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Selim Kermasha - One of the best experts on this subject based on the ideXlab platform.
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Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media
Applied Biochemistry and Biotechnology, 2007Co-Authors: Paula Arriagada-strodthoff, Salwa Karboune, Ronald J. Neufeld, Selim KermashaAbstract:The effects of selected reaction parameters, including solvent hydrophobicity, initial water activity, agitation speed, temperature and enzyme concentration, on the biocatalytic efficiency of a Chlorophyllase enzymatic extract from Phaeodactylum tricornutum in neat organic solvent media were investigated. The highest Chlorophyllase specific activity of 322 nmol hydrolyzed chlorophyll per gram of protein per minute and bioconversion yield of 91% were obtained in the reaction mixture of hexane/2-octanone (98.3:1.7, v / v ), at a controlled initial water activity of 0.90. R _O/A value, which is the ratio of the specific activity in the organic solvent to that in the aqueous/miscible organic solvent medium, was 1.5 × 10^−3. To reduce the substrate diffusional limitations, the appropriate agitation speed and enzyme concentration were determined. The optimum temperature for maximal enzymatic activity and activation energy were 35°C and 105.0 kJ/mol, respectively. Although the catalytic efficiency of chlorphyllase in the neat organic solvent mixture was lower than that in the aqueous medium, its half-life time in the first environment at temperature ranging from 35 to 50°C was increased by 5.0 to 15.0 times.
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Controlling sol-gel properties enhancing entrapped membrane protein activity through doping additives
Journal of Sol-Gel Science and Technology, 2007Co-Authors: Yunyu Yi, Ronald Neufeld, Selim KermashaAbstract:Chlorophyllase, a membrane protein, was entrapped in tetramethoxysilane (TMOS)—derived sol-gel and activity examined through a modification of sol-gel properties with doping additives. Polyvinyl alcohol or polyethyleneglycol as uncharged polymeric additives reduced the activity of entrapped Chlorophyllase by 60 and 70% respectively, explained by restricted accessibility of Chlorophyllase, as revealed by nitrogen desorption experiments. Entrapped Chlorophyllase demonstrated 10% activity yield in polyethylenimine-doped gel relative to a polyethylenimine-free control, explained by electrostatic interaction between positively charged polyethylenimine and negatively charged Chlorophyllase, considering that polyethylenimine led to a gel with a slightly reduced specific surface area and pore volume, but 20% larger pore size. When Chlorophyllase was introduced into sol-gel together with various amounts of glycerol, it demonstrated only slightly lower activity in those gels, even though sol-gel demonstrated decreased specific surface area and pore volume with increasing amount of glycerol. Lipase, as additive, reduced activity of entrapped Chlorophyllase, explained by its effect on gel formation, and thus gelation time and gel properties, whereas activity of sol-gel entrapped Chlorophyllase associated with monogalactosyl diglyceride (MGDG), was increased by 40%. This study provides an in-depth understanding of the change in sol-gel properties as affected by a wide variety of additives, and the consequent affect on the activity of the membrane protein Chlorophyllase.
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Characterization of sol-gel entrapped Chlorophyllase.
Biotechnology and bioengineering, 2006Co-Authors: Selim Kermasha, Ronald J. NeufeldAbstract:Immobilization of membrane proteins remains a challenge compared to soluble proteins. The membrane protein-Chlorophyllase was successful entrapped in tetramethoxysilane (TMOS)-based sol-gel in the presence of lipid. Activity was examined against mixing rate, incubation temperature, time, substrate, acetone, and canola oil concentration. The external mass transfer of chlorophyll is not the rate-limiting step at higher mixing rates. Stability against temperature and acetone as denaturant was enhanced. In spite of the fact that an initial reaction lag phase was observed, 20% more chlorophyll was hydrolyzed, compared to reaction with free enzyme by the end of a 12 h assay. The initial lower activity demonstrated by entrapped Chlorophyllase is likely due to the diffusion resistance of chlorophyll into and within the entrapment matrix. This hypothesis was substantiated by a low diffusion coefficient on the order of 10(-14) m(2)/s obtained for chlorophyll in nanoporous sol-gel particles. Pore size distribution of nanoporous wet TMOS-based sol-gel with or without protein was determined by thermoporometry. The change in pore morphology upon doping with Chlorophyllase suggests that protein acts as a template during the sol-gel process.
