CATALYTIC ACTIVITY OF DENATURED INVERTASE

Janella Ann Javenrie R. Calapit, Kristiana Marian E. Calupitan, Koleen G. Chavez, Valerie S. Co and Carlson G. David Group 2 2A Medical Technology Chem 600 Laboratory

Enzymes are proteins that act as catalysts by lowering the activation energy in specific chemical reactions. A specific type of enzyme, invertase, hydrolyses sucrose into glucose and fructose. Dinitrosalicylic acid works on reduction-oxidation method and due to its change in coloration; absorbance can be measured using spectrophotometric analysis. The absorbance was measured by setting the wavelength of the standard samples at 540 nm and zero for its corresponding blank solutions.

ABSTRACT

INTRODUCTION Enzymes are biomolecules with catalytic activity. They increase the rate of a biochemical reaction without undergoing chemical change. Virtually, all chemical reactions in a biological system are mediated by enzymes that allow these reactions to proceed at a rate sufficient to sustain life. Enzymes in general, are more effective catalysts than inorganic ones. The efficiency of enzymes is affected by a variety of factors, most notably the temperature of the surrounding environment. Enzymes can have molecular weights ranging from about 10,000 to over 1 million. A small number of enzymes are not proteins, but consist of small catalytic RNA molecules. Often, enzymes are multiprotein complexes made up of a number of individual protein subunits. Many enzymes catalyze reactions without help, but some require an additional nonprotein component called a co-factor. Cofactors may be inorganic ions such as Fe2+, Mg2+, Mn2+, or Zn2+, or consist of organic or metalloorganic molecules known as co-enzymes. Enzymes are classified according to the reactions they catalyze. The six classes are: oxidoreductases, transferases,

hydrolases, lyases, isomerases and ligases. Invertase (systematic name: betafructofuranosidase) is an enzyme that catalyzes the hydrolysis of sucrose. The resulting mixture of fructose and glucose is called inverted sugar syrup. Related to invertases are sucrases. Invertases and sucrases hydrolyze sucrose to give the same mixture of glucose and fructose. Invertases cleave the fructose bond, whereas the sucrases cleave the glucose bond. Invertase is usually derived from yeast. It is also synthesized by bees, who use it to make honey from nectar. Optimum temperature at which the rate of reaction is at its greatest is 60 C and an optimum pH of 5. Typically, sugar is inverted with sulfuric acid. EXPERIMENTAL A. Samples used: Bakers yeast, distilled water, sucrose standard solution, conc. HCl, 0.5M KOH, dinitrosalicylic acid (DNS) reagent, 0.1M buffer solutions B. Procedure: 1. Extraction of Invertase from Yeast In the act of drawing out the invertase, 0.25g of Bakers yeast was weighed and added with distilled water until a volume of 250 mL was achieved. The solution was allowed to stand for 20 minutes at room temperature and the collection of the

supernatant was followed upon the occurrence of sedimentation. The supernatant was then used as the enzyme stock solution for the succeeding experiments. 2. Preparation of Denatured Invertase Stock Solution 100mL of enzyme stock solution was incubated in a boiling water bath for 10 minutes. The solution was then cooled and the supernatant was collected. The collected supernatant served as the denatured enzyme stock solution for the succeeding enzymes. 3. Sucrose Assay Using Dinitrosalicylic Colorimetric Method 7 test tubes with the following contents were prepared:
Tube No. mL sucrose standar d solution mL distilled water Blank 1 2 3 4 5 6

bond is broken and results in formation of glucose and fructose. The hydrolysis reaction takes place at slower rate, but upon addition of enzyme invertase, the reaction took place at a rapid rate. Dinitrosalicylic acid reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540nm. The more red-brown color produced, the more glucose is released from sucrose. The more glucose released, the more hydrolyzed the sucrose is. For the spectrophotometric assay, the more intense color of the product, the higher absorbance at 540 nm wavelength.

0.25

0.50

0.75

1.00

1.25

1.50

Figure 1. Hydrolysis of Sucrose Figure 2. Graph of Absorbance Based on the data presented, we can infer that as the standard solution increases and as the amount of water in the solution decreases, the absorbance of light of the solution increases. It can be said that the preparation of the solution was accurate because the absorbance obtained was increasing and there was no negative absorbance.

1.50

1.25

1.00

0.75

0.50

0.25

Table 1. Content of prepared test tubes After preparing the test tubes, 3 drops of conc. HCl was added to each tube and was incubated at a 90C water bath for 5 minutes. After that, 0.15mL of 0.5M KOH and 2.80mL of 0.1M buffer solution (pH 5) were added to the test tubes. The last reagent added to the samples was 3mL of the DNS reagent and was immersed in a 95C water bath for 10 minutes to develop the characteristic red brown color. The samples were allowed to stand for a few minutes to cool and the absorbance was measured at 540 nm using a spectrophotometer. RESULTS AND OBSERVATION Sucrose is made up of glucose and fructose and is connected by glycosidic linkage. Upon hydrolysis, the glycosidic

REFERENCES From books Crisostomo, A.C. et al. (2010). Laboratory Manual in General Biochemistry. C&E Publishing, Inc.

Fry, M. (2005) Catch Up Chemistry. Scion From the Internet Enzymeshttp://www.wisegeek.com/what-areenzymes.htm

http://biotech.about.com/od/glossary/g/E nzyme.htm Invertasehttp://www.wisegeek.com/what-isinvertase.htm