located in the chloroplast,16,17 chloroplast membrane,14,15 thylakoid membrane,18 plastid,19,20 or vacuole10 or outside the chloroplast.12,13 The signal peptide (residues 1− 22) of CrCLH1 was predicted using the SignalP 4.1 server46 (Figure 3), and its location on the chloroplast membrane was predicted using the PredictProtein server.44 To identify the subcellular localization of CrCLH1, the C-terminal GFP fusion
Figure 4. Recombinant CrCLH1 overexpression in an E. coli system. The arrowhead indicates the recombinant CrCLH1 and its deduced protein molecular mass of 36.5 kDa in lane 1. M indicates the protein molecular markers, and lane 1 indicates the purified recombinant CrCLH1 in a 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis gel. The protein gel was stained with Coomassie blue.
protein (CrCLH1-GFP) was driven by a CAMV35S promoter and was transiently expressed in the Arabidopsis mesophyll protoplast. The CrCLH1-GFP fusion protein was localized outside the chloroplast, and free GFP (pBI221G) as a negative control was detected in the cytosol (Figure 5). The subcellular localization of CrCLH1 is similar to that of AtCLHs.12 Takamiya et al.4 supposed that the alternative extraplastidial Chl breakdown pathway is involved in Chl transfer through chloroplast-derived lipophilic globules, and these globules occur in a senescence-specific manner. During the degreening stage of Chlorella, the chloroplasts of the algae may perform Chl 9499
DOI: 10.1021/acs.jafc.5b02787 J. Agric. Food Chem. 2015, 63, 9496−9503
Article
Journal of Agricultural and Food Chemistry
Figure 5. Subcellular localization of CrCLH1. The CrCLH1-GFP fusion protein and free GFP driven by the CAMV35S promoter were transiently expressed in the Arabidopsis mesophyll protoplast. Free GFP was used as a negative control. The GFP fluorescence and autofluorescence of protoplasts were examined through confocal laser scanning microscopy. The bar length is 10 μm.
breakdown through the same pathway that is used in chloroplast-derived lipophilic globules.47 Optimal pH and Temperature for Recombinant CrCLH1 Activity. The optimal pH and pH stability of CrCLH1 were investigated over a pH range of 3−10. The optimal pH value of the recombinant CrCLH1 was 6, and >90% of the maximal activity occurred at pH 5 and 7 (Figure 6). Furthermore, the pH stability of the recombinant CrCLH1
Figure 7. Effect of temperature on the activity and stability of the recombinant CrCLH1. The optimal temperature (●) and temperature stability (■) of the recombinant CrCLH1 were measured according to the standard Chlase assay by using Chl a as a substrate. Data represent means ± the standard deviation of three independent experiments.
that the enzyme had low catalytic activity at high temperatures probably because of a conformational change in the enzyme. When the enzyme was gradually cooled and the temperature became closer to the optimal value, the enzyme showed conformational flexibility and its activity was restored.48 Furthermore, enzyme immobilization methods may enhance the thermal stability of CrCLH1 and allow it to tolerate high temperatures for industrial applications. Enzyme Kinetics of Recombinant CrCLH1. Three substrates, Chl a, Chl b, and BChl a, were prepared at five concentrations (100, 200, 300, 400, and 500 μM) to measure the initial rate (V0) of the recombinant CrCLH1. The production of Chlide a, Chlide b, and BChlide a was determined according to the absorbance of the aqueous acetone layer. The reactions were performed at 40 °C and pH 6. The structures of the three substrates differed only in the side chain and ring (Figure 1), thus affecting their interaction with the recombinant CrCLH1 and leading to different catalytic
Figure 6. Effect of pH on the activity and stability of the recombinant CrCLH1. The optimal pH (●) and pH stability (■) were measured according to the standard Chlase assay by using Chl a as a substrate. Data represent means ± the standard deviation of three independent experiments.
remained at approximately >96% of the maximum (100% at pH 6) in the pH range of 5−7. The optimal temperature for recombinant CrCLH1 activity was 40 °C (Figure 7); however, the recombinant CrCLH1 retained approximately 70 and 30% of its specific activity at 50 and 60 °C, respectively. The thermal stability curve of the recombinant CrCLH1 was similar to the optimal temperature curve and exhibited a maximal temperature of 40 °C. Moreover, after the recombinant CrCLH1 was incubated at 60 and 70 °C, the residual activity was approximately 93 and 52%, respectively. This result indicated 9500
DOI: 10.1021/acs.jafc.5b02787 J. Agric. Food Chem. 2015, 63, 9496−9503
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Journal of Agricultural and Food Chemistry efficiencies. Table 1 shows the steady-state kinetic parameters, maximal velocity (Vmax), Michaelis constant (Km), catalytic Table 1. Enzyme Kinetic Parameters of the Recombinant CrCLH1 substratea
Vmax (μM min−1 mg−1)
Km (μM)
kcat (s−1)
kcat/Km (s−1 μM−1)
Chl a Chl b BChl a
10.83 4.52 4.10
56.6 99.9 460.3
559.55 232.8 212.2
9.89 2.33 0.46
a
Substrates Chl a, Chl b, and BChl a can be hydrolyzed to produce Chlide a, Chlide b, and BChlide a, respectively. Enzyme analyses of three substrates were performed at 40 °C for 30 min.