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optimization of tetramethoxysilane derived sol gel entrapment protocol stabilizes highly active Chlorophyllase
Journal of Sol-Gel Science and Technology, 2006Co-Authors: Yunyu Yi, Ronald J. Neufeld, Selim KermashaAbstract:Entrapment of membrane proteins is a challenging task compared to that involving soluble proteins. Chlorophyllase, a membrane protein, was successfully entrapped in tetramethoxysilane-derived sol-gel. Pre-gel sol typically consists of an aqueous suspension of Chlorophyllase, precursors including tetramethoxysilane and/or methytrimethoxysilane, and sodium fluoride as catalyst. To obtain a highly active entrapped enzyme preparation, the effects of various immobilization parameters, including the chemical compositions of pre-gel sol (water/silane ratio, precursor type and proportions, enzyme loading, sodium fluoride concentration), and sol-gel process parameters (aging and drying time and approach) have been investigated. Chlorophyllase demonstrated the highest activity in gel derived from a pre-gel sol with water/silane ratio of 30 and enzyme loading of 0.257 mgprotein/ggel, and showed moderately lower activity in organically modified sol-gel than that in hydrophilic sol-gel. The effects of water/silane ratio and precursor combinations on the activity of entrapped Chlorophyllase were also studied by examining the pore morphology of gel via nitrogen adsorption-desorption. Longer aging time leads to an entrapped Chlorophyllase preparation with higher activity. Chlorophyllase preparation demonstrated negligible activity after air-drying for 12 h while lyophilized Chlorophyllase preparation demonstrated 8, 4 and 4 times higher activity than air-dried, vacuum-dried and solvent-dried preparations. Chlorophyllase demonstrated 30% higher activity in the improved sol-gel protocol than that from a non-optimized sol-gel protocol developed in a previous study.
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Immobilization and biocatalysis of Chlorophyllase in selected organic solvent systems.
Journal of biotechnology, 2005Co-Authors: Salwa Karboune, Ronald J. Neufeld, Selim KermashaAbstract:Abstract Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83–93%) and efficiency (51–53%) were obtained when Chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of Chlorophyllase as compared to that of the free one. The immobilization of Chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of Chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of Chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled Chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized Chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.
Ronald J. Neufeld - One of the best experts on this subject based on the ideXlab platform.
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Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media
Applied Biochemistry and Biotechnology, 2007Co-Authors: Paula Arriagada-strodthoff, Salwa Karboune, Ronald J. Neufeld, Selim KermashaAbstract:The effects of selected reaction parameters, including solvent hydrophobicity, initial water activity, agitation speed, temperature and enzyme concentration, on the biocatalytic efficiency of a Chlorophyllase enzymatic extract from Phaeodactylum tricornutum in neat organic solvent media were investigated. The highest Chlorophyllase specific activity of 322 nmol hydrolyzed chlorophyll per gram of protein per minute and bioconversion yield of 91% were obtained in the reaction mixture of hexane/2-octanone (98.3:1.7, v / v ), at a controlled initial water activity of 0.90. R _O/A value, which is the ratio of the specific activity in the organic solvent to that in the aqueous/miscible organic solvent medium, was 1.5 × 10^−3. To reduce the substrate diffusional limitations, the appropriate agitation speed and enzyme concentration were determined. The optimum temperature for maximal enzymatic activity and activation energy were 35°C and 105.0 kJ/mol, respectively. Although the catalytic efficiency of chlorphyllase in the neat organic solvent mixture was lower than that in the aqueous medium, its half-life time in the first environment at temperature ranging from 35 to 50°C was increased by 5.0 to 15.0 times.
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Characterization of sol-gel entrapped Chlorophyllase.
