EX.NO.

1 EXTRACTION AND ESTIMATION OF ENZYMES FROM

PLANT SOURCE
AIM:
To extract and characterise a plant enzyme called glucosidase.

PRINCIPLE:
Glucosidase is capable of hydrolysing a variety of glycosides and depending upon their
occurrence, localisation and substrate specificity, In this experiment we make use of an
artificial substrate, p-nitrophenyl -D-glycoside. In the presence of this enzyme, p-nitrophenol
and glucose will be released. Therefore, one can determine the enzyme activity by measuring
any of the two products, but it is more convenient to measure p-nitrophenol.

PROCEDURE:

1. p-Nitrophenol standard curve

Glucosidase catalyses the hydrolysis of p-nitrophenol--glucoside to p-nitrophenol and glucose.

Under alkaline conditions, the p-nitrophenolate anion absorbs light at 400-450 nm. The amount of
enzyme present is therefore determined by measuring the amount of p-nitrophenolate anion
produced in the reaction. But to make this estimate, you must prepare a standard curve so that the
amount of yellow-orange colour can be translated into the amount of p-nitrophenol produced.

Set up a series of tubes as given in Table 1.

Table 1. Preparation of Standard Curve

Tube No 1 2 3 4 5 6

p-nitrophenol (1
0 0.20 0.30 0.40 0.50 0.60
micromole/ml), ml

Distilled water, ml 0.6 0.40 0.30 0.20 0.10 0

100 mM Na citrate
0.6 0.60 0.60 0.60 0.60 0.60
pH 6, ml

To each tube add 5 ml of 100 mM Na2CO3. Read the absorbance at 420 nm. Blank the
Spectronic 20 using tube no. 1.

Prepare a standard curve by plotting the absorbance (at 420 nm) against umoles of p-nitrophenol.
Use this standard curve for the rest of the practical session.
 2. Enzyme extraction and measurement

Grind about 0.5 g cut leaf in a mortar with 10 ml of Na citrate pH 6.0 containing 1%
PVP.

Spin down the residue using the bench top centrifuge (full speed for 15 minutes) and
keep the supernatant on ice.

First you must determine how much enzyme activity is present in the extract, since this is
dependent upon the health of the leaf and how you grind the leaf material. For this purpose, you
must prepare several dilutions of your extract for enzyme assay. You may dilute your extract using
the extractions buffer. Remember to keep your extract in ice.

Table 2 shows you how to prepare your extract for measurement of enzyme activity. Using
Table 2, prepare the 10 tubes and incubate them in a water bath (37˚C) for 15 minutes, then add
5.0 ml 100 mM Na2CO3 into each of them. Measure the absorbance at 420 nm

Tube 1 contains the enzyme as you have prepared, while tubes 2 to 4 contain enzyme
preparation in the diluted form, namely 10x, 20x and 40x dilution. This step is necessary to obtain
a suitable dilution for enzyme assay. This can be taken as the enzyme dilution that gives you an
absorbance reading within the standard curve.

Table 2. Measurement of enzyme activity

Test tube 1* 2* 3* 4* 5

Dilution No dilution 10X 20X 40X Control

20 mM PNPG
0.6 0.6 0.6 0.6 0
(ml)

100 mM Na
citrate pH6 0.4 0.4 0.4 0.4 1.0
(ml)

Enzyme
0.2 0.2 0.2 0.2 0.2*
extract (ml)

* Separate controls are needed for the different dilutions as plant extracts may interfere with
colour development of the enzyme assay. Therefore, 4 controls are required here, one for each of
the different dilutions. Controls are prepared as shown by tube 5 using the respective diluted
enzyme preparation

Tube 5 will establish if the enzyme preparation gives any "coloration" that may interfere with
your enzyme assay. Controls must therefore be set up for the different enzyme preparations (i.e.
undiluted and diluted). If the controls (tube 5) give any positive /value at 420 nm, you must
subtract this value from the respective tests (tubes 1 to 4).
 Using the standard curve you have prepared earlier, calculate the enzyme activity of your
extract as umol PNP/15 mins at 37˚C/0.1 ml extract.

RESULT:

Enzyme activity of extracted plant enzyme is _____________________