A comprehensive evaluation of pectinase, pectinmethylesterase and pectolyase activity

Pectin polysaccharide has galacturonic acid with linear chains of α-(1–4)-linked D- galacturonic acid. Rhamnogalacturonan I pectins (RG-I) shows the existence of the repeating disaccharide 4-α-D-galacturonic acid-(1,2)-α-L- rhamnose, which acts as a backbone. Chiefly, D-galactose, L-arabinose, and D-xylose are the sugars types and its proportions of neutral sugars are varied according to the origin of pectin. Pectinase, pectinmethylesterase, and pectolyase enzymes have important applications in food, textile and agricultural industries. These enzymes play an important role in the breakdown of the central part of the plant cell wall. Pectin forms the center part of the plant cell wall. Pectins are termed as structural polysaccharide that has integrity for the steadiness of the plant cell wall. Citrate buffer of molarity 0.1 utilized to verify optimal pH along with temperature, for standardising enzyme activity of pectinase, pectolyase, and pectinmethylesterase by the dinitrosalicylic acid reagent method. Confirmatory check of enzyme’s activity was performed on plant leaves dried particles. Impact of catechin presence in enzyme reaction was too studied. Results delve into degradation of the plant polysaccharide by applying these enzymes. An increase in the monosaccharide galacturonic acid quantity was also significant. The highest release of the polyphenols was found due to pectolyase followed by pectinmethylesterase and pectinase. Pectinmethylesterase effect showed the maximum release of the flavonoids followed by pectinase and pectolyase which was remarkable.

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Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase

Biochemical Journal ◽

10.1042/bj2790343 ◽

1991 ◽

Vol 279 (2) ◽

pp. 343-350 ◽

Cited By ~ 81

Author(s):

J Nari ◽

G Noat ◽

J Ricard

Keyword(s):

Cell Wall ◽

Metal Ions ◽

Plant Cell ◽

Plant Cell Wall ◽

Direct Interaction ◽

Enzyme Reaction ◽

Pectin Methylesterase ◽

High Concentrations ◽

Enzyme Molecules ◽

Hydrolysis Of

The hydrolysis of p-nitrophenyl acetate catalysed by pectin methylesterase is competitively inhibited by pectin and does not require metal ions to occur. The results suggest that the activastion by metal ions may be explained by assuming that they interact with the substrate rather than with the enzyme. With pectin used as substrate, metal ions are required in order to allow the hydrolysis to occur in the presence of pectin methylesterase. This is explained by the existence of ‘blocks’ of carboxy groups on pectin that may trap enzyme molecules and thus prevent the enzyme reaction occurring. Metal ions may interact with these negatively charged groups, thus allowing the enzyme to interact with the ester bonds to be cleaved. At high concentrations, however, metal ions inhibit the enzyme reaction. This is again understandable on the basis of the view that some carboxy groups must be adjacent to the ester bond to be cleaved in order to allow the reaction to proceed. Indeed, if these groups are blocked by metal ions, the enzyme reaction cannot occur, and this is the reason for the apparent inhibition of the reaction by high concentrations of metal ions. Methylene Blue, which may be bound to pectin, may replace metal ions in the ‘activation’ and ‘inhibition’ of the enzyme reaction. A kinetic model based on these results has been proposed and fits the kinetic data very well. All the available results favour the view that metal ions do not affect the reaction through a direct interaction with enzyme, but rather with pectin.

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Genome-wide and enzymatic analysis reveals efficient D-galacturonic acid metabolism in the basidiomycete yeastRhodosporidium toruloides

10.1101/675652 ◽

2019 ◽

Author(s):

Ryan J. Protzko ◽

Christina A. Hach ◽

Samuel T. Coradetti ◽

Magdalena A. Hackhofer ◽

Sonja Magosch ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Galacturonic Acid ◽

Plant Cell Wall ◽

Rhodosporidium Toruloides ◽

Cell Wall Polysaccharide ◽

Aromatic Molecules ◽

Renewable Feedstocks ◽

Genome Wide ◽

A Genome

AbstractBiorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such asSaccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeastRhodosporidium toruloidesis a promising and robust host for lipid and terpene derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6-sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzedR. toruloidespotential to assimilate D-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. D-galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with D-glucose and D-xylose inR. toruloides. A genome-wide analysis by combined RNAseq/RB-TDNAseq revealed those genes with high relevance for fitness on D-galacturonic acid. WhileR. toruloideswas found to utilize the same non-phosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for D-galacturonic acid utilization. These results set the basis for use ofR. toruloidesas a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies in basidiomycetes.ImportanceThe switch from the traditional fossil-based industry to a green and sustainable bio-economy demands the complete utilization of renewable feedstocks. Many currently used bio-conversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. D-galacturonic acid is a plant component of bio-conversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeastRhodosporidium toruloideshas been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods, physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of D-galacturonic acid. This study identifies an efficient pathway for uronic acid conversion to guide future engineering efforts, and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus.

