Isolation of Starch degrading bacteria Enzymes in Action Introduction In this laboratory exercise, you will be playing the role of biotechnologists in search of a new amylase. Since most industrially used enzymes can found in soil organisms, you will isolate starch-degrading bacteria in pure culture from soil using a starch laden agar medium. In future exercises, you will measure amylase enzyme activity and you will use chromatography to purify amylase and to determine its molecular weight. Learning Objectives You should understand: Conceptual enzymes are proteins that catalyze biological reactions living organisms (particularly microorganisms) are a rich source of industrially and commercially useful substances and processes assays designed to detect enzymatic activity can also be used to screen for organisms that express that particular enzyme (isolation strategies for desired phenotypes). Practical isolation of bacteria from natural samples. aseptic technique serial dilution/spread plate technique for screening microbial populations proper technique to streak agar plates for subculture and pure culture isolation. Underlying Science An enzyme is a protein that catalyzes a specific reaction in the cell. In general, this reaction can be written as: enzyme S > P substrate product The action of enzymes depends on their ability to bind the substrate at a domain of the enzyme molecule called the active site. Enzymes themselves are not modified (or are only temporarily modified) in the reaction Therefore, a better way to think of an enzymatic reaction may be: E + S > ES > EP > E + P The binding site is usually specific for the substrate and depends on the three-dimensional conformation of the enzyme. This in turn is determined by the amino acid sequence of the protein and ultimately by the interactions of these amino acids when they fold into a complex three-dimensional structure. Some enzymes are not reactive on their own however and often
possess cofactors. Common cofactors include metals such as Zn and Fe and organic molecules such as vitamins. Millions of enzymes, each with a specific role, are required in nature to break down compounds during the decomposition process (and make compounds in catabolic processes). By purifying these enzymes and producing them on a large scale, they can be used for a vast range of applications in the service of mankind. Humans have used bacteria and the enzymes they produce for thousands of years (e.g. cheese making). Common exploitation of microorganisms for decomposition of substances include use of bacteria in sewage treatment plants and in composting. On a more experimental basis microbes have been used to decompose contamination from oil spills and other hazardous contaminants in ground water (bioremediation). Almost all enzymes used in these applications originate from microorganisms found in the soil. The search for a new enzyme or a biological mechanism for a desired industrial process can start by examining soil samples from the far corners of the world (or the far corners of East Lansing). Just one microorganism can contain over 1,000 different enzymes and a long period of trial and error in the laboratory can be needed to isolate the best microorganism for producing a particular type of enzyme. When the right microorganism has been found, the work is still not over. The microorganism has to be characterized and maybe even genetically-modified to produce the desired enzyme or process at high yields. Commercially, the microoganisms are grown in huge (four-story) tanks where they produce the desired product. This technique, referred generically to as fermentation (not to be confused with metabolic fermentation), has made it possible to produce enzymes, vitamins and other products economically and in virtually unlimited quantities. The end product of fermentation is a culture broth from which the desired products are extracted. The fermentation broth is centrifuged or filtered to remove all solid particles. The resulting biomass, or sludge, contains the residues of microorganisms and raw materials and is often used as fertilizer. In this and other exercises we will be studying the enzyme amylase, which breaks down starch, a large, often multi-branched polymer of glucose molecules. Amylase has several important industrial applications: it is present in dishwashing and laundry detergents to remove residues of starchy foods such as potatoes and pasta; is used in the brewing and baking industry to break down the starch present in grains; is used in the textile industry in the desizing of fabric, and is used in tandem with another enzyme, glucose isomerase, in the production of high fructose corn syrup (a sweetener used in products such as soft drinks that has twice the sweetening effect of glucose). Scenario You are an industrial microbiologist working for Proctor and Gamble Inc. Management wants to come out with a New and Improved version of your dishwashing detergent (and possibly improve the performance of all your various detergents). You ve been assigned the task. You know that removing starchy foods is one of the biggest dish washing problems because of its insolubility (same with starchy stains on clothing). An additive that improves starch removal would be a great leap forward so that is the strategy that you adopt. You know that starch can be hydrolyzed chemically but those chemical are harsh and would be skin irritants and/or damage fabrics. From your undergraduate days at MSU, you know that starch is efficiently broken down biologically by many organisms--from humans to bacteria and that that is accomplished using
enzymes called amylases. You look in the chemical catalogs and find a long list of available amylases but they are expensive and would not be cost effective to buy. You think, Why not just make it ourselves? You think about the problem Where do I get an organism that we can exploit to make amylase? Which organism and which amylase will be the most economically useful? You know bacteria are the most diverse and most easily manipulated organisms so your first step is to screen natural samples for microbes capable of degrading starch. Using your knowledge of biological systems you devise a fast and easy screening method to tell starch-degraders from starch-nondegraders using agar plates containing insoluble starch. In this week s lab, you will follow this strategy to isolate starch degraders from soil. In subsequent exercises, you will then characterize the amylase produced by these microbes and also see which will work under dishwashing conditions. Materials Sample of material that you believe will contain starch digesting organisms (i.e. soil) Petri dishs containing nutrient agar with 2% corn starch One sterile 99 ml dilution blank Sterile 9 ml dilution blanks Analytical Balance Micropipettors and tips Cell spreaders ( hockey sticks!) 95% ethanol Inoculating loops beaker Bunsen burner Ziplock bag 37 C and 50 C incubators Parafilm Iodine stain (if necessary) Controls and Variables Controls: Variables: Positive Controls: Starch plates with a known starch degrader will be provided to ensure starch plates are working properly and demonstrate what a positive reaction for starch degradation looks like. NegativeControls: Ain t got enny. Try to design one in your prelab write-up (hint: What might be the opposite of the positive control described above?) Independent variables: (the variables that are intentionally changed) Incubation temperatures 37 C and 50 C. Dependent variable: (or results or thing you are measuring) the growth of bacterial colonies and the degradation of starch. Controlled variables: The variables that are unchanged or constant throughout the experiment. Environmental sample (i.e. the same soil sample will be plated at two incubation temperatures). A single dilution scheme will be used in both
