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Extraction of Invertase from Yeast Essay Sample

Extraction of Invertase from Yeast Pages
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Enzymes are substances that are produced by living organisms and act as catalysts in order to speed up or chance a chemical reaction without changing itself at the end of the reaction. Invertase was extracted first from baker’s yeast. Determination of the effects of changes in pH on enzymatic activity was the main objective of this experiment. Dinitrosalicylic acid (DNS) colorimetric method was used in determining the enzymatic activity of invertase. Invertase was then mixed in different buffer solutions of varying pH. After subjecting the enzyme to the different buffers, absorbance of the invertase was measured under 540 nm using a spectrophotometer.

Invertase was subjected to different PH of buffer solution and was observed under 540 nmabsorbance using spectrophotometer. After observation and analysis, a peak was observed by plotting absorbanceversus ph and was known as optimum PH. Optimum PH is said to be the most favorable PH value or the point wherethe enzyme is most active. And that invertase exhibits high activity over a broad PH range of 3.5 – 5.5 with optimumPH near 4.5. But due to human errors, the acquired data was incorrect giving an unreliable basis for thedetermination of the effect of PH on enzymatic activity

INTRODUCTION
Enzymes are biological catalysts which speed up chemical reactions by lowering the activation energy, the energy required to break bonds. [1] Enzymes are also affected by factors such as temperature, cofactors, activators, inhibitors, and pH. Invertase, also known as β-D-Fructofuranoside fructohydrolase [2], is a glycoprotein which contains 50% mannan and 2-3% glucosamine. Invertase also can break peptide bonds. It hydrolyzes sucrose to produce glucose and fructose. [3] Dinitrosalicylic acid colorimetric method is a method wherein 3,5-Dinitrosalicylic acid reacts with reducing sugars and other molecules to form 3-amino-5-nitrosalicylic acid, a compound that strongly absorbs light at 540 nm. [4] Samples were also observed under the spectrophotometer to measure their absorbance at 540 nm. The main objectives of the experiment are: (1) to extract invertase from Baker’s yeast; (2) to determine the effects of varying pH on enzymatic activity.

EXPERIMENTAL
A. Compounds tested (or Samples used)
Baker’s yeast, distilled water, sucrose standard solution (100mg/L), concentrated HCl, DNS reagent, 0.1M buffer solutions (ph 3, 5, 7, 9, 10), sucrose solution (10g/L)

B. Procedure
1. Extraction of Invertase from Yeast
Dissolution of Baker’s yeast in 250mL distilled water was done after weighing 0.25g of Baker’s yeast. Incubation of the solution at room temperature for 20 minutes and decantation were the next steps. Collection of the supernatant was done after decantation.

2. Effect of pH on Invertase Activity
2.90 mL buffer solutions of varying pH (3, 5, 7, 9, 10) was put into six test tubes, one of them being the blank (pH = 5). 0.1mL invertase stock solution was then added followed by incubation at 60 °C for five minutes. Addition of 1.5mL sucrose solution and incubation at 60 °C for five minutes were done. Addition of 3mL DNS and incubation at 95 °C for 10 minutes followed. Finally, absorbance reading at 540 nm under the spectrophotometer was done.

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