Biotechnology and bioengineering, 2006Co-Authors: Selim Kermasha, Ronald J. NeufeldAbstract:Immobilization of membrane proteins remains a challenge compared to soluble proteins. The membrane protein-Chlorophyllase was successful entrapped in tetramethoxysilane (TMOS)-based sol-gel in the presence of lipid. Activity was examined against mixing rate, incubation temperature, time, substrate, acetone, and canola oil concentration. The external mass transfer of chlorophyll is not the rate-limiting step at higher mixing rates. Stability against temperature and acetone as denaturant was enhanced. In spite of the fact that an initial reaction lag phase was observed, 20% more chlorophyll was hydrolyzed, compared to reaction with free enzyme by the end of a 12 h assay. The initial lower activity demonstrated by entrapped Chlorophyllase is likely due to the diffusion resistance of chlorophyll into and within the entrapment matrix. This hypothesis was substantiated by a low diffusion coefficient on the order of 10(-14) m(2)/s obtained for chlorophyll in nanoporous sol-gel particles. Pore size distribution of nanoporous wet TMOS-based sol-gel with or without protein was determined by thermoporometry. The change in pore morphology upon doping with Chlorophyllase suggests that protein acts as a template during the sol-gel process.
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optimization of tetramethoxysilane derived sol gel entrapment protocol stabilizes highly active Chlorophyllase
Journal of Sol-Gel Science and Technology, 2006Co-Authors: Yunyu Yi, Ronald J. Neufeld, Selim KermashaAbstract:Entrapment of membrane proteins is a challenging task compared to that involving soluble proteins. Chlorophyllase, a membrane protein, was successfully entrapped in tetramethoxysilane-derived sol-gel. Pre-gel sol typically consists of an aqueous suspension of Chlorophyllase, precursors including tetramethoxysilane and/or methytrimethoxysilane, and sodium fluoride as catalyst. To obtain a highly active entrapped enzyme preparation, the effects of various immobilization parameters, including the chemical compositions of pre-gel sol (water/silane ratio, precursor type and proportions, enzyme loading, sodium fluoride concentration), and sol-gel process parameters (aging and drying time and approach) have been investigated. Chlorophyllase demonstrated the highest activity in gel derived from a pre-gel sol with water/silane ratio of 30 and enzyme loading of 0.257 mgprotein/ggel, and showed moderately lower activity in organically modified sol-gel than that in hydrophilic sol-gel. The effects of water/silane ratio and precursor combinations on the activity of entrapped Chlorophyllase were also studied by examining the pore morphology of gel via nitrogen adsorption-desorption. Longer aging time leads to an entrapped Chlorophyllase preparation with higher activity. Chlorophyllase preparation demonstrated negligible activity after air-drying for 12 h while lyophilized Chlorophyllase preparation demonstrated 8, 4 and 4 times higher activity than air-dried, vacuum-dried and solvent-dried preparations. Chlorophyllase demonstrated 30% higher activity in the improved sol-gel protocol than that from a non-optimized sol-gel protocol developed in a previous study.
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Immobilization and biocatalysis of Chlorophyllase in selected organic solvent systems.
Journal of biotechnology, 2005Co-Authors: Salwa Karboune, Ronald J. Neufeld, Selim KermashaAbstract:Abstract Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83–93%) and efficiency (51–53%) were obtained when Chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of Chlorophyllase as compared to that of the free one. The immobilization of Chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of Chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of Chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled Chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized Chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.
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Matrix physicochemical properties affect activity of entrapped Chlorophyllase
Journal of Chemical Technology & Biotechnology, 2005Co-Authors: Selim Kermasha, Ronald J. NeufeldAbstract:Chlorophyllase, a membrane glycoprotein, was entrapped in various matrices, including alginate, alginate–silicate mixed gel, TMOS-based sol–gel, and their hydrophobically-modified counterparts. Chlorophyllase activity was affected by the physicochemical properties of the matrix, demonstrating lower activity in organically-modified matrices compared with the corresponding hydrophilic matrices. The advantage of adopting organically-modified matrices to facilitate the transfer of hydrophobic substrate is likely compromised by detrimental interaction between Chlorophyllase and the hydrophobic components. This hypothesis is at least partly substantiated by the negligible activity demonstrated by free Chlorophyllase in hydrophobic micellar media. Even though activity yields exceeded 50% in alginate beads, the release profile reveals that alginate matrix is too porous to retain Chlorophyllase. Although alginate–silicate mixed gel more effectively confined Chlorophyllase in the matrix, only 16% of the activity was recovered. In contrast, inorganic sol–gel yielded a Chlorophyllase preparation with mass yield above 90%, and activity yield above 50%. Doping additives did not improve activity yield, which could be explained by the lower specific surface area and pore volume, and hence possible restricted accessibility of Chlorophyllase by its substrate. Water/silane ratio was found to affect the sol–gel-entrapped Chlorophyllase activity by influencing the gelation time and physical properties of the gel. Copyright © 2005 Society of Chemical Industry
