BIOL 2051 Lab Final Study Guide Fall 2014 Experiment 21 Catalase Production Background information Enzymes proteins that serve as biological catalysts very specific o Usually have ase suffix o Can be protein or RNA o Are not consumed during reactions o Lower the activation energy of the reaction Help reaction proceed quicker substance with which an enzyme reacts o Generally can only react with one substrate and can only catalyze one reaction They are Extracellular enzymes Substrate degrade large molecules into units that are small enough to be transported across the cell membrane and into the cell where they can be useful enzymes that act outside of the host cell in which they are produced They o Can reach substances in agar Endoenzymes act inside the cell to degrade molecules into simpler molecules releasing energy or are only produced if the appropriate substrate is available synthesizing molecules that are needed by the cell Inducible adaptable enzymes conserves energy the cell size shape and arrangement as observed by microbiologists how the organism functions in its environment takes into account metabolic capabilities which includes an examination of the enzymes it produces and how it reacts biochemically essential to the cell and are produced at all times Constitutive enzymes Morphology Physiology Hydrogen peroxide H2O2 is a product of aerobic metabolism but it is toxic So organisms must be able to detoxify H2O2 in order to survive in aerobic environments breaks H2O2 into water and molecular oxygen Catalase o Oxygen produced can be observed due to the production of bubbles 2 H2O2 2 H2O O2 Catalase Reaction o Produced by almost all microorganisms that use oxygen for respiration Catalase test can be used to distinguish between different genera of bacteria o Staphylococcus and Micrococcus are catalase positive o Streptococcus Lactococcus and Enterococcus are catalase negative Add hydrogen peroxide to slide and mix in organism Procedure Results Staphylococcus epidermidis is catalase positive and therefore aerobic Streptococcus mutans is catalase negative and therefore anaerobic Lab Notebook Questions 1 What is the reagent that serves as the substrate in the catalase test 2 What type of metabolism is associated with catalase production H2O2 Aerobic 3 For what product is one testing when using the catalase test Oxygen 4 Would you expect anaerobes to produce catalase Explain your answer No The enzyme is not necessary and it would be a waste of energy to make it 5 Describe the appearance of a positive test and a negative test Positive bubbles produced Negative nothing happens 6 What does a positive or negative test indicate about the metabolism of the cells tested Positive aerobic respiration negative anaerobic respiration fermentation 7 If your catalase test was positive to what 2 possible genera could the microorganism belong Staphylococcus or Micrococcus 8 What is the equation catalyzed by the enzyme catalase 2 H2O2 2 H2O O2 9 Would you expect your cells to produce catalase Explain your answer Yes Mammals have catalase in mitochondrial intermembrane space Moodle Quiz Questions 1 What is the visible product in the catalase test 2 Choose the genera with which you d expect a positive result with the catalase test 3 Enzymes that are essential to the cell and are produced at all times are called constitutive 4 Which of the following enzymes can break down hydrogen peroxide into water a Hydrogen peroxide b Air bubbles c Water d Oxygen a Staphylococcus b Micrococcus c Lactococcus d Streptococcus enzymes a Peroxidase b Superoxide dismutase c Catalase d Hexokinase e Primase 5 What metallic process positively correlates with the production of catalase by a microorganism a Anaerobic respiration b Aerobic respiration c Both aerobic and anaerobic respiration 6 To perform the catalase add a drop of water to the slide to mix with the bacteria a True b False add a drop of hydrogen peroxide to the slide Experiment 22 Cytochrome c Oxidase Production Background Information Series of redox reactions provides energy to build the H gradient that leads to ATP production Oxidase enzymes are made by organisms that produce energy using aerobic respiration Aerobic respiration oxygen serves as the electron acceptor Cytochrome oxidase catalyzes the oxidation of a cytochrome a member of the membrane electron transport chain and the subsequent reaction of molecular oxygen to either H2O or H2O2 o H2O2 is toxic to cells What is the connection between cytochrome c oxidase and catalase o Both are made and used by aerobic organisms o Cytochrome c oxidase can make hydrogen peroxide as a byproduct o Catalase is used to detoxify the cell getting rid of that H2O2 Organisms that use oxygen can be identified using oxidase enzyme activity o Aerobes facultative anaerobes and microaerophiles o Neisseria and Moraxella are oxidase positive o Acinetobacter are oxidase negative o Pseudomonads and Vibrionaceae are oxidase positive o Enterobacteriaceae are oxidase negative Cytochrome c oxidase takes electrons from cytochrome c oxidizing it and transfers the electrons to molecular oxygen reducing it Procedure Tetramethyl p dihydrochloride is added directly to colonies on a TSA plate o Serves as an artificial substrate to be oxidized by cytochrome oxidase in the presence of oxygen o Turns blue purple if cytochrome oxidase is present o Results must be observed immediately If you wait too long it will be oxidized by the air and lead to false positives Results Pseudomonas putida is catalase positive and therefore aerobic Escherichia coli is catalase negative and therefore anaerobic Lab Notebook Questions 1 What is the function of cytochrome oxidase Catalyzes the oxidation of cytochromes and reducing oxygen in the electron transport chain helping make the H gradient for the production of ATP 2 Why are strict aerobes oxidase positive Because oxygen is their final electron acceptor and cytochrome oxidase is necessary to catalyze the reduction of oxygen 3 The oxidase test is used to differentiate among which groups of bacteria Neisseria Moraxella pseudomonads and Vibrionaceae oxidase positive and Acinetobacter and Enterobacteriaceae oxidase negative Aerobes are oxidase positive and anaerobes are oxidase negative 4 What is the function of the test reagent in this procedure Serves as an artificial substrate in the presence of oxygen Moodle Quiz Questions 1 The oxidase reagent must be mixed added to the bacteria on the plate and the