Part A platings. The same culture medium (starch agar plates) will be used for both platings. Methods 1. Weigh out 1 gram of your soil and add this to 99 ml of sterile distilled water (1/100 dilution). Mix well. 2. While still suspended, further dilute the suspension by removing 1 ml with a P-1000 micropipettor and add it to a test tube containing 9 ml of sterile distilled water(1/10 dilution 1/1000 total dilution). 3. Label the agar side of your Petri dish with your group and your section numbers. 4. Remove 50 µl of your mixture with a P200 (a further dilutiion) and transfer it to the center of a starch-agar plate. 5. Using sterile technique, dip a cell spreader in alcohol, hold it in a flame briefly and briefly air cool it then by touching it to the outer edge of the agar. Use this to spread the 50 µl sample evenly around the plate. (Carefull: If your spreader is not cool enough, it will kill the bacteria as you spread them around!). try to use the entire agar surface. The available turntable makes this easier though it has to be shared between teams. 6. Repeat steps 4 and 5 to make a second plate. 7. Incubate one plate agar-side up in the 37 C incubator. 8. Place your other plate in a ziplock bag with and incubate agar-side up in the 50 C incubator. (Why in a zip-lock bag?) 9. The next day, come back and examine the plates. Look to see if clear areas are developing around certain colonies. You may have to use an Iodine stain to visualize starch degradation on you plates. 10. Photograph the colonies on your plates in the results section in your notebook. Do not forget to write in your notebook additional notes and observations that may not be obvious on the photograph. If possible, compare your plate with several other groups' plates in terms of degree of starch digestion. Summarize these observations in your notebook. 11. Go to the Part B Methods section below before going on to step 12. 12. Wrap your plates in parafilm and place in the refrigerator. Part B Obtaining pure cultures of starch degrading microbes In a later exercise, you will be investigating the activity of purified amylase enzymes. In order to obtain purified enzymes, you must first have the organisms that produce a particular enzyme in pure culture. One way to do this is by streak plating the organism on solid medium to get well separated (i.e.pure culture) colonies. Each colony (in theory) is derived from a single microbial cell This is done by essentially diluting the microbes present in a sample by spreading the bacterial cells across an agar surface with a wire loop. Cells are diluted by zigzagging the loop
across four quadrants of the medium, heat sterilizing and cooling the loop between quadrants. The method works because a small number of bacteria from a previous quadrant are pulled to the next quadrant during each phase of the technique. Ultimately the original population cells is so diluted that only single cells are spread out in the final (4th) quadrant. These individual cells then replicate over the next 24-36 hours to become visible colonies. All members of a colony are genetically identical because they originated from a single cell (assuming no mutations took place during the incubation period). In order to maintain a pure culture, the medium, all equipment, and working surfaces must be kept free from contamination from foreign bacteria and fungi. The overall procedure for maintaining contamination-free conditions is called aseptic technique (a = without, and septic = contamination). Method 1. Obtain starch agar plates. Write your section and team numbers on agar-side of the plate. Label one plate 37 C and the other 50 C. 2. have your instructors/tas help you identify colonies on each plate (both 37 C and 50 C) that appear to be degrading starch. 3. Before you begin, wash your hands and wipe down the working surface. Make sure there is a bunsen burner and inoculating loop available. 4. First, flame the inoculating loop by holding its end in the flame until the loop glows red. This will kill any microorganisms and their spores found on the loop or handle end. DO NOT LAY THE LOOP DOWN OR LET IT TOUCH ANYTHING BEFORE YOU DO THE TRANSFER. 5. Let the loop cool for 10 sec. Briefly touch the loop to a sterile agar surface to ensure it is cool then pick a starch degrading colony on the 37 C plate. 6. Carefully move the loop over to an uninoculated starch plate. Holding the agar side of the dish in your hand, gently streak the loop over the medium in area #1 (i.e. first quadrant) taking care not to cut into the agar surface. Reflame your loop as described in step 4. Streak in areas 2, 3 and 4, reflaming the loop between each quadrant. See Figure 1. #1#2#3#4
FIGURE 1._ Plate streaking pattern 7. Replace the agar onto the petri cover. Label the bottom of the plate with your section and team numbers and the name of the isolate. You can name your isolate whatever you wish, like A, small/white, or Elvis. Incubate the plate at 37 C. 8 Repeat the above steps to isolate starch degraders growing at 50 C. Place the plates in a ziplock bag and place in the 50 C incubator. 9. Come back the next day to record colony color and morphology in your notebook. Do all the colonies on your plate look the same? If you do not have well isolated colonies (i.e. You should be able to easily pick a colony without worry of touching another colony), streak out one or two new plates with your starch degrading isolate and incubate at the appropriate temperature. Cleanup When you have completed the exercise be sure to dispose of your plates in the biohazard waste container. However do not throw out your final plate containing your pure starch degrading isolate, you may whish to use it in your inquiry project. Questions to be addressed in conclusions and discussion 1. Given the industrial microbiologist scenario of this exercise, what might be an advantage of having a starch degrading bacteria that grows at high temperatures (i.e. 50 C)? 2. How might you isolate a new strain of bacteria to degrade, or decompose oil from spills in marine environments.