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Biochemical characterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2017.03.012 ◽

2017 ◽

Vol 486 (1) ◽

pp. 130-136 ◽

Cited By ~ 6

Author(s):

Yohei Uehara ◽

Shunsuke Tamura ◽

Yusuke Maki ◽

Kenta Yagyu ◽

Tadashi Mizoguchi ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Biochemical Characterization ◽

Rhamnogalacturonan I

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Genomewide and Enzymatic Analysis Reveals Efficient D-Galacturonic Acid Metabolism in the Basidiomycete Yeast Rhodosporidium toruloides

mSystems ◽

10.1128/msystems.00389-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 5

Author(s):

Ryan J. Protzko ◽

Christina A. Hach ◽

Samuel T. Coradetti ◽

Magdalena A. Hackhofer ◽

Sonja Magosch ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Galacturonic Acid ◽

Plant Cell Wall ◽

Agricultural Waste ◽

Rhodosporidium Toruloides ◽

Cell Wall Polysaccharide ◽

Content Type ◽

Aromatic Molecules ◽

Renewable Feedstocks

ABSTRACT Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid- and terpene-derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6 sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed the potential of R. toruloides to assimilate d-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. d-Galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with those of d-glucose and d-xylose in R. toruloides. A genomewide analysis by combined transcriptome sequencing (RNA-seq) and RB-TDNA-seq revealed those genes with high relevance for fitness on d-galacturonic acid. While R. toruloides was found to utilize the nonphosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for d-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies with basidiomycetes. IMPORTANCE The switch from the traditional fossil-based industry to a green and sustainable bioeconomy demands the complete utilization of renewable feedstocks. Many currently used bioconversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. d-Galacturonic acid is a plant component of bioconversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeast Rhodosporidium toruloides has been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods and physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of d-galacturonic acid. This study identified an efficient pathway for uronic acid conversion to guide future engineering efforts and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus.

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Characterisation of Fiber Obtained from Pumpkin (cucumis moschata duch.) Mesocarp Through Enzymatic Treatment

Food Science and Technology International ◽

10.1177/1082013207077914 ◽

2007 ◽

Vol 13 (2) ◽

pp. 141-151 ◽

Cited By ~ 21

Author(s):

E.N. Fissore ◽

N.M. Ponce ◽

C.A. Stortz ◽

A.M. Rojas ◽

L.N. Gerschenson

Keyword(s):

Cell Wall ◽

Enzymatic Hydrolysis ◽

Galacturonic Acid ◽

Cold Water ◽

Side Chains ◽

Cell Wall Degrading Enzymes ◽

Citrate Buffer ◽

Rhamnogalacturonan I ◽

Enzyme Substrate ◽

Fiber Fractions

Cell wall-enriched pumpkin ( Cucumis moschata Duch.) powder was submitted to enzymatic hydrolysis by cellulase or hemicellulase in order to evaluate the performance of these cell wall-degrading enzymes on that substrate. Different enzyme-substrate ratios were evaluated and the effect exerted by the buffer on cell wall polysaccharides. Cellulase produced the release of pectin macromolecules which include homogalacturonans side chains, the rhamnogalacturonan I core and rhamnogalacturonan II, in conjunction with xylogalacturonans. The content of galacturonic acid in product obtained ranged from 545 to 781 g/kg of fiber. Hemicellulases produced intense pectin hydrolysis leading to fiber-fractions with galacturonic acid contents ranging from 390 to 444 g/kg of fiber and enriched in glucose polymers as the enzyme proportion increased. Few rhamnogalacturonan-I was present.The acidic citrate buffer (pH 5.2) used for allowing enzyme activity could per se remove noncovalent cross-links like ionic bonds. As a consequence, pectin-in-extensin entanglements, pectins joined by Ca2+-bridges through the homogalacturonan side chains, and some pectins that are originally soluble in cold water due to little or no binding to the cell wall, could be removed by this citrate buffer. Enzymatic hydrolysis as well as buffer extraction produced fiber-products with an important thickening effect of aqueous systems. This effect was smaller as the ratio enzyme-substrate was increased and, in general, the fiber fractions isolated produced an in vitro glucose diffusion retardation.

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