P. Marsot - One of the best experts on this subject based on the ideXlab platform.
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Purification and characterization of Chlorophyllase from algaPhaeodactylum tricornutum by preparative native electrophoresis
Applied Biochemistry and Biotechnology, 1995Co-Authors: A. Khalyfa, S. Kermasha, P. Marsot, M. GoetghebeurAbstract:The partially purified Chlorophyllase, obtained from the alga Phaeodactylum tricornutum , was further purified by preparative native gel electrophoresis. The purification procedure provided the recovery of large amounts of a single purified Chlorophyllase fraction. However, the electrophoretic analyses of the purified enzymatic fraction under denaturing conditions demonstrated the presence of two bands with mol wt of 43 ±3 and 46 ±3 kDa. The purification procedure resulted in 2-and 195-fold increases in Chlorophyllase activity compared to that of the partially purified and crude enzymatic extracts, respectively. The optimum pH for Chlorophyllase hydrolytic activity was found to be 8.0. The optimum incubation time and temperature for the hydrolytic activity of the purified Chlorophyllase were found to be 2 h and 31°C, respectively. The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were 4 and 5 mM, respectively. The addition of individual plant membrane lipids, including phosphatidylcholine, phosphatidylglycerol, and β-carotene, to the reaction media increased the enzyme activity markedly. The purified enzyme fraction displayed preferential specificity toward selective substrates with an order of activity as follows: purified chlorophyll b > purified chlorophyll a > partially purified chlorophyll > crude chlorophyll. Diisopropyl fluorophosphate and phytol, respectively, showed noncompetitive and competitive inhibitory effects on Chlorophyllase activity with K_i, values of 0.78 mM and 3.75 μM , respectively.
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Purification and characterization of Chlorophyllase from alga Phaeodactylum tricornutum by preparative native electrophoresis
Applied Biochemistry and Biotechnology, 1995Co-Authors: A. Khalyfa, Selim Kermasha, P. Marsot, M. GoetghebeurAbstract:The partially purified Chlorophyllase, obtained from the algaPhaeodactylum tricornutum, was further purified by preparative native gel electrophoresis. The purification procedure provided the recovery of large amounts of a single purified Chlorophyllase fraction. However, the electrophoretic analyses of the purified enzymatic fraction under denaturing conditions demonstrated the presence of two bands with mol wt of 43 ±3 and 46 ±3 kDa. The purification procedure resulted in 2-and 195-fold increases in Chlorophyllase activity compared to that of the partially purified and crude enzymatic extracts, respectively. The optimum pH for Chlorophyllase hydrolytic activity was found to be 8.0. The optimum incubation time and temperature for the hydrolytic activity of the purified Chlorophyllase were found to be 2 h and 31°C, respectively. The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were 4 and 5 mM, respectively. The addition of individual plant membrane lipids, including phosphatidylcholine, phosphatidylglycerol, and β-carotene, to the reaction media increased the enzyme activity markedly. The purified enzyme fraction displayed preferential specificity toward selective substrates with an order of activity as follows: purified chlorophyllb > purified chlorophylla > partially purified chlorophyll > crude chlorophyll. Diisopropyl fluorophosphate and phytol, respectively, showed noncompetitive and competitive inhibitory effects on Chlorophyllase activity with Ki, values of 0.78 mM and 3.75μM, respectively.
