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Specific Activity of major pectinases reported in literature
Protopectinases
Title: Microbial pectinolytic enzymes: A review Journal: Process Biochemistry, Volume : 40
Year: 2005, Page No.: 2931–2944 Authors: Ranveer Singh Jayani, Shivalika Saxena,
Reena Gupta Protopectinase activity (PPase) is assayed by measuring
the amount of pectic substance liberated from protopectin by the carbazole–
sulphuric acid method. The pectin concentration is measured as D-galacturonic
acid from its standard curve. One unit of PPase activity is defined as the
enzyme that liberates pectic substance corresponding to 1 mmol of
Dgalacturonic acid per millilitre of reaction mixture under assay
conditions. Carbazole - Sulphuric Acid Method
http://www.jbc.org/cgi/reprint/163/2/511.pdf Pectin Methyl Esterase
(EC 3.1.1.11) Title: Application of Doehlert design for water
activity, pH and fermentation time optimization for Aspergillus niger
Pectinolytic activities production in solid-state and submerged fermentation Journal: Enzyme and Microbial Technology, Volume:
25 Year: 1999, Page No.: 411-419 Author: Viviana M. Taragano, Ana M.R. Pilosof Pectinesterase activity was determined by mixing 1.9 ml
of a pectin 0.5%, bromophenol green 0.017% solution (pH 5) with 0.1 ml of
the appropriate extract dilution and monitoring the decrease in the
absorbance at 617 nm due to the generation of free corboxyl groups. The
calibration curve was made by using galacturonic acid as standard and 1 Unit
was defined as the amount of enzyme that liberated one carboxyl group per
minute. Pectin Methyl Esterase (EC 3.1.1.11): Title:
Journal: Process Biochemistry, Volume: 40 Year: 2005 Page No.: 2885–2889 Authors: Denis Silva, Kivia Tokuioshi, Eduardo da Silva Martins, Roberto Da Silva, Eleni GomesPectin methylesterase (PE) was assayed by measuring the methanol liberated from pectin solution, colorimetrically. The reaction mixture, containing 0.8 mL of a 1% solution of 67% methoxylated citric pectin (Braspectina) in 0.2 M acetate buffer (pH 5.0) and 0.2 mL of crude enzyme solution, was incubated at 50 0C for 10 min. One unit of the PE was defined as the activity that released 1 mmol of methanol released per minute under defined conditions. Sigma Aldrich Company has given the enzymatic activity of pectinesterase in the following link: http://www.sigmaaldrich.com/sigma/enzyme%20assay/pectinesterase.pdf Pectin Esterase Assay: Title: Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems Journal: Journal of Industrial Microbiology & Biotechnology Volume: 20, Year: 1998, Page No.:34–38 Authors: MC Maldonado and AM Strasser de Saad Pectinesterase assay (PE): To 10 ml of 0.5% (w/v) pectin in 0.1 M NaCl, 2 ml of filtrate was added. The pH was adjusted to 4.5 with 0.1 M NaOH and the mixture was incubated for 60 min at 35°C. PE activity was measured by determining the carboxyl groups released by titration with 0.02 N NaOH. One unit of PE was defined as the amount of enzyme releasing one milliequivalent of ester hydrolyzed (carboxyl group) per minute.
