Scouting for a biotech product!
Amylase Adventure

(Developed from Kreuzer and Massey’s 2nd Edition Recombinant DNA and Biotechnology, the New York State Center for Advanced Technological Education’s web site, and Paul Patev’s Advanced Biotechnology Education Project at Middlesex Community College, Bedford, MA.)

PROBLEM CONTEXT

A small developing country with very limited resources has decided to encourage development of a biotechnology industry using local resources. A representative from the Country’s Economic Development Committee has approached your company to develop a process to make and sell amylase (an enzyme found in many organisms that breaks down starch) for commercial purposes. Amylase can be sold for producing corn syrup from cornstarch, as an additive in laundry detergents to better clean clothes, for sizing in the fabric industry and in the making of bread, beer and pizza dough.

You have recently left a large pharmaceutical firm where you have been moderately successful and have developed an excellent reputation for your biotechnical skills. You have decided, however, that you wish to become an entrepreneur, starting your own company with some risk, but a solid scientific background and some financial resources. This would be your new company’s first “product” so it is important that you make good showing in order to attract new investors.

It is important that your product be unique to the Country hiring your firm, so you have decided to screen native soil organisms and plants to find one that produces amylase in abundant quantities that can be isolated and developed cheaply.

Before beginning product development, you must know the following:

· Starch is a complex storage form of glucose (a simple sugar) used by many life forms, especially plants.

· When an organism needs to break down starch into glucose for energy purposes, it uses enzymes such as amylase. They are naturally occurring.

· Amylase works to break down covalent bonds between glucose molecules in starch by inserting a water molecule (hydrolysis) converting the big starch molecules into smaller pieces or shorter chains like maltose (a double sugar.)

Phase One. Research: Be prepared or it will cost time and money!

Before investing your money and time, you need to do some research. The internet will provide you with information you need. Develop teams within your company (classmates) to investigate the existing industrial uses of amylase in business/industry. Divide up the work between team members then summarize the findings of your group and present them to your current investors.

Phase One Research Projects:

Prepare a poster or powerpoint presentation about uses or manufacture of amylase.
Present an oral report on current uses of amylase.
Bring in samples of products on the market that contain amylase.
Prepare formal "stockholders report" listing several products you might manufacture.

Recognize constraints on product development.
The country is underdeveloped and poor, resources are limited, money scarce. Your start-up company cannot take on risk beyond your means. You will have to do a lot of the work yourself. You must make your own media and solutions. Keeping excellent records so that any experiment is repeatable is necessary to avoid costly time and reagents. You must develop a set of SOP’s (Standard Operating Procedures) for using tools and processes of production for when you go into commercial development. Remember that you want to guard your trade secrets closely and that you must keep track of costs.

Phase Two. Development: Testing Food sources for amylase activity.

Materials:

Starch agar & sterile starch-nutrient agar plates
Metric Balances
Spectrophotometer (optional)
0.1 M Tris Buffer pH 7.5-8
Test Tubes
Coffee Filters or Cheese cloth
Disposable transfer pipettes or droppers
3 oz. plastic cups
Test tubes or 15 mL centrifuge tubes with caps
Iodine test solution
Ice
Plastic disposable pipettes or droppers
1% starch solution
Mortar and pestle
distilled water
marker pens/pencils

Making Food Extracts: Collect any/all of the following:
Two half-dollar size washed/peeled sweet potato slices
Two half-dollar size potato slices
Four quarter-sized carrot pieces
a wedge of apple (peeled)
10- dried beans soaked overnight in water 10-12
germinated beansBaker’s yeast activated with sugar
Plant leaves or roots
Yogurt made with active cultures

Procedure
Prepare extracts (1 class period)
1. Weigh out your first food source (when making extracts from other foods, use the same weight sample for each food, about 1-2 g.).
2. Place food pieces into mortar and grind with pestle into paste.
3. Add 10 ml of cold 0.1 M Tris Buffer pH 7.5-8.0. Stir and let set for 10-15 min to bring enzyme into solution, stirring occasionally.
4. Obtain 15 mL centrifuge tube with cap and put your group number on the cap and the name of the food extracted on the side of the tube. Alternately, use and label glass test tubes.
5. Pour mash and liquid onto surface of a coffee filter to remove pulp, gently squeeze to collect liquid extract into a plastic cup, then transfer to labeled test tube. Hold on ice for later use, or refrigerate overnight. These are your stock extract solutions.
6. Formulate hypotheses on which foods will have the most amylase activity and record in question one below.

