Determination of Km and Vmax of Amylase

BACKGROUND

Of any biological system enzymes are the catalyst and they are very efficient as well as specific like that of catalysts. The most important function of an enzyme is to accelerate the reaction rate by million factors as compared to the rate of that reaction when enzymes are not present.

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According to Michaelis-Menten equation¹

V_o = Initial reaction velocity

V_max = Maximum velocity

K_m = Michaelis constant

[S] = Substrate concentration.

K_m is enzyme characteristics and is particular substrate that reflects the affinity of the enzymes for the substrate. It is numerically equal to the concentration of the substrate at which the reaction velocity is same as half value of V_max.
It is not dependent on enzyme concentration.

Small K_m indicates the enzymes high affinity for the substrates low concentration of the substrate is required for saturating enzymes. Large K_m is the low affinity of the enzymes for substrate.²

REQUIREMENTS

Chemicals:      Enzymes

Starch solution – 2% stock and 0.1-2% working solution.

0.1 HCl

Iodine solution- Prepare it by adding 5 g of potassium iodide to 100 ml of water.

Then this dissolved potassium iodide is added to 1 g of iodide and then allows to completely dissolve.

         Amylase solution

         Potassium phosphate buffer having pH-7

Apparatus:     Spectrophotometer

Glasswares:    Test tubes

PROCEDURE

At first prepare different concentration of soluble starch substrate from the stock solution. The different concentration is 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml.³ Then to it add about 1 ml of the amylase solution to every test tube to initiate the reaction. Then keep it for incubation at 37^oC for about 10 min.^4,5 

Then stop this reaction by adding 0.1N HCl. Then add 0.1 ml of iodine solution. Take absorbance at 546 nm. Now draw graph with absorbance with V in Y axis and substrate concentration in X axis and then determine the value of V_max and K_M^.3.

CONCLUSION

It is vital to have a good knowledge about the performance enzymes characteristics if they are to be utilized efficiently. The parameters K_m and V_max are therefore very important to determine.

REFERENCES

1. Brigidi P, González-Vera A, Rossi M (1997). Study of stability of recombinant plasmids during the continuous culture of Bacillus stearothermophilus NUB3621 in nonselective medium. Biotechnol. Bioeng. 53: 507-514.
2. Castro GR, Baigori MD, Sineriz F (1999). Studies on -amylase production by Bacillus licheniformis MIR-61. Acta Biotechnol. 19: 263-272.
3. Bajpai P (1989) High temperature alkaline -amylase from Bacillus licheniformis TCRDC-B13. Biotech. Bioeng. 33: 72-78.
4. Becker P, Abu-Resh I, Markossian S, Antranikian G, Mürkl H (1997). Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. 1H1-91 on olive oil. Appl. Microbial. Biotechnol. 48: 184-190.
5. Bernfeld P (1955). Amylases alpha and beta. Methods in enzymology. Academic Press, New York, pp. 140-146.

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