Understanding Enzyme Activity and Metabolism in Environmental Microbiology Labs

1. Environmental Microbiology-Laboratory Manual-prepared for Environmental MicrobiologyIVBiochemical Activity of microorganism Environmental Management technology Faculty of civil and environmental engineering Itb, 2010

2. Terms • Metabolism • Anabolism • Catabolism • Catalytic • Catalyst • Enzyme • Apoenzyme • Holoenzyme • Coenzym • Endoenzyme • Exoenzyme • Enzyme activity • Enzyme system • Substrate • Active Site • Oxidation • Reduction • Hydrolysis • Glycolysis • Fermentation • Aerobic respiration • Anaerobic respiration • Peptide bonding • Peptonization • Starch • Carbohydrate • Amino acid • Lipid • Fatty acid • Protein • Casein • Pyruvic acid • ATP, ADP

3. Metabolism • Metabolism refers to all chemical reactions that occur within a living organism.

4. Anabolic and Catabolic Reactions are Linked by ATP in Living Organisms

5. Catalyst = an agent that accelerates chemical reaction without itself being destroyed or used up ENZYME

6. Enzyme • Organic catalyst (elaborated by living cell) • Protein mollecular • Thermolabile (denaturated by heat) • Precipitated by ethanol and high concentration inorganic salts • Non-dialyzable (does not go through semi-permeable membrane)

7. CHARACTERISTIC OF ENZYMES

8. Energy of Activation • Energy of activation: The amount of energy required to trigger a chemical reaction. • Enzymes speed up chemical reactions by decreasing their energy of activation without increasing the temperature or pressure inside the cell.

10. Enzyme Components • Some enzymes consist of protein only. • Others : Holoenzyme= Apoenzyme + Cofactor • Enzyme cofactors may be a metal ion, an organic molecule, or derived from vitamins. Examples: • NAD+:Nicotinamide adenine dinucleotide • NADP+:Nicotinamide adenine dinucleotide phosphate are both cofactors derived from niacin (B vitamin). • Coenzyme A is derived from panthotenic acid.

11. Components of a Holoenzyme

12. Mechanism of Enzymatic Action Surface of enzyme contains an active site that binds specifically to the substrate. 1. An enzyme-substrate complex forms. 2. Substrate molecule is transformed by: • Rearrangement of existing atoms • Breakdown of substrate molecule • Combination with another substrate molecule 3.Products of reaction no longer fit the active site and are released. 4. Unchanged enzyme is free to bind to more substrate molecules.

13. Mechanism of Enzymatic Action

14. Factors that Affect Enzyme Activity: pH, Temperature, and Substrate Concentration

15. Denaturation of a Protein Abolishes its Activity Denaturation: Loss of three-dimensional protein structure. Involves breakage of H and noncovalent bonds.

16. REDOX RedoxReactions:Reactions in which both oxidation and reduction occur.

17. Oxidation-Reduction Reactions

18. Aerobic Respiration is a Redox Reaction C6H12O6 + 6 O2 -----> 6 CO2 + 6 H2O + ATP Glucose oxygen oxidized reduced

19. Hydrolysis • Hydrolysis is a chemical reaction during which molecules of water (H2O) are split into hydrogen cations (H+) (conventionally referred to as protons) and hydroxide anions (OH−) in the process of a chemical mechanism.

20. Carbohydrate Catabolism • Most microorganisms use glucose or other carbohydrates as their primary source of energy. • Lipids and proteins are also used as energy sources. • Two general processes are used to obtain energy from glucose: • cellular respiration • fermentation

21. Cellular Respiration : AEROBIC • ATP generating process in which food molecules are oxidized. • Final electron acceptor is oxygen. Aerobic Respiration C6H12O6 + 6 O2-----> 6 CO2 + 6H2O + ATP Glucose oxygen oxidized reduced Aerobic Respiration occurs in three stages: 1. Glycolysis 2. Kreb’s Cycle 3. Electron Transport & Chemiosmosis

22. Three Stages of Aerobic Respiration

23. Fermentation • Releases energy from sugars or other organic molecules. • Does not requireoxygen, but may occur in its presence. • Does not require an electron transport chain. • Final electron acceptor is organic molecule. • Inefficient: Produces a small amount of ATP for each molecule of food. • End-products are energy rich organic compounds: • Lactic acid • Alcohol

24. Aerobic Respiration versus Fermentation

25. CELLULAR RESPIRATION : ANAEROBIC • Final electron acceptor is not oxygen. • Instead it is an inorganic molecule: • Nitrate (NO3-):Pseudomonas and Bacillus. Reduced to nitrite (NO2-):, nitrous oxide, or nitrogen gas. • Sulfate (SO42-):Desulfovibrio. Reduced to hydrogen sulfide (H2S). • Carbonate (CO32-): Reduced to methane. • Inefficient (2 ATPs per glucose molecule). • Only part of the Krebs cycle operates without oxygen. • Not all carriers in electron transport chain participate. • Anaerobes tend to grow more slowly than aerobes.

