Pectate Lyase

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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CAS RN: 9015-75-2.-An enzyme obtained from Aspergillus sp. Light brown, viscous liquid. Specific gravity is about 1.5. It is readily soluble in water. It is supplied at approximately 14 units per mL at pH 8.0 in Tris-HCl buffer [50 mM of Tris(hydroxymethyl)aminomethane containing 1 mM of CaCl, pH 8.0] in a solution of 50% Glycerol and 0.02% sodium azide. One unit is defined as the enzyme activity that produces 1 µmol of unsaturated product per minute.

1 Activity

1.1 Pectin solution

Transfer a quantity of Pectin, equivalent to 0.05 g on the dried basis, to a 100-mL volumetric flask. [NOTE-Pectin has a molecular weight of 103,000 Da; its degree of esterification (percentage of galacturonic acid groups substituted with methyl) is 12.]

Moisten with 0.1 mL of 2-propanol. Add 50 mL of water to the flask, and mix the solution with a magnetic stirrer. Use 0.5 N sodium hydroxide to adjust the solution to a pH of 12. Stop the stirrer, and allow the solution to stand undisturbed at room temperature for 15 minutes. Adjust the solution with 0.5 N hydrochloric acid to a pH of 8.0. Dilute with water to volume.

1.2 Tris buffer solution

Transfer 6.055 g of Tris (hydroxymethyl)aminomethane and 0.147 g of calcium chloride to a 1000-mL volumetric flask containing 950 mL of water, and mix. Adjust the solution with 1 N hydrochloric acid to a pH of 8.0. Dilute with water to volume.

1.3 Diluted pectate lyase

Transfer 0.5 mL of Pectate Lyase to a 50-mL volumetric flask, dilute with Tris buffer solution to volume, and mix.

1.4 Procedure

Add the solutions set forth in the table below to quartz cuvettes.

Perform the test on the solutions so obtained, using a suitable UV-Vis spectrophotometer (see Ultraviolet-Visible Spectroscopy (857)) and using water as the blank. Mix the solutions well at time 0, and immediately measure the absorbances at 235 nm. Record the value for the Enzyme blank, A_0-EB for the Test blank, A_0-TB and for the Test solution, A_0-TS. After incubation at room temperature for 30 minutes, determine the absorbance again at 235 nm for the Enzyme blank, A_30-EB: for the Test blank, A_30-TB and for the Test solution, A3_0-TS. One unit is defined as the enzymatic activity that produces 1 µmol of unsaturated product from pectin per minute. Calculate the Pectate Lyase activity, in units per mL, using the following formula:

50(10³) [(A_30-TS-A_30-EB-A_30-TB)-(A_0-TS-A_0-EB-A_O-TB)]/30E₂₃₅L

in which 50 is the volume, in mL, of Diluted pectate lyase; 10³ is the unit conversion factor, 30 is the time, in minutes, of the reaction; E₂₃₅ is the molar extinction coefficient, in M^-1cm^-1, of the reaction product (4600 M^-1cm^-1); and L is the path length, in cm, of the reaction cuvette (1 cm). Alternatively, these solutions, after being mixed in the cuvettes, can be immediately measured at 235 nm continuously in a recording UV-Vis spectrophotometer set up for kinetic assays. The result is obtained by correcting the blank determination, using the Enzyme blank and the Test blank.