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Biocatalysis of Chlorophyllase from Phaeodactylum tricornutum in organic solvent media
Process Biochemistry, 1995Co-Authors: Ali Khamessan, Selim Kermasha, P. MarsotAbstract:Abstract The biocatalysis of partially purified Chlorophyllase from Phaeodactylum tricornutum in an organic solvent medium was investigated. The optimum amounts of water and organic solvent required for the Chlorophyllase-catalyzed hydrolytic activity were measured in a wide range of solvents, including acetone, ethanol, propanol, butanol, pentanol, hexanol, toluene, pentane, hexane, octane, and heptane as a function of their hydrophobicities. The logarithm of partition coefficient (log P ) was used to define the quantity of solvent polarity. The amount of aqueous buffer required for the enzymic activity was lower in non-polar solvents (log P >1.8) than polar ones (log P V max values for the hydrolytic activity of Chlorophyllase in water-miscible solvents (log P P > 0.8). The hydrolytic activity was decreased by approximately 12 times by increasing the carbon chain of alcohol from two to five. The results also indicated that phytol has a non-competitive inhibitory effect on the Chlorophyllase activity in reaction media containing acetone, and activatory effect in that containing hexane.
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effects of polar organic solvents on the biocatalysis of Chlorophyllase in a biphasic organic system
Bioscience Biotechnology and Biochemistry, 1994Co-Authors: Ali Khamessan, Selim Kermasha, P. MarsotAbstract:The effects of polarity of various organic solvents, including acetone, ethanol, and propanol, used in a biphasic organic system, on the hydrolytic activity of a partially purified Chlorophyllase from Phaeodactylum tricornutum were investigated. The different concentrations of each polar organic solvent, from 0 to 40%, were added to a mixture (45:55, v/v) of hexane and a buffer solution of Tris–HCl (20 mm, pH 7.5). The most appropriate concentrations of acetone, ethanol, and propanol for the hydrolytic activity of Chlorophyllase were 12.5, 5.0, and 2.5%, respectively. The results indicated that the optimum reaction time for the Chlorophyllase activity in the biphasic system decreased from 7.0 h to 3.0, 5.0, and 5.0 h, respectively, upon the addition of an appropriate amount of acetone, ethanol, or propanol. The Vmax and Km as well as the inhibitory effect of phytol on the Chlorophyllase activity in the biphasic organic system containing a polar organic solvent were also investigated.
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Biocatalysis of Chlorophyllase from Phaeodactylum tricornutum in a biphasic organic system
Journal of Chemical Technology AND Biotechnology, 1994Co-Authors: Ali Khamessan, Selim Kermasha, Ashraf A. Ismail, P. MarsotAbstract:The hydrolytic activity of a partially purified Chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was determined in a biphasic organic system using different mixtures of hexane and Tris–HCl buffer solution (20 mmol dm−3, pH 7·5). The most appropriate kinetic parameters were determined for the optimum hydrolytic activity of Chlorophyllase including hexane concentration (45%), optimum pH (7·5), time of incubation (7 h), incubation temperature (25°C) and enzyme concentration (10 μg cm−3). The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were determined to be 4 mmol dm−3 and 5 mmol dm−3, respectively. The results also indicated that diisopropyl fluorophosphate (DIFP) had a mixed competitive–non-competitive inhibitory effect on Chlorophyllase activity. The end-products of the Chlorophyllase hydrolytic reaction were identified and confirmed, using thin-layer and high-performance liquid chromatographies, spectrophotometric scanning and Fourier transform infrared spectroscopy.
Ali Khamessan - One of the best experts on this subject based on the ideXlab platform.
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BIOCATALYSIS OF Chlorophyllase IN CANOLA OIL USING ORGANIC SOLVENT SYSTEMS
Journal of Food Biochemistry, 1996Co-Authors: Ali Khamessan, Selim KermashaAbstract:The hydrolytic activity of a partially purified Chlorophyllase, obtained from an algal source, was investigated in a refined-bleached-deodorized (RBD) canola oil model system using various organic solvent media, including water/miscible-organic, biphasic organic, and micellar ternary systems. Although the use of a a ternary micellar system containing Span 85 was found to be the most appropriate medium for the hydrolysis of chlorophyll, the hydrolytic activity of Chlorophyllase declined as oil content increased. The effects of various parameters, including surfactant concentration, hexane/buffer proportion, enzyme content, reaction time, incubation temperature, shaker speed, and activator concentration, on Chlorophyllase activity in the micellar ternary systems containing 20% RBD oil were also investigated. In addition, phytol displayed a noncompetitive inhibition in reaction media containing polysorbate 80 and Span 85. The plot of 1/v versus oil concentrations revealed that the oil had K i values of3. 73 and 4.56% in the micellar systems containing polysorbate 80 and Span 85, respectively. Moreover, the presence of 10% oil in the micellar system containing span 85 and polysorbate 80 decreased V max values of Chlorophyllase activity by 6.2 and 9.6-fold, respectively.