Endo - polygalacturonase (EC 3.2.1.15)
Endo polygalacturonase (EC 3.2.1.15): Title: Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation Journal: Applied Microbiology and Biotechnology, Volume: 58 Year: 2002, Page No.: 164–169 Authors: A. Blandino, T. Iqbalsyah, S.S. Pandiella, D. Cantero, C. Webb Endo-Polygalacturonase(Endo-PG) was determined at 45 °C by viscometry method. For endo- PG, 5 ml of enzyme extract were mixed with 15 ml of 1% (w/v) pectin in 0.1 M acetate buffer (pH 5.0). The reduction in viscosity was monitored with an Ostwald viscometer (Technico, BS/U, UK). A unit of endo-PG activity (U) was defined as the amount of enzyme that reduced the viscosity of the pectin solution by 50% per minute under the conditions described above. All measurements were made in triplicate. Endo polygalacturonase (EC 3.2.1.15): Sigma Aldich is giving the activity in the following link http://www.sigmaaldrich.com/sigma/enzyme%20assay /polygalacturonase.pdf
Exo-Polygalacturonase (EC 3.2.1.67)
Title: Production of pectinase by solid-state fermentation with Penicillium viridicatum RFC3Journal: Process Biochemistry, Volume: 40 Year: 2005 Page No.: 2885–2889 Authors: Denis Silva, Kivia Tokuioshi, Eduardo da Silva Martins, Roberto Da Silva, Eleni GomesExo-Polygalacturonase activity, 1 ml of enzyme extract was added to a solution containing 2 ml of 0.5% pectin in 0.1 M acetate buffer (pH 5.0). Samples were incubated at 45 °C for 10 min and the Reducing Sugars were determined by a modification of the dinitrosalicylic acid method. A unit of exo-PG activity (U) was defined as the amount of enzyme that produced 1 μmol galacturonic acid/min under the conditions described above. All measurements were made in triplicate. Exo-Polygalacturonase (EXO-PG) Title: Carbon sources effect on pectinase production form Aspergillus japonicus 586 Journal: Brazilian Journal of Microbiology Volume: 31(4), Year: 2000 Authors: Maria F. S. Teixeira; José L. Lima Filho; Nelson Durán The EXO-PG activity was measured by quantification of reductant groups liberated from the pectin solution by the DNS method. To 250 ml 0.5% (w/v) of citric pectin in 0.025 M acetate buffer, pH 5.0, 1 mM EDTA, 250 ml of the dialyzed enzymatic extract were added. The blank was prepared as described above. After 10 min at 50°C, 0.5 ml DNS solution was added to each tube and boiled for 5 min. After cooling 5 ml of destilled water was added, agitated and the absorbance at 575 nm was measured. The reductant sugar values were determined from a standard curve of monogalacturonic acid. One enzymatic unit (U) was defined by the enzyme quantity which liberates a mmol of monogalacturonic acid per min under the described experimental conditions. The enzymatic activity was expressed in mmol/ml. Available online: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822000000400009&lng=es&nrm=iso
exo-poly-a -galacturonosidase(EC 3.2.1.82)
Enzyme Activity Not Available
Oligogalacturonate hydrolase
Enzyme Activity Not Available
Unsaturated oligogalacturonate hydrolases
Enzyme Activity Not Available
Endo polymethylgalacturonases
Title: Hydrolytic enzymes and ability of arbuscular mycorrhiza fungi to colonize roots Journal: Journal of Experimental Botany, Volume: 51, Year: 2000. Page No. 1443-1448 Authors: J. M. Garcia Garrido, M. Tribak, A. Rejon Palomares, J.A. Ocampo and I. Garcia Romera Endo polymethylgalacturonase (Endo - PMG) was assayed by the viscosity method using citrus pectin as substrates. The reduction in viscosity was determined as 0-30 min intervals. Approximately 0.5 ml of teh reaction mixture was sucked into a 1 ml syringe and the time taken for the meniscus to flow from the 0.70 ml to 0.2 ml mark was recorded. The reaction mixture contained 1 ml of 0.5% substrate in 50 mM citrate -phosphate buffer (pH 5) and 0.2 ml enzyme. Viscosity reduction was determined at 370C. One unit of enzyme activity was expressed as specific activity (U mg-1 protein) (U reciprocal of time in hours for 50% viscosity loss x 103). Available at: http://jxb.oxfordjournals.org/cgi/reprint/51/349/1443.pdf Endo Polymethylgalacturonase (PMG): Title: Rotating simplex method of optimization of physical parameters for higher production of extracellular pectinases in bioreactor. Journal: Bioprocess Engineering, Volume: 23, Year: 2000, Page No. 47-49. Authors: T. Panda, G.S.N. Naidu. PMG has been assayed by conducting enzymatic reaction at 300C using pectin as a substrate. This reaction was conducted by adding 0.074 cm3 of culture filtrate containing enzyme to 0.2 cm3 of substrate solution (2.4 kg/m3, at pH 5.3). The reaction was terminated by adding 0.5 cm3 of copper reagent. The resulting solution was placed in vigorously boiling water bath for 10 min. After cooling to 300C 1 cm3 of arsenomolybdate reagent was added to the reaction mixture. The absorbance was read on a spectrophotometer at 500 nm against a suitable blank. Standard solutions of galacturonic acid were prepared in suitable concentration range and a standard plot was prepared to estimate the activity of PMG. The end product is oligomers of galacturonic acid and oligomers as high as pentamer give the same absorbance of monomer. Hence, galacturonic acid was used as the standard. The enzyme activity was expressed in terms of unit (U). One unit of enzyme activity was defined as the amount of enzyme that catalyzes the release of 1 micro mole of product per unit volume of culture filtrate per unit time at standard assay conditions.
