EXPERIMENT 1 EFFECT OF SUBSTRATE CONCENTRATION ON ENZYME KINETICS STUDY.

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1 E R T B I O C H E M I C A L E N G I N E E R I N G S E M 1, / EXPERIMENT 1 EFFECT OF SUBSTRATE CONCENTRATION ON ENZYME KINETICS STUDY

2 Let s Understand it More based on your first Experiment ERT 317

3 OBJECTIVES 1. To develop a suitable standard curve for enzyme assay 2. To analyze the effect of substrate concentration on the activity of enzyme 3. To determine V max and K m from the enzyme reaction using enzyme kinetics plots.

4 COURSE OUTCOMES CO1 Ability to develop enzyme reactions based on its kinetics study and applied catalysis INTRODUCTION The enzyme α-amylase can catalyze the hydrolysis of internal α -1,4-glycosidic bond present in starch with the production of reducing sugars. In the study of substrate concentration on enzyme kinetics, the enzyme is kept constant where as the concentration of starch is taken in increasing order. As the substrate concentration increases, the amount of products produced in every successive tube also increases. This enzyme-substrate reaction can be determined by measuring the increase in reducing sugars using the 3,5-dinitrosalicylic acid reagent. In an alkaline condition, the pale yellow coloured the 3,5-dinitrosalicylic acid undergo reduction to yield orange coloured 3-amino-5-nitrosalicylic acid. The absorbance of resultant solutions is read at 540nm. The intensity of colour depends on the concentration of reducing sugars produced.

5 Starch + α-amylase hydrolysis Maltose + glucose Amylose comprises 15-30% of the common starches. Amylose is a linear polymer containing up to 6000 glucose units, connected by α (1, 4) linkages. Therefore, we use α-amylase to hydrolyzed starch in this hydrolysis process. The hydrolysis of starch with a low molecular weight, catalyzed by an α- amylase, is one of the most important commercial enzyme processes.

6 Starch + α-amylase hydrolysis Maltose + glucose What does alpha amylase do? α-amylase is a protein enzyme EC that hydrolyses alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. The individual subunits that make up maltose are glucose

7 Starch + α-amylase hydrolysis Maltose + glucose Your Task: 1) Develop of maltose standard curve why? How? -to measure the product of reducing sugar (after hydrolysis process), we NEED a standard. Pure Sugar : Maltose (main reducing sugar produced from starch after enzymatic hydrolysis) Reagent needed: DNS reagent (the intensity of colour in DNS depends on the concentration of reducing sugars produced)

8 Simple Method for Development Standard Curve 1. Prepare at least 5 different concentration of pure sugar (Maltose) 2. Prepare 1 Blank (no sugar/no maltose) 3. All samples + DNS reagent (follow DNS method) 4. Read 540 nm (for blank: Zeroing) 5. Plot Absorbance reading (y-axis) VS Maltose conc (x-axis) 6. Linear graph with an equation Y=mx+c (make sure R 2 for any standard curve is 0.999)

9 Your Task: 2) Study effect of substrate concentration on enzyme activity -Vary substrate (starch) concentration but fix the enzyme concentration. SIMPLE METHOD: 1. Run the hydrolysis process (different starch concentration react with an enzyme) at 37oC, 3 min. Produced product (hydrolysate) containing reducing sugars (maltose) Blank: no starch 1. All product samples contain unknown concentration of maltose. Therefore, we need to run DNS method. 2. Samples + DNS reagent (follow DNS method). 3. Cek concentration of samples using MALTOSE Standard Curved.

10 Starch concentration, [S] (%) Abs at 540 nm Amount of maltose (µg) Velocity [V] in µmoles/min 1/[V] 1/[S] 1.Draw the Michaelis-Menten s plot and Line Weaver Burk plot using the data in Table 1.3 and find the V max and K m from the plot. 1.Compare the value of V max and K m from both plot and give your reason of their differences.

11 Michaelis-Menten s plot

12 Lineweaver-Burk Plot From equation 6 (Quasi-steady-state ), v V K Double reciprocal plot slope m m [ S ] [ S ] Y-intercept Lineweaver-Burk plot gives good estimates on V m but not necessarily on K m (error relates with substrate conc)

13 Thank You

14 Example : Standard Curve Abs vs Glucose Concentration YOUR curve should be Abs vs MALTOSE conc