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Biocatalysis of Chlorophyllase from Phaeodactylum tricornutm in micellar ternary system containing spans
Journal of Biotechnology, 1996Co-Authors: Ali Khamessan, Selim KermashaAbstract:Abstract The hydrolytic activity of a partially purified Chlorophyllase, obtained from alga Phaeodactylum tricornutum, was dramatically increased in a micellar ternary system, hexane/Tris-HCl buffer/surfactant, using sorbitan surfactants of different hydrophobic chains, including sorbitan-monolaurate (Span 20), -monopalmitate (Span 40), -monostearate (Span 60), -monooleate (Span 80), and -trioleate (Span 85). The optimum concentrations of Spans 20, 40, 60, 80, and 85, required for the hydrolytic activity of Chlorophyllase, were 15, 15, 25, 50, and 75 μM, respectively. The results demonstrated that the maximum partitioning, at the interface, for both enzyme and substrate was obtained with Span 85. The Vmax value for the hydrolytic activity of Chlorophyllase in the hexane/aqueous medium containing Span 85 was about 288-times higher than that obtained in the absence of the surfactant. The addition of 1.0% alcohols, with different carbon chains, to the micellar system inhibited the enzyme activity by 25 to 100%; the degree of inhibition increased as the carbon-chain length of alcohols increased. The optimum values of pH, enzyme content, incubation temperature and shaker speed, required for the maximum hydrolytic activity, were 8.0, 0.5 μg protein/ml, 35 °C and 300 rpm, respectively. The stability of Chlorophyllase activity in the enzymatic extracts stored at 4, 25 and 35 °C and shaken in a mixture of buffer solution and hexane (70:30, v/v) was temperature dependent.
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Optimization of Chlorophyllase-catalysed hydrolytic activity in a micellar ternary system
Biotechnology and Applied Biochemistry, 1995Co-Authors: Ali Khamessan, Selim Kermasha, L. MollimardAbstract:The hydrolytic activity of a partly purified Chlorophyllase (EC 3.1.1.14), obtained from the alga Phaeodactylum tricornutum, was determined in a micellar ternary system [hexane/(Tris/HCl buffer)/surfactant]. The surfactants, of various hydrophobic chain lengths, included polysorbates 20, 40, 60, 80 and 85 ; their optimum concentrations for the hydrolytic activity of Chlorophyllase were determined to be 2.50, 1.00, 0.75, 5.00 and 7.50 μM, respectively. The maximum partitions of both Chlorophyllase and substrate were obtained at the interface, using the optimum concentrations of polysorbates for the enzyme biocatalysis activity. The results showed that the addition of polysorbate 80 to the hexane/aqueous medium increased the enzyme activity 139-fold. However, the V max. of Chlorophyllase activity in the micellar system was dependent on the hydrophobicity of the alkyl chain of the polysorbate. In addition, the effects of various parameters, including pH, enzyme and substrate concentrations, reaction time, incubation temperature, propanol concentration and shaker speed on the Chlorophyllase activity were investigated as a function of the alkyl chain of polysorbate. The stability of Chlorophyllase activity in the enzymic extracts was temperature-dependent when suspended in the Tris/HCl buffer solution containing 5 μM polysorbate 80 or shaken in a mixture of hexane/buffer (12.5 : 87.5, v/v), with or without the presence of the surfactant, and stored at 4-35°C.
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Biocatalysis of Chlorophyllase from Phaeodactylum tricornutum in organic solvent media
Process Biochemistry, 1995Co-Authors: Ali Khamessan, Selim Kermasha, P. MarsotAbstract:Abstract The biocatalysis of partially purified Chlorophyllase from Phaeodactylum tricornutum in an organic solvent medium was investigated. The optimum amounts of water and organic solvent required for the Chlorophyllase-catalyzed hydrolytic activity were measured in a wide range of solvents, including acetone, ethanol, propanol, butanol, pentanol, hexanol, toluene, pentane, hexane, octane, and heptane as a function of their hydrophobicities. The logarithm of partition coefficient (log P ) was used to define the quantity of solvent polarity. The amount of aqueous buffer required for the enzymic activity was lower in non-polar solvents (log P >1.8) than polar ones (log P V max values for the hydrolytic activity of Chlorophyllase in water-miscible solvents (log P P > 0.8). The hydrolytic activity was decreased by approximately 12 times by increasing the carbon chain of alcohol from two to five. The results also indicated that phytol has a non-competitive inhibitory effect on the Chlorophyllase activity in reaction media containing acetone, and activatory effect in that containing hexane.