pectate lyase (EC 4.2.2.2)
Title: Kinetic behaviour and thermal inactivation of pectinlyase used in food processing Journal: International Journal of Food Science and Technology Year: 2004, Volume: 39, Page No.: 631–639 Authors: Natividad Ortega, Silvia de Diego, Jose M. Rodrı Guez Nogales, Manuel Perez-Mateos and Marıa D. Busto1 The Pectate Lyase activity was determined spectrophotometrically by monitoring the increase of absorbance at 235 nm during 120s. The reaction mixture contained 1 mL of a 5 mg mL-1 apple pectin solution as substrate and 1 mL of enzyme solution. Both solutions were dissolved in 0.1 m McIlvaine buffer at pH 6.0. Preincubations were at 40 0C for 10min, and adding the enzyme started the reaction. Control tubes contained distilled water instead of enzyme solution. One unit of enzyme activity was defined as the amount of enzyme that produced an increase of one unit of A235 per minute. The increase in the A235 originates from the formation of unsaturatedmethyloligosaccharides and can be transformed into l moles of product by using an extinction coefficient of 5500 M -1 cm-1).McIlvaine buffer Preparation: http://www-saps.plantsci.cam.ac.uk/records/rec412.htm Molar Extinction Coefficient definition: http://www.biology-online.org/dictionary/Molar_extinction_coefficient http://www.ktf-split.hr/periodni/en/abc/a.html http://ccc.chem.pitt.edu/wipf/Courses/1140_05_files/Extinction-coefficients.pdf Pectate lyase activity: Title: Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome Journal: Proceedings of National Academy of Sciences of the United States of America, Volume: 98, Year: 2001, Page No. : 4125-4129 Authors: Yutaka Tamaru, and Roy H. DoiAvailable online: http://www.pnas.org/cgi/reprint/98/7/4125.pdf The reaction mixture consisted of 0.1 ml of Pectate lyase A solution, 1 ml of 0.4% sodium polygalacturonic acid (Sigma), 0.5 ml of 200mMTriszHCl buffer (pH 8.0), and 0.4 ml of 0.25 mM CaCl2 in a total volume of 2 ml. After reaction at 40°C for 10 min, 2 ml of 50 mM HCl was added to terminate the reaction, and the absorbance in the solution was measured at 235 nm (19). One unit of enzyme activity was defined as the amount of enzyme that liberates 1 mmol of unsaturated oligogalacturonides, equivalent to 1 mmol digalacturonideymin under the above conditions. The molar extinction coefficient value was assumed to be 4,600 M-1cm-1 for unsaturated digalacturonide.
Pectate disaccharide-lyase EC 4.2.2.9
Enzymatic assay not available
Oligogalacturonide lyase EC 4.2.2.6
Enzymatic assay not available
Oligogalacturonide lyase EC 4.2.2.6
Enzymatic assay not available
Pectin lyase EC 4.2.2.10
Title: Pectin lyase from fungi Journal: Methods of Enzymology, Volume: 8, Year: 1966, Page No.: 628- 631 Author: P. Albersheim Pectin lyase (Pl) activity was determined by measuring the increase in absorbance at 235 nm of substrate solution (0.8 ml 1% citric pectin in 0.2M tris-HCl buffer, pH 8.5) hydrolyzed by 0.2 ml enzyme solution, at 50ºC. One unit of enzymatic activity (U) was defined as the amount of enzyme which releases 1 μmol of unsaturated uronide per minute, based on the molar extinction coefficient (ε=5500) of the unsaturated products. Molar Extinction Coefficient definition Pectin Lyase Activity: Title: Rotating simplex method of optimization of physical parameters for higher production of extracellular pectinases in bioreactor. Journal: Bioprocess Engineering, Volume: 23, Year: 2000, Page No. 47-49. Authors: T. Panda, G.S.N. Naidu. PL was assayed by conducting the enzymatic reaction with pectin (5 kg/m3) in citrate-phosphate buffer (pH 4.9, 0.7 cm3), of culture filtrate containing enzyme was added to 0.63 cm3 of substrate and incubated for 60 min at 300C. The reaction was stopped by adding 3.5 cm3 of 0.5 M HCl and contents were estimated spectrophotometrically for 4-5 unsaturated galactuonosyl residues at 235 nm against suitable blank. A molar extinction coefficient of 5550 M-1cm-1 was used to calculate the enzyme activity. Molar Extinction Coefficient definition
Buffers for Enzyme Activity (In internet you can find online buffer calculations) |
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