experiment 1 effect of substrate concentration on enzyme...

EXPERIMENT 1

EFFECT OF SUBSTRATE

CONCENTRATION ON ENZYME

KINETICS STUDY

hafizashukor@unimap.edu.my

E R T 3 1 7 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 , 2 0 1 6 / 2 0 1 7

Let’s Understand it Morebased on your first

Experiment ERT 317

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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 Vmax and Km from the enzyme reaction

using enzyme kinetics plots.

OBJECTIVES

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COURSE OUTCOMES

CO1 – Ability to develop enzyme reactions based on its kinetics study and applied catalysis

INTRODUCTIONThe enzyme α-amylase can catalyze the hydrolysis of internal α -1,4-glycosidicbond present in starch with the production of reducing sugars. In the study ofsubstrate concentration on enzyme kinetics, the enzyme is kept constant where asthe concentration of starch is taken in increasing order. As the substrateconcentration increases, the amount of products produced in every successivetube also increases.

This enzyme-substrate reaction can be determined by measuring the increase inreducing sugars using the 3,5-dinitrosalicylic acid reagent. In an alkalinecondition, the pale yellow coloured the 3,5-dinitrosalicylic acid undergo reductionto yield orange coloured 3-amino-5-nitrosalicylic acid. The absorbance ofresultant solutions is read at 540nm. The intensity of colour depends on theconcentration of reducing sugars produced.

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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.

Maltose + glucoseStarch + α-amylase

hydrolysis

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What does alpha amylase do?

α-Amylase is a protein enzyme EC 3.2.1.1 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

Maltose + glucoseStarch + α-amylasehydrolysis

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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)

Maltose + glucoseStarch + α-amylasehydrolysis

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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 absorbance @ 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 R2 for any standard curve is 0.999)

Simple Method for Development Standard Curve

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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.

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1.Draw the Michaelis-Menten’s plot and Line WeaverBurk plot using the data in Table 1.3 and find the Vmax andKm from the plot.

1.Compare the value of Vmax and Km from both plot andgive your reason of their differences.

Starch concentration,

[S] (%)

Abs at 540 nm

Amount of maltose

(µg)

Velocity [V] in

µmoles/min

1/[V] 1/[S]

0.020.040.060.080.10

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Michaelis-Menten’s plot

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Lineweaver-Burk plot gives good estimates on Vm but not necessarily on Km (error relates with substrate conc)

From equation 6 (Quasi-steady-state ),

Double reciprocal plot

Y-intercept

]S[K

]S[Vv

m

m

slope

Lineweaver-Burk Plot

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Thank You

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Example : Standard Curve Abs vs Glucose Concentration

YOUR curve should be Abs vs MALTOSE conc