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effects of polar organic solvents on the biocatalysis of Chlorophyllase in a biphasic organic system
Bioscience Biotechnology and Biochemistry, 1994Co-Authors: Ali Khamessan, Selim Kermasha, P. MarsotAbstract:The effects of polarity of various organic solvents, including acetone, ethanol, and propanol, used in a biphasic organic system, on the hydrolytic activity of a partially purified Chlorophyllase from Phaeodactylum tricornutum were investigated. The different concentrations of each polar organic solvent, from 0 to 40%, were added to a mixture (45:55, v/v) of hexane and a buffer solution of Tris–HCl (20 mm, pH 7.5). The most appropriate concentrations of acetone, ethanol, and propanol for the hydrolytic activity of Chlorophyllase were 12.5, 5.0, and 2.5%, respectively. The results indicated that the optimum reaction time for the Chlorophyllase activity in the biphasic system decreased from 7.0 h to 3.0, 5.0, and 5.0 h, respectively, upon the addition of an appropriate amount of acetone, ethanol, or propanol. The Vmax and Km as well as the inhibitory effect of phytol on the Chlorophyllase activity in the biphasic organic system containing a polar organic solvent were also investigated.
Gustavo A Martinez - One of the best experts on this subject based on the ideXlab platform.
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effect of hot air uv c white light and modified atmosphere treatments on expression of chlorophyll degrading genes in postharvest broccoli brassica oleracea l florets
Scientia Horticulturae, 2011Co-Authors: Agustin M Buchert, Pedro M. Civello, Gustavo A MartinezAbstract:Abstract Several treatments were applied in order to delay postharvest degreening in broccoli florets and investigate their effects on genes associated with chlorophyll catabolism. Degradation of chlorophylls is the most evident visual manifestation of broccoli postharvest deterioration, occurring rapidly due to the immature stage in which the material is harvested. Treatments such as storage in modified atmosphere, exposure to hot air, UV-C and white lamps were employed in the current work to induce a delay in degreening and chlorophyll degradation. Expression of genes possibly related to chlorophyll catabolism was analyzed in these samples and discussed. Chlorophyllases, the enzymes traditionally believed to remove the phytol side chain from chlorophyll appear to have a gene expression that was not regulated by postharvest treatments. Pheophytinase, a recently discovered new enzyme in this metabolic pathway, correlated chlorophyll loss accurately in heat, UV-C and white-light treatments, but not in modified atmospheres. Results presented in this work indicate that postharvest treatments that delay chlorophyll degradation have a higher effect on the expression of pheophytinase rather than on Chlorophyllase genes.
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Chlorophyllase versus pheophytinase as candidates for chlorophyll dephytilation during senescence of broccoli
Journal of plant physiology, 2010Co-Authors: Agustin M Buchert, Pedro M. Civello, Gustavo A MartinezAbstract:Degradation of chlorophylls during senescence is a highly regulated process which requires the concerted action of several enzymes. Traditionally, it has been stated that the dismantling process of the chlorophyll molecule begins with a dephytilation step, followed by Mg(2+) removal and other breakdown reactions. Recently, new evidence suggests the possibility of a rearrangement in the first two steps of this process, occurring Mg(2+) removal prior to the loss of the phytol side chain. With the purpose of approximating to the real sequential order of these reactions and to assess if dephytilation occurs on intact (catalyzed by Chlorophyllase) or Mg-free (catalyzed by pheophytinase) chlorophyll, expression of both genes was analyzed in broccoli tissue during senescence. Samples of broccoli florets treated with plant hormones, such as cytokinin and ethylene were utilized, as to assess the effect of such compounds on the expression of these genes. Results showed that Chlorophyllase expression did not correlate to typical expression patterns for genes related to senescence, since a decrease in expression during senescence was found for one of the two Chlorophyllase genes analyzed, and the hormonal-treatment effects on gene expression did not match those observed on chlorophyll content for both Chlorophyllase genes. Pheophytinase expression patterns, on the other hand, displayed an increase in the first 3 days of induced senescence, followed by lower expression values towards the end of the experiment. Samples subjected to postharvest treatments mostly showed an inhibition of pheophytinase expression, especially in samples in which degradation of chlorophylls had been delayed. These results suggest that pheophytinase expression correlates to the visual manifestation of postharvest treatments, supporting the possibility that this enzyme is responsible for the dephytilation step in chlorophyll breakdown.