2nd Class Period.
A.Testing for amylase Activity: Starch-agar plate method.
1. Test your food extracts for amylase activity by using a pipette to paint the extract onto the surface of starch-agar plates. Label plate and let stand at room temperature for 30 minutes. During wait period, do part B below.
2. After 30 minute wait period, test for the presence of amylase enzyme on the starch agar plates as follows: flood plates with iodine test solution and observe color. If starch is present, the iodine will react to form blue-black color. If amylase is present in the extract, it breaks down or digests the starch and clear areas will appear in the agar. (Thus a positive test for starch is a negative result for amylase and vice versa!). Record results in the chart. Rank amylase activity from 1 (least) to 5 (most).

B. Testing for amylase activity: Test tube method.
1. Pour 1 ml of 1% starch solution into a test tube, one tube for each food.
2. Add 5 drops of extract to be tested. Label each tube.
3. Set up one tube with starch solution only, no added extract.
4. Let sit for 30-45 minutes to allow enzyme to work on the starch if any is present.
5. Add one drop of iodine solution to each tube.If no starch is broken down, the solution will turn blue. If starch is broken down, indicating amylase presence, the solution will turn from blue to purple to avocado colored to brown. Rank on scale of 1 to 5 (least to most amylase).
6. Record your results in the chart. Return to part A and complete step 2 (test).

Results: Record information on amylase activity in the following table:

Food extract

Tube #

Time processed

Starch agar Plate amylase results from part A

Test Tube amylase results from part B

Questions
1. Hypotheses: Which foods do you think will have the most amylase activity in the extracts?


2. Based on your results, which food extracts produced the most amylase activity?

3. With what foods were the results different from the starch agar plate method versus the test tube method?

4. What might account for differences if any?

5. If you determined that an extract had amylase activity but only a small amount, what infromation might explain that result, using your knowledge of starch and sugars.

Conclusions:
Every lab experiment involves looking at all the results to determine what you learned, and summarizing what you found out in the conclusion section. Moreover, often an experiment produces unexpected results, so that your initial questions were not answered by the experiment. Sometimes the experiment brings up new questions. Hence, a good conclusion also describes briefly what further experiments need to be done to address new or unresolved questions.

Look over your results and write a good scientific conclusion as defined above:
(Teachers: use this rubric for assessment of this laboratory activity)

Part C. Isolating an Amylase-Producing Microorganism from Soil
The Corn Starch Biodegradable Packing Peanut Activity could be a substitute for part C because it also involves soil microbes but eliminates the need for sterile starch-nutrient agar.

1. Make or obtain the sterile starch-nutrient agar media needed to identify amylase-producing microorganisms.
2. Collect soil samples from different sources, being careful to record where the samples came from and describing the area around the samples.
3. Put 1 teaspoon of soil sample into a bottle or container with 100 ml of water. Mix well and allow to settle out.
4. Pipette 0.5 mL of supernatant (liquid above sediment) onto starch-nutrient agar plate and spread over surface of plate.
5. Repeat with 0.1 mL of supernatant onto another starch-nutrient agar plate and again spread over surface of plate.
6. Incubate for one or two days.
7. Flood with iodine solution to search for amylase activity. Record results.
8. Handle growth plates safely: view results with minimal opening the lid of plates; dispose of plates by placing in biohazard bags and autoclaving used plates before disposal.

Results

Sample

Location found

Description of location

Amylase activity

1. Which soil sample provided the most amylase activity? ____________________

2. List characteristics of soil or similarities of locations that produce abundant amylase activity after comparing with other students results. ________________________________________________________________ ________________________________________________________________

3. Suggest some unique places where soil microorganisms might be collected that would be of benefit in developing a new amylase product. __________________ __________________________________________________________________

4. Besides amylase that breaks down starch, suggest some other enzyme activities that might be worth investigating for future product development. ________________________________________________ _________________________________________________

C. Optional Activity: Quantify your results on the Food Extracts!

Use a spectrophotometer to measure absorbance. First, dilute samples 10X (after the iodine was added) by removing one ml from each tube and adding it to a new tube with 9 ml of distilled water. Be sure to label each of these new tubes. Record absorbance with the wavelength set at 620 nm. Refer to SOP (Standard Operating Procedure) for the Spectrophotometer for use. Record your results in the table:

Food sample 1 in 10 diln

Absorbance at 620 nm

SOPs for making media and solutions for this lab can be found on our mock biotech website: click here.