activity (kcat), and catalytic efficiency (kcat/Km). A comparison of the Km values of Chl a, Chl b, and BChl a for substrate binding affinity indicated that the recombinant CrCLH1 had the highest affinity for Chl a (56.6 μM), followed by Chl b (99.9 μM), and the lowest affinity for BChl a (460.3 μM). Thus, the recombinant CrCLH1 had the highest kcat/Km value for Chl a (9.89 s−1μM−1), which was approximately 4 and 22 times higher than those for Chl b (2.33 s−1 μM−1) and BChl a (0.46 s−1 μM−1), respectively. Previous studies have reported that the Chlases of different plant species exhibited catalytic activity in Chl a degradation. The recombinant CrCLH1 had a catalytic efficiency higher than that of BoCLH1 (15.51 × 10−5 s−1 μM−1), BoCLH2 (0.78 × 10−5 s−1 μM−1),16 and TaCLH (0.137 s−1 μM−1).22 The Km value of the recombinant CrCLH1 indicated that it had a binding affinity for Chl a higher than those of BoCLH1 (465.92 μM), BoCLH2 (307.93 μM),16 and TaCLH (69 μM).22 These results indicate that the recombinant CrCLH1 was a more favorable enzyme than BoCLH1, BoCLH2, and TaCLH in catalyzing Chl degradation to produce Chlide. Pheide Formation during Chl Degradation under Acidic Conditions. Chlase removes phytol in both Chl and Phein degradation6 and exhibits carboxylesterase activity in ester compound hydrolysis in vitro.2,22 Chl and Phein metabolites, such as Chlide and Pheide, have been demonstrated to have physiological functions, including antioxidant, anticancer, and antivirus functions in vitro.23−27 According to the HPLC analysis (Figure 8), Chl a and b were catalyzed by the recombinant CrCLH1 to produce Chlide a and b at pH 5 and 6. However, the minor products of Pheide a and b were detected at pH 5. This indicates that the recombinant CrCLH1 exhibits dephytylation in Chl degradation, inducing Chlide formation. Subsequently, Chlide can be converted to Pheide in an acidic environment. In conclusion, the recombinant CrCLH1 contains a conserved GHSRG lipase motif and a Ser-Asp-His catalytic triad that belongs to the α/β hydrolase superfamily. The transient experiment evidenced subcellular localization of CrCLH1-GFP was localized outside the chloroplast. The recombinant CrCLH1 is more efficient for Chl degradation than BoCLH1, BoCLH2, and TaCLH. In addition, when the recombinant CrCLH1 catalyzed Chl hydrolysis under an acidic condition (pH 5), it yielded both Chlide and Pheide. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce Chlide and Pheide. Chlide and Pheide can be developed for medicinal and pharmaceutical applications in the future.
Figure 8. HPLC analysis of products of Chl degradation by CrCLH1. Chl degradation was catalyzed by the recombinant CrCLH1 at pH 5.0 and 6.0 at 40 °C for 30 min. The products were separated by HPLC and detected at 665 nm from 0 to 40 min.
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AUTHOR INFORMATION
Corresponding Authors
*Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan. Telephone: +886-2-2787-1184. Fax: +886-2-27827954. E-mail:
[email protected]. *Department of Biological Science and Technology, I-Shou University, Kaohsiung City 82445, Taiwan. Telephone: +886-76151100, ext. 7302. Fax: +886-7-6151959. E-mail: shawjf@isu. edu.tw. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported in part by the Ministry of Education, Taiwan, R.O.C., under the ATU plan and a grant (104-2311-B214-001-) from the National Science Council of Taiwan to J.F.S.
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ABBREVIATIONS USED BChl a, bacteriochlorophyll a; Chl, chlorophyll; Chl a, chlorophyll a; Chl b, chlorophyll b; Chlase, chlorophyllase; Chlide, chlorophyllide; GFP, green fluorescent protein; HPLC, high-performance liquid chromatography; IPTG, isopropyl β-Dthiogalactopyranoside; Pheide, pheophorbide; Phein, pheophytin; PPH, pheophytin pheophorbide hydrolase
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REFERENCES
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