Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media

Arriagada-Strodthoff, Paula; Karboune, Salwa; Neufeld, Ronald J.; Kermasha, Sélim

doi:10.1007/s12010-007-0021-8

Cited by 11 publications

(4 citation statements)

References 26 publications

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“…The specific activity of Chlase was defined as nanomole of substrate (Chl a) hydrolyzed per minute per mg of protein (Arriagada-Strodthoff et al, 2007). The concentration of protein was measured using the method of Lowry et al (1951).…”

Section: Plant Materials Leaves Of Three Representatives Ofmentioning

confidence: 99%

Biochemical characterisation of chlorophyllase from leaves of selected Prunus species--a comparative study.

Sytykiewicz

Sprawka²,

Czerniewicz³

et al. 2013

Acta Biochim Pol

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Despite senescence-induced chlorophyll depletion in plants has been widely studied, the enzymatic background of this physiologically regulated process still remains highly unclear. The purpose of this study was to determine selected biochemical properties of partially purified fractions of chlorophyllase (Chlase, chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from leaves of three Prunus species: bird cherry (Prunus padus L.), European plum (Prunus domestica L.), and sour cherry (Prunus cerasus L.). Secondarily, this report was aimed at comparing seasonal dynamics of Chlase activity and chlorophyll a (Chl a) content within investigated plant systems. Molecular weight of native Chlase F1 has been estimated at 90 kDa (bird cherry) and approximately 100 kDa (European plum and sour cherry), whereas molecular mass of Chlase F2 varied from 35 kDa (European plum) to 60 kDa (sour cherry). Furthermore, enzyme fractions possessed similar optimal pH values ranging from 7.6 to 8.0. It was found that among a broad panel of tested metal ions, Hg(+2), Fe(+2), and Cu(+2) cations showed the most pronounced inhibitory effect on the activity of Chlase. In contrast, the presence of Mg(+2) ions influenced a subtle stimulation of the enzymatic activity. Importantly, although Chlase activity was negatively correlated with the amount of Chl a in leaves of examined Prunus species, detailed comparative analyses revealed an incidental decrement of enzymatic activity in early or moderately senescing leaves. It provides evidence that foliar Chlase is not the only enzyme involved in autumnal chlorophyll breakdown and further in-depth studies elucidating this catabolic process are required.

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Section: Plant Materials Leaves Of Three Representatives Ofmentioning

confidence: 99%

Biochemical characterisation of chlorophyllase from leaves of selected Prunus species--a comparative study.

Sytykiewicz

Sprawka²,

Czerniewicz³

et al. 2013

Acta Biochim Pol

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“…PPAs are used routinely to quantify rates of reactions that release PP i as a byproduct, such as single nucleotide polymorphism (SNP) genotyping reactions (10-12), RNA synthesis by viral RNA-dependent RNA polymerases (13), and aminoacyl-tRNA synthetase activity (14,15). The advantage of PPA-coupled assays is that PPA hydrolyzes PP i to a product (2P i ) which is readily detected by colorimetric assay (16,17).PPAs that operate in a wide variety of organic solvents and salt concentrations are desirable in bioindustry to increase the solubility of hydrophobic substrates, allow for novel synthetic chemistry, alter substrate specificity, ease product recovery, and reduce microbial contamination (18)(19)(20)(21)(22)(23)(24). Biotechnological applications of solvent-tolerant PPAs can be envisioned in the biosynthesis of hydrophobic compounds derived from carbon skeletons such as cholesterol and rubber.…”

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confidence: 99%

“…PPAs that operate in a wide variety of organic solvents and salt concentrations are desirable in bioindustry to increase the solubility of hydrophobic substrates, allow for novel synthetic chemistry, alter substrate specificity, ease product recovery, and reduce microbial contamination (18)(19)(20)(21)(22)(23)(24). Biotechnological applications of solvent-tolerant PPAs can be envisioned in the biosynthesis of hydrophobic compounds derived from carbon skeletons such as cholesterol and rubber.…”

