COMPOSITIONS AND METHODS FOR MEASURING 3,6-L-AHG TRANSFERASE ACTIVITY AND 3,6-L-AHG

• US 20110306059A1
• Filed: 02/24/2011
• Published: 12/15/2011
• Est. Priority Date: 06/15/2010
• Status: Active Grant

• Current Assignee: Korea University Research and Business Foundation, Samsung Electronics Co. Ltd.
• Original Assignee: Korea University Research and Business Foundation, Samsung Electronics Co. Ltd.
• Inventors: KOO, Hyun Min, PARK, Sung Min, PARK, Jae Chan, KIM, Kyoung Heon, CHOI, In Geol
• Granted Patent: US 8,486,654 B2
• Time in Patent Office: Days
• US Class Current: 435/7.4
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 2333/91102 with definite EC number (2.4.1.-)

Abstract

Provided herein are a composition for measuring 3,6-anhydro-L-galactose (3,6-L-AHG) transferase activity by reduction of NADP to NADPH, and a method of measuring 3,6-L-AHG transferase activity using the same. The composition and method are useful for determining 3,6-L-AHG in a material containing 3,6-L-AHG such as algae biomass and industrial applications.

Claims

1. A composition for measuring 3,6-anhydro-L-galactose(3,6-L-AHG) transferase activity, comprising

NADP⁺ as a coenzyme,

3,6-L-AHG as a substrate, and

a buffer.

2. A method of measuring activity of 3,6-L-AHG transferase in an enzyme extract, comprising:

contacting an enzyme extract with the composition for measuring 3,6-L-AHG transferase activity according to claim 1; and

measuring amount of NADPH produced.

3. The method of claim 2, wherein amount of NADPH is determined by measuring absorbance at a wavelength of about 339 to 340 nm.

4. A composition for quantitative analysis of 3,6-anhydro-L-galactose(3,6-L-AHG), comprising

a purified 3,6-L-AHG transferase,

NADP⁺ as a coenzyme, and

a buffer.

5. A method for determining the presence or absence of 3,6-anhydro-L-galactose(3,6-L-AHG) in a sample, comprising

adding 3,6-L-AHG transferase and NADP to a sample;

and

measuring amount of NADPH produced in the sample to determine the presence or absence of 3,6-L-AHG in the sample.

6. The method of claim 5, wherein the amount of NADPH produced in the sample is directly proportional to the amount of 3,6-L-AHG in the sample.

7. The method of claim 5, wherein the amount of NADPH is determined by measuring absorbance at about 339 to 340 nm.

8. The method of claim 7, wherein the 3,6-L-AHG in the sample is determined from a calibration curve of absorbance at about 339 to 340 nm as a function of 3,6-L-AHG at the reaction conditions of the sample.

9. The method of claim 5, wherein the 3,6-L-AHG transferase is obtained from Saccharophagus degradans.

10. A method of purifying 3,6-anhydro-L-galactose(3,6-L-AHG) transferase from Saccharophagus degradans, —

comprising;

obtaining a crude extract of Saccharophagus degradans;

adding ammonium sulfate ((NH₄)₂SO₄) to the crude extract at a saturation level of about 0 to 50% to induce precipitation;

adding ammonium sulfate ((NH₄)₂SO₄) to the resultant supernatant from the induced precipitation at a saturation level of about 50 to 70% to induce a second precipitation; and

fractionating the resultant precipitant from the second precipitation by anion exchange chromatography to obtain 3,6-anhydro-L-galactose(3,6-L-AHG) transferase.

11. The method of claim 10, wherein the 3,6-anhydro-L-galactose(3,6-L-AHG) transferase has a specific activity of about 2.5 to 3.0 U/mg.

12. Purified Saccharophagus degradans 3,6-anhydro-L-galactose(3,6-L-AHG) transferase with a specific activity of about 2.5 to 3.0 U/mg.