Exercise 31: Casein Protease PDF

w VI u cc w >< w CASEIN PROTEASE LEARNING OBJECTIVES------------ l. List and describe the four basic functions of proteins in living organisms. 2. Differentiate between the four levels of complexity of protein structure. 3. Explain...

w VI u cc w >< w CASEIN PROTEASE LEARNING OBJECTIVES------------ l. List and describe the four basic functions of proteins in living organisms. 2. Differentiate between the four levels of complexity of protein structure. 3. Explain the importance of proteases in microbial metabolism. 4. Explain the biochemistry involved in the utilization of casein by casein protease. 5. Investigate the utilization of casein by the environmental isolate. 6. Interpret the results found on the casein agar plates. INTRODUCTION--------------- After studying an organism's structure, growth characteristics, and susceptibility to chemo- therapeutics, microbiologists may next choose to study biochemical or metabolic characteristics that make an organism unique. As we have previously discussed, each organism has adapted to its particular niche. Some of these adaptations may include the development of physical structures that aid in growth, but many more adaptations correspond to the metabolism of nutrients that exist within the niche. In this exercise we will begin to study some of the unique biochemical adaptations possessed by microorganisms and how these adaptations can aid in specific identification of an unknown microbe. Proteins As you learned in Exercise 3, microbes are classified based on the substrate they use as a source of carbon-heterotrophs use organic chemicals and autotrophs use CO2 Additionally, recall that microbes are classified based on the substances from which they derive energy- organic chemicals, inorganic chemicals, or sunlight. Previous experiments with the environ- mental isolates have indicated that they are all chemoheterotrophic organisms. Over the course of the next several lab periods, we will examine some of the metabolic capabilities of these fascinating organisms. Proteins are composed of amino acids, which are known as the "building blocks of proteins." There are twenty individual naturally-occurring amino acids, and proteins are composed of combinations of these amino acids. Amino acids are joined together by peptide bonds that covalently link the amino group of one amino acid with the carboxyl group of another amino acid. Many amino acids joined in this manner form a polypeptide. Proteins are classified by their major function within living organisms. Structural proteins contribute to the three-dimensional structure of the organism, as well as to the various components and membranes of the cells. Motility proteins, as their name implies, play a role in movement and motility in living cells. These proteins are involved with muscle contraction in animal cells and with structure and movement in bacterial cells. Enzymes are an extremely important class of proteins. Enzymes are referred to as catalysts because they increase the rate at which chemical reactions take place in living cells. They can be used over and over again, but are destroyed by extremes in temperature and pH. Enzymes may be active within the cell proper (intracellular), or they may be excreted from the cell (exoenzymes). Finally, a few proteins are not structural, motile, or enzymatic. These proteins are involved in the immune response to foreign material in higher organisms and are known as antibodies. Proteins have varying levels of structure referred to as primary, secondary, tertiary, and qua- ternary. Primary structure consists of the specific amino acids and the order in which they are joined. Various amino acids within a protein interact to form the secondary structure. Two major patterns of secondary structure are typically observed-a-helices and -sheets. Tertiary structure is formed when the protein folds into a three-dimensional shape as a result of the helices and sheets folding back on themselves. Both globular (tightly coiled and folded into a somewhat spherical shape) and fibrous (long chains of intermittently linked polypeptides) proteins result from these interactions. Finally, a protein may assume quaternary structure if it is composed of more than one polypeptide chain held together by many weak bonds. All microorganisms require carbon and energy sources to survive. Many proteins are an excel- lent source of both, but proteins are too large to be transported inside the cell. If organisms are to use proteins as a carbon and energy source, they must have a mechanism for breaking down the protein and transporting the usable components into the cell. Some microbes excrete extracellular peptidases and proteases which can degrade an extracellular protein into smaller polypeptides. The smaller polypeptides can be transported into the cell and then be digested by endopeptidases. Casein Proteases Casein is a milk protein and is the protein that gives milk its cloudy, white color. In fact, approximately 80% of the proteins in cow's milk are caseins and most exist in colloidal molecules known as casein micelles. The function of casein micelles is to carry large amounts of calcium and phosphate from mother to offspring. Interestingly, casein is the most commonly used milk product in the food industry and is included in foods ranging from coffee whiteners to infant formulas. Casein itself is insoluble, so casein salts, known as caseinates, are listed as ingredients in foods. Cheese is produced by the fermentation of the milk sugar lactose by lactic acid bacteria found in milk. The acidic end-products of this fermentation produce an acidic environment which causes casein molecules to coagulate and form curds. The yellow liquid remaining after the formation of curds is known as whey. Cheese is produced when all liquid is removed from the curds and a secondary fermentation takes place. When milk is included in an agar preparation, the resultant casein agar plates are cloudy and opaque. To test for the presence of a casein protease or peptidase, microbes are spot inoculated onto the medium. If the organism produces extracellular proteolytic enzymes that break down the substrate casein into smaller polypeptides, the medium surrounding the microbial growth will become clear. This is known as a zone of proteolysis and it is clear evidence that the intact casein protein is no longer present in the medium. On the other hand, if the casein protein is not broken down, the medium around the microbial growth will remain cloudy.