Mananase

PRINCIPLE of Mananase

The substrate employed is Azurine-crosslinked carob galactomannan (AZCL-Galactomannan).  The substrate is prepared by dyeing and crosslinking highly purified carob galactomannan to produce a material, which hydrates in water but is water insoluble.  Hydrolysis by endo-1,4-β-mannanase produces water soluble dyed fragments, and the rate of release of these (increase in absorbance at 590 nm) can be related directly to enzyme activity.
One Unit of activity is defined as the amount of enzyme required to release 1 μmol of mannose reducing-sugar equivalents per minute under the defined assay conditions.

REAGENTS of Mananase
1. 2M Stock Buffer (pH 4.0) - Add 107.0 mL of Glacial Acetic Acid (CH3COOH) to 800 ml of distilled water. This solution is adjusted to pH 4.0 by the addition of 2N NaOH solution. The volume is adjusted to 1 Liter.
2.50 mM Dilution Buffer (pH 4.0) - Use 25 mL Stock buffer and added to 900 ml of water and the pH is adjusted to pH 4.0 by dropwise addition of 1N HCl solution. The volume is adjusted to 1 Liter.
3.2% Trizma Base (Same as Arabinase/Beta Glucanase)  - Dissolve 2 g of Trizma Base ≥99%  (Sigma T-1503) in DI water and bring to volume 100 ml.
4.Substrate  - Use ready-to-use tablet form as Beta-Mannazyme Tablets.

PREPARATION OF ENZYME SOLUTION
Weigh all samples to an accuracy of 4 digits. Triturate all solid samples. Prepare Enzyme solutions in Sodium Acetate Buffer.

PROCEDURE of Mananase
1.Pre-equilibrate an aliquot 0.5 ml of suitably Enzyme preparation in medium test tubes at 40C.
1.Initiate the reaction by adding a Beta-Mannazyme tablet. The tablet hydrated rapidly.  The suspension should not be stirred.  
2.Terminate the reaction exactly 10 minutes after addition of the tablet by adding 10 ml of Trizma Base solution with vigorous stirring on a vortex mixer.
3.Leave the tubes at room temperature for about 5 min, and then stir them again.
4.Filter the tube contents through a filter paper.
5.Prepare a substrate/enzyme blank by adding a β-Mannazyme tablet to 0.5 ml of extraction buffer. Incubate at 40C for 10 min, add 10 ml of Trizma Base 2% and filter after 5 minutes.
6.The absorbance of the reaction solutions is measured at 590 nm against the blank. If the absorbance is above 1.4 dilute an aliquot of the enzyme extract with an equal volume of extraction/dilution buffer and repeat the assay.

CALCULATION of Mananase
endo--Mananase activity in the sample being assayed is determined by reference to the standard curve to convert absorbance values to milli-Units per assay (ie. per 0.5 ml).Alternatively, for absorbance values in the range for 0.1 to 1.5, these values can be calculated by reference to the equation:
Y = MX + C.
Y = endo--Mannanase activity (in milliUnits / assay, ie per 0.5 ml).        M = Slope of the calibration graph.
X = Absorbance of the reaction at 590nm (minus the reaction blank,            C = Intercept on the Y-axis.             
       or read against the reaction blank).
Values for M and C vary slightly between batches of Beta-Mannazyme tablets.  M and C values for the particular batch of tablets are provided with the tablets.

endo--Mannanase activity per ml or g or original preparation:

          U/g   =        Y   x   100  x   2     
                                 Wt   x  1000
Where:
Y         =   Endo--Mananase activity (in milliUnits /assay, ie per 0.5 ml).
100      =  1 g or ml of enzyme extracted in 100 ml of buffer.
2          =   Conversion from 0.5 ml (as assayed) to 1.0 ml.
1/1000 =   Conversion from milliUnits to Units.

Wt       =   Final weight of enzyme dilution (g/mL)

CERTIFICATE OF ANALYSIS

Packaging and storage

Solid food packaging bag, 25 kg / barrel.

Mananase should be stored in dry, cool and well ventilated places away from the sun, heat.

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