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effect of ethephon and 6 benzylaminopurine on chlorophyll degrading enzymes and a peroxidase linked chlorophyll bleaching during post harvest senescence of broccoli brassica oleracea l at 20 c
Postharvest Biology and Technology, 2005Co-Authors: Maria L Costa, Pedro M. Civello, Alicia R Chaves, Gustavo A MartinezAbstract:Effects of ethephon and 6-benzylaminopurine (BAP) treatments on enzyme degrading enzymes and a peroxidase-linked chlorophyll bleaching were investigated in broccoli (Brassica oleracea L.) florets. The florets were dipped in solutions containing either BAP or ethephon and then incubated in darkness at 20 °C. The hue angle values and chlorophyll contents of ethephon-treated florets declined the most of the five-day experiment. In contrast, hue angle and chlorophyll content declined the least in BAP-treated florets. Pheophytin levels increased the most in ethephon-treated florets and the least in BAP-treated florets. Chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching increased over the five days in control (untreated) florets. Ethephon treatment enhanced Chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching levels as compared with control. BAP treatment reduced Chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching levels. It was concluded that the activities of Chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching could be regulated by external application of either ethylene (applied as ethephon) or a cytokinin (applied as BAP). In the case of ethephon treatment, accelerated rates of chlorophyll degradation were found and in the case of BAP-treatment, reduced rates of chlorophyll degradation were found.
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Effect of exogenous application of gibberellic acid on color change and phenylalanine ammonia-lyase, Chlorophyllase, and peroxidase activities during ripening of strawberry fruit (Fragaria x ananassa Duch.)
Journal of Plant Growth Regulation, 1996Co-Authors: Gustavo A Martinez, Alicia R Chaves, María Cristina AñónAbstract:The effect of exogenously applied gibberellic acid (GA3) on the postharvest color change of strawberry fruit was evaluated through their external color and surface color parameters. A significant delay on color evolution was observed in fruits treated with GA3. The evolution of activities of phenylalanine ammonia-lyase (PAL), Chlorophyllase, and peroxidase was also analyzed. PAL activity increased during strawberry ripening, but in fruits treated with GA3 the increase in such activity was slower, and, probably as consequence, the development of red color was delayed. Moreover, the activity of Chlorophyllase and peroxidase, enzymes possibly involved in chlorophyll metabolism, decreased during strawberry ripening. However, a delay was observed in the decrease of such activities in GA3-treated fruits.
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PARTIAL CHARACTERIZATION OF Chlorophyllase FROM STRAWBERRY FRUIT (FRAGARIA ANANASSA, DUCH.)
Journal of Food Biochemistry, 1994Co-Authors: Gustavo A Martinez, Pedro M. Civello, Alicia R Chaves, María Cristina AñónAbstract:In this article we report the existence of Chlorophyllase in strawberry fruit (Fragaria ananassa, Duch.). The enzyme was extracted from either fresh fruit or from acetone powder in the presence of Triton X-100 to improve its solubilization. The composition of the reaction mixture as well as the enzymatic activity conditions were optimized. At test conditions the temperature of maximum enzymotic activity was 40C and optimum pH 7.8. The Chlorophyllase showed a considerable resistance to thermal treatment. The enzyme was stable for incubation times of 30 min up to 50C. Chlorophyllase showed a preferential action on chlorophyll a at pHs 6.0, 7.0 and 8.0. The specific enzyme activity, just as did chlorophyll level, decreased during ripening.