The effect of pH on amylase activity HO KP (2008)

1. The effect of pH on amylase activity HO KP (2008)

2. How does the degree of acidity / alkalinity affect the rate of enzymatic reaction ? pH

3. Germinating Mung bean – source of the enzyme AMYLASE

4. Amylase is • a digestive enzyme that helps break down starch • found in (a) saliva • (b) some digestive • juices

5. Sprouting mung beans is a rich source of amylase and other enzymes. (Shall we remove the seed coat ?)

6. Remove the seed coat – but don’t throw them into the sink.

7. Preparation of amylase extract

8. Extract amylase from the mung bean seedling.

10. Sit down, hold the mortar and pestle tight.Press on the seedlings to release (釋放) cell sap. Grind for a few minutes. A mistake

11. The mortar must be put on a heat-proof mat or wet cloth to reduce wear. X A mistake

12. Mortar Pestle Make an enzyme extract – use only a few ml of water for grinding

13. Pour the enzyme extract into a clean test tube.

15. Get the top portion of the mung bean 綠豆enzyme extract by using a clean dropper. Filtering may be done by a muslin cloth.

16. No need to have so much ! Enzyme extract Less than a few ml of extract is required.

17. Preparation of starch agar plate

18. Agar (瓊脂), (大菜糕) dissolves in hot water Preparing for the starch - agar suspension

19. Petri dish Solidified starch agar Starch(substrate) is mixed with agar (a jelly like semi-solid)

20. The reaction : 不是 substance, substrate 指 enzyme reaction 的 reactant.

21. Chemicals in reagent bottle with dropper.

22. Independent variable – pH ! Read the bottle label carefully !

23. Which chemical provides for an acidic medium ? Hydrochloric acid Which chemical provides for an alkaline medium ? Sodium carbonate

24. Preparation of reaction mixtures

25. Preparing for the reaction mixture –

26. C3 C1 C2 C5 C6 C4 Into each of six cavities, Prepare a mixture of “ enzyme + pH medium ”

28. Dropper – Hold it vertically ! White cavity tile Or use a spotting tile

29. Make sure that each drop of chemical is of the same size. Why is this important ? Use clean dropper.

30. Let the paper disc(s) soak up the mixture

32. Soak the paper disc into the reaction mixture for at least 30 seconds. Do not dip the forceps into the reaction mixtures. If so, clean the forceps every time !

33. Rinse and clean.

34. Put six labels on the side of the Petri dish, in sequence.(Not on the lid 蓋)

35. At this stage, the “ enzyme - soaked paper discs ” may be put onto the starch agar plate by a pair of clean forceps.

39. Soak the 6 paper disc(s) in the 6 respective solutions for 30seconds. Label the six positions on the starch-agar plate. Transfer轉移 the disc(s) onto the starch-agar plate by using cleanforceps. Wash the forceps after every transfer to avoid contamination.

40. Class, name The set-up just before putting into the incubator. Remember to label the group name on the lid.

41. Incubation stage for enzyme reaction

42. Incubator 恆溫器 Label your name Incubation time : 60 – 120 min. Incubation temperature – at about 37 deg C

43. Do this step after the plate has been put into the incubator. Clearly label each of the conditions Find out the pH of each mixture !!!

44. Testing for the disappearance of substrate (starch)

45. Use brown Iodine solution to test for starch. Pour in a little to cover the agar surface.

46. Flush away excess Iodine immediately – be gentle !!!

47. Look against light Measure the diameter of the clear zone and write down the degree of clearing

48. Put the plate against a light source (light box) to record results.

49. A clear zone shows that starch has been digested

50. For each condition, measure (1) the diameter of the clear zone, and (2) how clear the spot is.