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    High DP Xyloglucan Oligosaccharide: Preparation

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    andirio7486
    This document describes the preparation and use of high degree of polymerization xyloglucan oligosaccharides (HDP-XGO) as a substrate for the enzyme isoprimeverose-producing xyloglucan hydrolase (IP-P-XGH). HDP-XGO is produced through exhaustive hydrolysis and purification of tamarind seed xyloglucan. It has a molecular weight of 3000-4000 daltons and serves as a useful and specific substrate for IP-P-XGH in enzymatic assays. The document provides a suggested assay procedure and Lineweaver-Burke plot showing IP-P-XGH kinetics on HDP-XGO.

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    High DP Xyloglucan Oligosaccharide: Preparation

    Uploaded by

    andirio7486
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    This document describes the preparation and use of high degree of polymerization xyloglucan oligosaccharides (HDP-XGO) as a substrate for the enzyme isoprimeverose-producing xyloglucan hydrolase (IP-P-XGH). HDP-XGO is produced through exhaustive hydrolysis and purification of tamarind seed xyloglucan. It has a molecular weight of 3000-4000 daltons and serves as a useful and specific substrate for IP-P-XGH in enzymatic assays. The document provides a suggested assay procedure and Lineweaver-Burke plot showing IP-P-XGH kinetics on HDP-XGO.

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    nelson somogy 1

    Original Title

    nelson 33

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    This document describes the preparation and use of high degree of polymerization xyloglucan oligosaccharides (HDP-XGO) as a substrate for the enzyme isoprimeverose-producing xyloglucan hydrolase (IP-P-XGH). HDP-XGO is produced through exhaustive hydrolysis and purification of tamarind seed xyloglucan. It has a molecular weight of 3000-4000 daltons and serves as a useful and specific substrate for IP-P-XGH in enzymatic assays. The document provides a suggested assay procedure and Lineweaver-Burke plot showing IP-P-XGH kinetics on HDP-XGO.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    28 views2 pages

    High DP Xyloglucan Oligosaccharide: Preparation

    Uploaded by

    andirio7486
    This document describes the preparation and use of high degree of polymerization xyloglucan oligosaccharides (HDP-XGO) as a substrate for the enzyme isoprimeverose-producing xyloglucan hydrolase (IP-P-XGH). HDP-XGO is produced through exhaustive hydrolysis and purification of tamarind seed xyloglucan. It has a molecular weight of 3000-4000 daltons and serves as a useful and specific substrate for IP-P-XGH in enzymatic assays. The document provides a suggested assay procedure and Lineweaver-Burke plot showing IP-P-XGH kinetics on HDP-XGO.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 2

    High DP XYLOGLUCAN OLIGOSACCHARIDE

    06/2002

    PREPARATION:
    High DP (degree of polymerisation) Xyloglucan oligosaccharides (HDP-XGO) is produced by
    exhaustive hydrolysis of tamarind seed xyloglucan with endo-cellulase followed by β-galactosidase. The
    reaction mixture is then treated with sodium borohydride to give complete reduction of reducing-
    sugars to sugar alcohols.The high DP material is then recovered by precipitation with 66% ethanol.The
    approximate MW of this material is 3,000-4,000 daltons.

    HDP-XGO is a useful and relatively specific substrate for the enzyme isoprimeverose-producing
    xyloglucan hydrolase (IP-P-XGH).

    Suggested Assay Procedure for IP-P-XGH:


    Incubate 0.2 ml of suitably diluted enzyme preparation wth 0.5 ml of HDP-XGO (5 mg/ml) in 0.1 M
    sodium acetate buffer (pH 4.5) at 40oC.Terminate reaction after 3, 6, 9 nad 12 min by the addition of
    0.5 mL of Nelson-Somogyi reducing sugar Solution D. Prepare blank by adding Nelson-Somogyi
    solution D to substrate solution before adding enzyme preparation. Develop colour by the modified
    Nelson-Somogyi procedure. Glucose standards (50 µg) are run concurrently.

    Lineweaver-Burke plot of isoprimeverose-producing xyloglucan hydrolase (IP-P-XGH) on


    HDP-XGO.

    6
    1/V

    0
    0 0.5 1 1.5 2 2.5 3

    1/[HDP-XGO], (mg/ml) -1

    Km = 0.80 mg/ml
    Reducing Sugars by the Nelson-Somogyi Procedure

    NOTE: This method is derived from the published literature. Poisonous chemicals are used. It is the
    responsibility of the analyst to ensure that the chemicals are used safely and disposed of
    properly. Megazyme takes no responsibility for injuries incurred during the correct or
    incorrect use of this method.

    METHOD:

    Reagents:
    A. Dissolve 25 g anhydrous sodium carbonate
    25 g sodium potassium tartrate, and
    200 g sodium sulphate
    in 800 ml of distilled water and diluted to 1 litre. Filter if necessary.

    B. Dissolve 30 g of copper sulphate pentahydrate in 200 ml of distilled water containing 4 drops


    concentrated sulphuric acid.

    C. Dissolve 50 g of ammonium molybdate in 900 ml of distilled water and add 42 ml of concentrated


    sulphuric acid. Separately, dissolve 6 g of sodium arsenate heptahydrate in 50 ml of water
    and add this solution to the solution of ammonium molybdate. Adjust the volume to 1 litre.
    Warm the solution to 55 °C if necessary for complete dissolution.

    D. Add 1 ml of reagent B to 25 ml of reagent A.

    E. Dilute solution C, 5-fold (50 ml to 250 ml) with distilled water before use (stable at 4°C for about
    one week).

    Procedure:
    To 0.5 ml of aqueous sample (containing up to 50 µg of reducing sugar equivalents)
    add:
    0.5 ml of solution D and mix well on a vortex stirrer.

    Heat the tubes in a boiling water bath for 20 min and then cool them to room temperature (5 min). Stir the
    tubes for 10 sec (until carbon dioxide is completely released).

    Add: 3.0 ml of Solution E to each tube. Stir the tubes well (10 sec on vortex mixer), and allow them to
    stand for 10 min and mix again.

    Measure the absorbance at 520 nm.

    A blank is prepared by using water (0.5 ml) in place of the sample (0.5 ml).

    A standard curve is prepared using appropriate amounts of reducing sugar (e.g. glucose 0-50 µg/test).

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