3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2 Unit Definition = One unit will liberate 1.0 μmole of reducing sugar (measured as D-(+)glucosamine equivalents) from Chitosan per minute at pH 5.5 at 37ºC.
3.3 p-HBAH = p-Hydroxybenzoic Acid Hydrazide Reagent
T = 37°C, pH = 5.5, A405nm, Light path = 1 cm
Spectrophotometric Stop Reaction
7.3.1 55 mM Sodium Acetate Buffer, pH 5.5 at 37ºC(Buffer) Prepare in purified water at 7.49 mg/ml using Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number , S8625. Adjust to pH 5.5 at 37ºC with 1 M NaOH.
7.3.2 50 mM Sodium Acetate Buffer and 10%(v/v) Glycerol Solution, pH 5.5 at 37ºC (Enzyme Diluent) Prepare in Reagent 7.3.1 (Buffer) at 0.1 ml/ml using Glycerol, Sigma-Aldrich Product Number , G9012.
7.3.3 1M Acetic Acid (Acetic) Prepare in purified water at 0.058 ml/ml using Acetic Acid, ACS Reagent, Sigma-Aldrich Product Number , 242853.
7.3.4 0.1%(w/v) Chitosan (Chitosan)
18.104.22.168 Use Chitosan from Crab Shells, Sigma-Aldrich Product Number , C3646. Obtain a minimum of 60 mg, 40 mesh sieve size.
22.214.171.124 If a minimum of 60 mg, 40 mesh sieve size is not available, obtain a mortar and crucible. Place mortar and crucible in dry ice. Allow to sit for a minimum of thirty minutes. Place a teaspoon of Chitosan, Sigma-Aldrich Product Number , C3646, in the crucible. Using thick leather gloves, fill the crucible with liquid nitrogen and grind the Chitosan as the liquid nitrogen is boiling off. Repeat this process until a minimum of 60 milligrams of Chitosan at 40 mesh is obtained.
126.96.36.199 In a four dram vial, add a suitable stir bar (approximate length equivalent to the diameter of a four dram) and accurately weigh a 55 mg to 80 mg of Chitosan at 40 mesh. Add the appropriate volume of Reagent 7.3.3 (Acetic) to obtain a 10.0 mg/ml solution.
188.8.131.52 Stir vigorously until dissolution (approximately 1 hour). Dissolution can be facilitated to less than one hour by using a water bath with immersible stirrer at 37ºC.
184.108.40.206 After dissolution, dilute 5.0 ml of the 1.0%(w/v) chitosan solution from step 220.127.116.11 to 50.0 ml with Reagent 7.3.1 (Buffer). Adjust to pH 5.2 at 37ºC with 0.05 ml aliquots of 10 N NaOH and then use 1N NaOH to adjust to pH 5.5 at 37ºC.
7.3.5 0.5N Sodium Hydroxide Solution (NaOH) Prepare a 0.5 ml/ml solution in purified water using Sodium Hydroxide, Standard Solution, 1.0N, Sigma-Aldrich Product Number , S2567 or at 20 mg/ml in purified water using Sodium Hydroxide, Reagent Grade, Sigma-Aldrich Product Number , S5881.
7.3.6 0.25%(w/v) p-Hydroxybenzoic Acid Hydrazide Solution (p-HBAH) Prepare in Reagent 7.3.5 (NaOH) at 2.5 mg/ml using p-Hydroxybenzoic Acid Hydrazine, Sigma-Aldrich Product Number , H9882.
7.3.7 1.5 mM D-Glucosamine (STD)
18.104.22.168 Prepare 100 ml in purified water using D(+)-Glucosamine Hydrochloride, Sigma-Aldrich Product Number , G4875.
22.214.171.124 Concentration must be adjusted for hydrochloride content.
7.3.8 Chitosanase Solution (Enzyme) Immediately before use, prepare a solution containing approximately 0.5 units/ml in cold purified Reagent 7.3.2 (Enzyme Diluent).
7.4.1 Enzymatic Assay
126.96.36.199 Pipette (in milliliters) the following reagents into suitable glass vessels in triplicate for control and/or sample:
|Reagent 7.3.4 (Chitosan)
188.8.131.52 Mix by swirling and equilibrate to 37ºC. Then add:
|Reagent 7.3.8 (Enzyme)
184.108.40.206 Mix by swirling and incubate Test and Blank for exactly 10 minutes at 37ºC. Then remove 0.40 ml from test and add to a cold microcentrifuge tube, Sigma-Aldrich Product Number , T6524, containing the following:
|Reagent 7.3.6 (p-HBAH)
220.127.116.11 Mix by inversion. For blank, boil an aliquot of the Reagent 7.3.8 (Enzyme) for exactly 2 minutes. Then add:
|Reagent 18.104.22.168 (Boiled Enzyme)
22.214.171.124 Remove 0.40 ml from blank and add to a cold microcentrifuge tube, Sigma-Aldrich Product Number , T6524, containing the following:
|Reagent 7.3.6 (p-HBAH)
126.96.36.199 Make sure all caps of microcentrifuge tubes are vented. Boil all blank and test reaction mixtures for exactly five minutes and cool to room temperature.
188.8.131.52 Centrifuge all test and blank reaction mixtures for a minimum of five minutes. Remove supernatant, transfer to suitable cuvette and measure/record the A405nm for all blank and test reactions versus purified water using a suitable spectrophotometer.
7.4.2 Standard Curve:
184.108.40.206 A standard curve is made by pipetting (in milliliters) the following reagents into microcentrifuge tubes, Sigma-Aldrich Product Number , T6524 with vented caps:
|Reagent 7.3.7 (STD)
|Reagent 7.3.6 (p-HBAH)
220.127.116.11 Mix by inversion and boil for exactly five minutes. Cool to room temperature. Centrifuge for exactly five minutes. Remove supernatant and transfer to suitable cuvette and measure/record the A405nm for all blank and test reactions versus purified water using a suitable spectrophotometer.
7.5.1 Standard Curve
ΔA405nm Standard = A405nm Std - A405nm Std Blank
Plot the ΔA405nm of the Standards vs μmoles of D(+)-Glucosamine. Calculate and record the slope, y-intercept, and linear regression(r-square).
7.5.2 Sample Determination
ΔA405nm Sample = A405nmTest - A405nmTest Blank
Determine the µmoles of reducing sugar liberated as D(+)- Glucosamine equivalents using the Standard Curve.
||Units/ml enzyme =
||(µmoles of D(+)- Glucosamine released)(df)
df = Dilution Factor
0.025 = Volume (in milliliter) of enzyme used in enzymatic reaction
0.40 = Volume (in milliliter) of enzyme reaction used in p-HBAH reaction
10 = Time of incubation of assay per unit definition
||Units/mg protein =
|mg solid/mL enzyme
7.6 FINAL ASSAY CONCENTRATION :
In a 1.00 mL reaction mix, the final concentrations are 98 mM acetic acid, 50 mM sodium acetate, 0.25%(v/v) glycerol, 0.098%(w/v) chitosan, and approximately 0.01 units of chitosanase.
8. References & Attachments
8.1 Boucher, I. et. al., (1995) Journal of Biological Chemistry. 270, 31,077.
8.2 Aretz, W. et. al., (1989) FEMS Microbiology Letters . 65, 31.
8.3 Lever, M. A. (1972) Journal of Analytical Biochemistry 47, 273-279.