Substrate Specificity of Invertase

Topics: Enzyme, Sucrose, Fructose Pages: 7 (1796 words) Published: January 26, 2011
Enzymes are highly specific and can distinguish isomers of the same molecule. The enzyme invertase specifically catalyzes the reaction of the conversion of sucrose to its individual carbohydrates glucose and fructose. It does not catalyse the reaction of maltose to 2 glucose or lactose to galactose. In this experiment, titrimetric and spectrophotometric methods were used to determine the specificity of invertase by determining the amount of glucose converted from the given disaccharides. The results show that sucrose yielded the least amount of glucose and got the lowest absorbance reading.

Enzymes are globular proteins. Their folded conformation creates an area known as the active site. The nature and arrangement of amino acids in the active site make it specific for only one type of substrate.

Enzymes catalyze an unfathomable number of reactions by using a combination of only six basic mechanisms: (1) acid-base catalysis; (2) covalent catalysis; (3) metal ion catalysis; (4) electrostatic catalysis; (5) proximity and orientation effects; and (6) preferential binding of the transition state complex. Independent of the mechanistic characteristics taken to generate product, the initial reaction rates of every enzyme can be analyzed in order to quantify their overall efficiency.

Enzymes are specific for:
a) substrate
b) reaction
It means that they catalyze the transformation of just one substrate or a family of substrates that are structurally related, catalyzing only one of the possible reactions of the substrate(s). The enzyme specificity of action is related to the fact that the enzyme only catalyzes one of the possible transformations of a substrate.

Invertase is beta-fructofuranosidase (EC3.2.1.26), which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal nonreducing beta-fructofuranoside residues in beta-fructofuranosides.

The objective of this experiment is to investigate specificity of enzyme activity by exposing the enzyme to other disaccharides.


Samples used
0.1% and 1% of: Maltose, lactose, cellulose, sucrose, and distilled water

Titrimetric method:
In four different Erlenmeyer flasks, ten milliliters of the samples (1% maltose, 1% lactose, 1%sucrose, 1% cellulose and water) were introduced. Five milliliters of acetate buffer (ph 5) and five milliliters of the invertase were then added to the four flasks. The solutions were then incubated at 55 degrees Celcius. After incubating, an aliquot portion of each solutions (10ml) were transferred again into four different flask. Sodium carbonate was then added to each flask. In each flask, fifteen milliliters of iodine solution (0.0235M) was rundown from the burette and the flasks were then kept in the dark for twenty-five minutes. After twenty-five minutes the solutions were acidified with sulfuric acid and then titrated with standard sodium thiosulfate( 0.0971M) using two milliliters of starch solution as the indicator. After the experiment, the amount of glucose in each samples were computed.

Spectrophotometric method:
First, the samples (1% maltose, 1% lactose, 1%sucrose, 1% cellulose and water) were diluted 100 milliliters of distilled water to make it 0.1% sample solutions. Then in four different test tubes one milliliter of the said samples were introduced. One milliliter of acetate buffer and invertase were then added. The test tubes were then incubated for five minutes at 55 degrees Celcius. When incubation was done, 0.5 milliliters of both Fehling’s A and B were added and boil the solutions for 5-10 minutes. After cooling, two milliliters of arsenomolybdate and ten milliliters of distilled water were added. Transfer the four solutions into the cuvettes and read the absorbance at 620 nm. After the absorbances were read, the amounts of the glucose were calculated. Glucose standard curve was also prepared.

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