26. EXPERIMENTS

27. BASIC PRINCIPLES

29. Exp. 18 – Acivities of Extracelullar Enzymes Hydrolysis of Starch and Casein

30. Hydrolysis of Starch • Starch dextrin /monohydrates • Amylase • Starch Agar  the presence of starch in medium • Iodine  indicator, if starch is still present, blue (-), if does not present, transparent (+)

31. Hydrolysis of Casein • Protein  various amino acids linked together in long chains by means of peptide bonds • Protein  peptide  amino acid • Prior use as nutrition material  needs to be degraded into simpler substances through peptonization or proteolysis process using protease enzyme (breaking CO-NH bonding) • Proteolytic zone  transparent

32. Hydrolysis of Starch & Casein

33. Exp. 19 – Acivities of Intracelullar Enzymes : Fermentation Test and Oxidation 19.A. Fermentation of Carbohydrates 19.B. Reduction of Nitrate 19.C. Catalase Reaction 19.D. Oxidation Test

34. 19.A. Fermentation of Carbohydrates • A wide variety of carbohydrates may be fermented in order to obtain energy and the types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism. • End products of fermentation. • acid end products. • acid and gas end products. • Red phenol  red in normal pH, yellow in acid condition

35. 19.A. Fermentation of Carbohydrates

37. 19.B. Reduction of Nitrate • Bacteria can reduce nitrate • Anaerobic condition • Nitrate reductase enzyme • NO3- + 2e- + 2H-  NO2- + H2O • Reagent A Sulfanilic acid + Reagen B alphanaphtylamine, if nitrite is presence (+), red • If (-), zinc will reduce nitrate, bring red color,indicates that nitrate did not reduced before, if transparent (+)

38. 19.B. Reduction of Nitrate

39. 19.C. Catalase Reaction • Aerobic reaction  hydrogen peroxide, reactive, destructing enzyme • Catalase  preventing damage, turning H2O2 into free H20 and O2 • Superoxide dismutase  in species which has no catalase

40. 19.C. Catalase Reaction

41. 19.D. Oxidase Test • Oxidase enzyme  Electron transport system in aerobic resp. • p-aminodimethylanyline oxalate  artificial substrate, donating electrone and be oxidized into black substances if oxydase and free oxygen are present • (+)  pink – maroon-black • (-)  no color change

42. 19.D. Oxidase Test

43. Exp. 20 – Acivities of Intracelullar Enzymes : IMViC and TSI Test 20.A.1 Indol Reaction Test 20.A.2 Methyl Red Reaction 20.A.3 Vogus-Proskauer Reaction 20.A.4 Use of Citrate 20.B. Triple Sugar Iron Agar Test

44. A. IMVic Test • Enterobactericeae G.I tract • Identification is important in preventing contamination to food and water supply • Pathogenic, sometimes pathogenic, normal flora

45. 20.A.1 Indol Reaction Test • Indol =a component of tryptophane, an essential amino acid • Will not occur if carbohydrate that needs to be degraded is exsist  low pH • Specific characteristic of intestinal bacteria • Indol + Kovac reagent  red cherry on the surface of the test tube

46. 20.A.1 Indol Reaction Test

47. 20.A.2 Methyl Red Reaction • Glucose  primary energy source for enteric • Some turn glucose into acid (glucose fermentation)  low pH • Important in differentiating E.coli and E.aerogenes • Methyl red • red : pH 4 • Yellow : pH6

48. 20.A.2 Methyl Red Reaction The Methyl Red Test: Left to Right: positive, positive, negative, control.

49. 20.A.3 Vogus-Proskauer Reaction • Some fermentative organisms do not produce enough stable acids to lower the pH of the medium. • To detect the ability of m.o. In producing non acid substance acetymethylcarbinol • Characteristic of E. aerogenes

50. Voges-Proskauer Test Left: uninoculated controlRight: negative (copper color) Left: uninoculated controlRight: positive (red color)