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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Cellulases (C1 and Cx)
(1,4-(1,3:1,4)-b-D-Glucan 4 glucanohydrolase EC 3.2.1.4)
 
Hydrolysis of crystalline cellulose is a complex process. A minimum of three different types of enzymes are believed to be involved
i) Endo-b,4 glucanase (Cx-cellulase).
ii) Exo-b,4 glucanase (C1-cellulase).
iii) b-glucosidase (cellobiase).
Initiation of Hydrolysis of native cellulose is effected by C1 enzyme. This enzyme is an exo-b-1,4 glucanase. Exo-glucanase splits alternate bonds from the non-reducing end of cellulose chain yielding cellobiose. The endo-glucanase is distinguished by the mechanism of their attack on carboxy methyl cellulose. It does not act on native cellulose. b-glucosidases play an important function in the degradation of cellulose by hydrolysing cellobiose which is an inhibitor of exo-glucanase. Only organisms producing C1­-cellulose (exo-glucanase) are capable of hydrolysing native cellulose (filter paper, cotton etc.)
 


 
Content
i)   Viscometric method
ii)  Colorimetric method
iii) Combined assay
 
 
i) Assay of Cx- cellulase (Endo-b-1,4 glucanase) (viscometric method)
Principle
Endo-b-1,4 glucanase acts on carboxymethyl cellulose (CMC) and hydrolyses the b-1,4 glycosidic bonds in a random manner. As a result, the viscosity of CMC solution is reduced. This is measured in an Ostwald viscometer.
 
 



Materials
Citrate-phosphate Buffer 0.1M (pH 6.0)
Carboxymethyl Cellulose 0.5% Solution
   Dissolve 0.5g sodium carboxymethyl cellulose in hot water. Adjust to pH 6.0.
Chloramphenicol-cyclohexamide Solution
   Dissolve 25mg each of chloramphenicol and cyclohexamide in 20mL water.
Ostwald Viscometer
 
 
Procedure
1.
Pipette   out   3mL   carboxymethyl   cellulose   solution   and   1mL citrate-phosphate buffer into a test tube.
2.
Add 1mL of enzyme extract.
3.
Add 0.1mL chloramphenicol-cyclohexamide solution to prevent microbial contamination.
4.
Incubate at 37°C for 16h.
5.
After incubation, heat the solution in boiling water for 3min, cool and then centrifuge at 8000g for 20min.
6.
Run a control which contains denatured enzyme (heat the enzyme extract for 3min in boiling water).
7.
Draw 5mL portion of control and test supernatant solution and measure the viscosity in an Ostwald viscometer.
 
 


Calculation
The percent loss of viscosity is interpreted as proportional to the cellulase activity. Calculate the percent reduction in viscosity as below:
V =
T0 – T
x 100
T0 - TH2O
 
where, V = percent loss in viscosity, To - flow time in seconds of zero time, T -flow time after incubation and TH2O — flow time of water.
 
 
References
1.  Hinton, D M and Pressey, R (1974) J Food Sci 39 783.


ii) Cx b (l-4) glucanase assay (colorimetric)
Principle
The production of reducing sugar (glucose) due to cellulolytic activity is measured by dinitrosalicylic acid method.
 
 



Materials
Sodium Citrate Buffer 0.1M (pH 5.0)
Carboxymethyl Cellulose 1%
   Dissolve 1g carboxymethyl cellulose in 100mL Sodium Citrate Buffer 0.1M (pH5.0)
Dinitrosalicylic Acid (DNS) Reagent (Refer carbohydrate section)
40% Rochelle salt solution (Potassium sodium tartrate)
 
 
Procedure
1.
Pipette out 0.45mL of 1% CMC solution at a temperature of 55°C and 0.05mL of enzyme extract.
2.
Incubate the mixture at 55°C for 15 min.
3.
Immediately after removing the enzyme substrate mixture from the bath add 0.5mL DNS reagent.
4.
Heat the mixture in a boiling water bath for 5 min.
5.
While the tubes are warm, add 1.0mL potassium sodium tartrate solution.
6.
Cool to room temperature.
7.
Add water to make 5mL volume.
8.
Measure the absorbance at 540nm.
9.
Prepare a standard graph with glucose in the concentration range 50mg to 1000mg/mL.
 
 
Calculation
Express the enzyme activity as the mg glucose released per min per mg protein.
 
 
References
1. Denison, D A and Koehn, R D (1977) Mycologia LXIX 592.
 
 
iii) C1 and Cx Cellulase (Combined Assay)
Principle
The principle is same as that of Cx cellulase.  The substrate used is filter paper instead of CMC
 
 


Reagents
Citrate-phosphate buffer 0.1M (pH 5.8)
Filter paper disc.
Cut the Whatman filter paper No.1 with a paper punch (7mm diameter) to ensure the same surface area of substrate in reaction tube.
 
 
Procedure
1.
Add 0.5mL enzyme extract to 32mg of dry Whatman No.1 filter paper.
2.
Incubate the mixture for 1h at 50°C.
3.
Follow steps 3 - 9 in the Cx cellulase colorimetric assay.
 
 
References
1.   Denison, D A and Koehn, R D (1977) Mycologia LXIX 592.
 
     
 
 
     




     
 
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