Catalase is a common enzyme found in nearly all living organisms exposed to oxygen. It catalyzes the decomposition of hydrogen peroxide to water and oxygen. It is a very important enzyme in reproductive reactions. Likewise, catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide. The optimum pH for human catalase is approximately 7, and has a fairly broad maximum (the rate of reaction does not change appreciably at pHs between 6.8 and 7.5). The pH optimum for other catalases varies between 4 and 11 depending on the species. The optimum temperature also varies by species. History
Catalase was first noticed in 1811 when Louis Jacques Thénard, who discovered H2O2 (hydrogen peroxide), suggested its breakdown is caused by an unknown substance. In 1900, Oscar Loew was the first to give it the name catalase, and found it in many plants and animals. In 1937 catalase from beef liver was crystallised by James B. Sumner and Alexander Dounce and the molecular weight was worked out in 1938. In 1969, the amino acid sequence of bovine catalase was worked out. Then in 1981, the three-dimensional structure of the protein was revealed. Action
The reaction of catalase in the decomposition of living tissue: 2 H2O2 → 2 H2O + O2 The presence of catalase in a microbial or tissue sample can be tested by adding a volume of hydrogen peroxide and observing the reaction. The formation of bubbles, oxygen, indicates a positive result. This easy assay, which can be seen with the naked eye, without the aid of instruments, is possible because catalase has a very high specific activity, which produces a detectable response. Cellular role
Hydrogen peroxide is a harmful byproduct of many normal metabolic processes; to prevent damage to cells and tissues, it must be quickly converted into other, less dangerous substances. To this end, catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less-reactive gaseous oxygen and water molecules. The true biological significance of catalase is not always straightforward to assess: Mice genetically engineered to lack catalase are phenotypically normal, indicating this enzyme is dispensable in animals under some conditions. A catalase deficiency may increase the likelihood of developing type 2 diabetes. Some humans have very low levels of catalase (acatalasia), yet show few ill effects.
The predominant scavengers of H2O2 in normal mammalian cells are likely peroxiredoxins rather than catalase. Human catalase works at an optimum temperature of 37°C, which is approximately the temperature of the human body. In contrast, catalase isolated from the hyperthermophile archaea Pyrobaculum calidifontis has a temperature optimum of 90°C. Catalase is usually located in a cellular, bipolar environment organelle called the peroxisome. Peroxisomes in plant cells are involved in photorespiration (the use of oxygen and production of carbon dioxide) and symbiotic nitrogen fixation (the breaking apart of diatomic nitrogen (N2) to reactive nitrogen atoms). Hydrogen peroxide is used as a potent antimicrobial agent when cells are infected with a pathogen. Catalase-positive pathogens, such as Mycobacterium tuberculosis, Legionella pneumophila, and Campylobacter jejuni, make catalase to deactivate the peroxide radicals, thus allowing them to survive unharmed within the host. Catalase contributes to ethanol metabolism in the body after ingestion of alcohol, but it only breaks down a small fraction of the alcohol in the body.
Catalase test The catalase test is also one of the main three tests used by microbiologists to identify species of bacteria. The presence of catalase enzyme in the test isolate is detected using hydrogen peroxide. If the bacteria possess catalase (i.e., are catalase-positive), when a small amount of bacterial isolate is added to hydrogen peroxide, bubbles of oxygen are observed. The catalase test is done by placing a drop of hydrogen peroxide on a microscope slide. Using an applicator stick, a scientist touches the colony, and then smears a sample into the hydrogen peroxide drop. If the mixture produces bubbles or froth, the organism is said to be ‘catalase-positive’. Staphylococci and Micrococci are catalase-positive. Other catalase-positive organisms include Listeria, Corynebacterium diphtheriae, Burkholderia cepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia, Pseudomonas), Mycobacterium tuberculosis, Aspergillus, and Cryptococcus. If not, the organism is ‘catalase-negative’.
Streptococcus and Enterococcus spp. are catalase-negative. While the catalase test alone cannot identify a particular organism, combined with other tests, such as antibiotic resistance, it can aid identification. The presence of catalase in bacterial cells depends on both the growth condition and the medium used to grow the cells. Capillary tubes may also be used. A small amount of bacteria is collected on the end of the capillary tube (it is essential to ensure that the end is not blocked, otherwise it may present a false negative). The opposite end is then dipped into hydrogen peroxide which will draw up the liquid (through capillary action), and turned upside down, so the bacterial end is closest to the bench. A few taps of the arm should then move the hydrogen peroxide closer to the bacteria. When the hydrogen peroxide and bacteria are touching, bubbles may begin to rise, giving a positive catalase result.
Materials Required: Cultures: 24-48 hour tryptic soy broth cultures of bacteria Media: Tryptic soy agar Reagent: 3% hydrogen peroxide (Storage:-Upon receipt, store at 2-8˚C away from direct light. Reagents should not be used if there are signs of deterioration or if the expiration date has passed.) Equipments:
* Bunsen burner * Inoculating loop * Test tubes * Test tube rack * Microscopic slides
Procedure: The test can be done by two methods. a) Slant method b) Slide method a) Slant Method: 1. Using a sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of a streak inoculation. 2. Incubate all cultures for 24-48 hours at 37˚C. 3. Allow three or four drops of the 3% hydrogen peroxide to flow over the entire surface of each slant culture. 4. Examine each culture for the presence or absence of bubbling or foaming.
b) Slide Method: 1. Divide a clean glass slide into two sections with grease pencil. One should be labeled as “test” and the other as “control”. 2. Place a small drop of normal saline on each area. 3. With a sterilized and cooled inoculating loop, pick up a small amount of the culture from the nutrient agar slant or Petri plate. 4. Emulsify one or two colonies on each drop to make a smooth suspension. The smear should be about the size of a pea. 5. With a Pasteur pipette, place one drop of hydrogen peroxide over the test smear. Be careful not to run the drops together. 6. Do not put anything in the other drop that serves as control. 7. Observe the fluid over the smears for the appearance of gas bubbles. 8. Discard the slide in a jar of disinfectant.
Limitations: 1. Hydrogen peroxide is unstable and should undergo a control check daily prior to use. 2. Growth for catalase testing must be taken from an 18-24 hour culture. Organisms lose their catalase activity with age, resulting in a false-negative reaction. 3. Catalase activity is a function of aerobic process. Organisms incubated anaerobically must be exposed to atmospheric oxygen for a minimum of 30 minutes before a catalase test is performed. Failure to complete this step may produce false- negative results. 4. A positive catalase reaction with anaerobic organisms may be delayed for up to a minute after addition of the reagent. 5. A weak catalase or pseudocatalase reaction may be produced by some strains of Aerococcus species and Enterococcus species.
Hints and Precautions: 1. Dispose the hydrogen peroxide slides in the appropriate container filled with disinfectant. 2. Nichrome wire should be used when testing the organism. Platinum wires may cause a false-positive reaction. 3. When using a slant for other purposes in the same laboratory period, it is possible to save material by adding H2O2 to the slant after finishing with it. 4. Extreme care must be taken if a colony is taken from a blood agar plate. Erythrocytes contain catalase, and a transfer of only a few blood cells can give a false-positive reaction. 5. Always use fresh hydrogen peroxide, since it is unstable. 6. Do not add organism to reagent, particularly if iron- containing inoculating loops are used. Iron containing loops will cause false positive test results if exposed to hydrogen peroxide.