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confidence: 99%

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

McMillan

Hepowit

Maupin-Furlow

2016

Appl Environ Microbiol

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Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PP i ) to orthophosphate (P i ) are commonly used to accelerate and detect biosynthetic reactions that generate PP i as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PP i -hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a V max of 465 U · mg ؊1 for PP i hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PP i and Mg 2؉ . Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PP i by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PP i under conditions of reduced water activity that are a hurdle to current PPA-based technologies. Inorganic pyrophosphatases (PPAs) (EC 3.6.1.1) catalyze the hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate (PP i ) (P 2 O 7 4Ϫ ) to form 2 mol of orthophosphate (P i ) (PO 4 3Ϫ ) (1). PP i is a common by-product of metabolism, including the biosynthesis of DNA, RNA, protein, peptidoglycan, lipids (e.g., cholesterol), cellulose, starch, and other biopolymers (2). PP i is also formed during the posttranslational modification of proteins, including adenylation, uridylation, and ubiquitylation (2).The hydrolysis of PP i by PPA releases a considerable amount of energy (⌬G=°ϭ Ϫ19.2 kJ/mol) that can drive unfavorable biochemical transformations to completion. One example is in the synthesis of DNA by DNA polymerase. In this endergonic (⌬G=°ϭ ϩ2.1 kJ/mol) reaction, the 3=-hydroxyl group of the nucleotide that resides at the 3= end of the growing DNA strand serves as a nucleophile in the attack of the ␣ phosphorus of the incoming deoxynucleoside 5=-triphosphate (dNTP), thus releasing PP i (2). The polymerization of DNA is highly dependent on PPA to hydrolyze the energy-rich PP i to 2P i and to drive the synthesis reaction forward (2). Under standard conditions, DNA polymerase alone converts DNA to dNTPs.PPAs are used in a wide variety of biotechnology applications based on the ability of these enzymes to drive reactions f...

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“…If the reaction temperature was continuously raised to 70 and 80 • C, the yield of pheophorbide was not parallel to lower temperatures, which suggested some degradation process occurred at these high temperatures. Previous studies have found that the optimum reaction temperature for the enzymatic activity of chlorophyllase and pheophytinase is around 35 to 50 • C (Arriagada-Strodthoff et al, 2007;Guyer et al, 2018). So, high temperatures will affect enzyme activity, and thus, reduce the yield of pheophorbides.…”

Section: Effect Of Temperature and Time On The Enzymatic Reaction Of ...mentioning

confidence: 99%

Screening of dephytinization reaction of chlorophyll pigments with citrus acetone powder by UPLC–DAD–MS

Agarry

Ding

et al. 2022

Journal of Food Science

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The preparation of dephytylated chlorophyll standards is inefficient and the process is complicated, which hinders chlorophyll determination and related bioactive property investigation. In this paper, chlorophyll derivatives from four phytylated chlorophylls (chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b) before and after the enzymatic reaction were qualitatively and quantitatively characterized by UPLC–DAD–MS. A simple index was proposed to characterize chlorophyll pigments from their oxidized counterparts by the λmax of the typical peak of chlorophyll derivatives in UV–visible spectrum and their signal intensity ratios. The optimal reaction conditions for the enzymatic reaction of four chlorophyll pigments were optimized, and kinetic models were fitted. The results showed that the optimal temperatures for the enzymatic reactions of chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b were 30, 30, 60, and 60°C, respectively, and their optimal reaction time was 2, 3, 1, and 3 h, respectively. Kinetic models were fitted under optimal reaction conditions to study the Km and Vm values of the enzymatic reactions. Practical Application Dephytylated chlorophylls, such as chlorophyllide and pheophorbide, are frequently determined in food industry and are always required to be prepared in lab with acetone powder from plant tissue. Moreover, chlorophyll pigments are easy to undergo oxidations, which make the characterization of dephytylated chlorophyll pigments more complicated and difficult. In this paper, four types of phytylated chlorophylls were investigated respectively about the dephytinization process with the citrus acetone powder, and the reaction mixture was analyzed with UPLC–DAD–MS, which can provide an important reference for relevant chlorophyll determination studies and the development of chlorophyll identification protocols.

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Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media

Cited by 11 publications

References 26 publications

Biochemical characterisation of chlorophyllase from leaves of selected Prunus species--a comparative study.

Biochemical characterisation of chlorophyllase from leaves of selected Prunus species--a comparative study.

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

Screening of dephytinization reaction of chlorophyll pigments with citrus acetone powder by UPLC–DAD–MS

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