Science Buddies: "Ask an Expert"

I'm attempting to continue my research on my science fair project, but I'm a little behind than in previous years.

I'm trying to find the effect of different proteases (papain, bromelain, trypsin, lactoferrin, and pepsin) on the effectiveness of denaturation.

I'm putting together some details because I'm trying to delve into the world of proteolysis with limited background knowledge, but my main problem is trying to find an amateur way of extracting the enzyme without complicated equipment.

Also, I need to figure out a way to test this. I'm thinking I'll test it through one of the release of one of the products.

If anyone has more knowledge about any of this science, please comment and help me out.

Thank you.

Hi,

Isolating and purifying proteins requires a lab with specialized equipment and reagents, but you can prepare an extract of bromelain from fresh pineapple which contains the enzyme:
http://www.google.com/url?sa=t&rct=j&q= ... 9g&cad=rja

The protein-digesting enzyme papain comes from the papaya fruit and you can make an extract from the fresh fruit and test it: http://foodrecap.net/health/fruits/extract-papain/2/

Rather than just comparing one enzyme with another, I would suggest comparing the fresh extracts with commercial meat tenderizers. Adolph’s brand contains bromelain (http://www.mccormick.com/Lawrys/Flavors ... Tenderizer) and Badia brand contains papain (http://www.foodfacts.com/ci/nutritionfa ... --oz/57612). Make sure you buy unseasoned.

To test the activity of the enzymes you can use gelatin digestion: http://www.google.com/url?sa=t&rct=j&q= ... XY&cad=rja

The above test just shows that there is digestive activity—it doesn’t measure how much. To do that you need a different assay such as this one where you can actually measure the amount of gelatin dissolved: http://www.central.rcs.k12.tn.us/Teache ... Enzyme.pdf

I hope these suggestions are helpful. Other experts may have additional ideas. Please post again if you have questions about the experiments or procedures.

Good luck!

Sybee

Thank you so much for assisting me with this. Delving into the world of proteolysis has been a little overwhelming for me, but the links you posted have really helped me.

Background Research: I am currently compiling it into a paper.

Question: What is the effect of different proteases (papain, bromelain, trypsin, and pepsin) on the effectiveness of gelatin digestion/denaturation in their own optimal environments?

Hypothesis: I am not really sure yet about which one will be the most effective yet. I will have to finish compiling all of my research and make a justified hypothesis.

Variables and Controls:
Independent Variable: Protease used in its optimal conditions: Papain, Bromelain, Trypsin, Pepsin
Dependent Variable: Change in gelatin mass every ______ minutes. I need to think of a time period, probably 10-30 minutes.
Control Group: I don't think this is necessary. Please tell me if it is.
Constants: Method for Collecting Data, Percentage/Concentration of Protease Present in each solution, Time Periods, Equipment,

Procedure:
Papain will be extracted from papaya latex using the method you provided me, except a heating oven will be used to dry the latex. Bromelain will be extracted using a similar method to the one you suggested. Trypsin is going to be extracted from beef pancreas that I will find at a local butcher shop. Pepsin will be derived from ?. I know it is a digestive enzyme, but I need to find a way to extract it that is not very expensive.

I will make solutions diluted with water to test it in. Then, pH buffers will be added, and the experiment will take place in water baths to maintain the optimal temperature for denaturation to take place specific to each enzyme. The gelatin will be taken out in ______ minute increments, dried, and massed. After 2 hours, or so, the experiment will be ended and final data collections will be made.


This is a brief version of my research plan. Do you see any major issues with it that I am missing? My main concern is if my procedure is doable, and if the results I achieve can be accurate enough to analyze my data. Any assistance that you could give me would be tremendous.

Thank you.

Hi,

You’ve done a great job of planning and mapping out the project ahead of time and that’s just what a scientist has to do before embarking on a new series of experiments. Makes things go a lot smoother!

As to your hypothesis—I get your hesitation because you really don’t know which of these enzymes will work best. I think that is why I suggested comparing fresh bromelain and papain to store-bought product because then your hypothesis can be: Fresh meat tenderizers work better than commercial.

If you want to compare the different kinds of enzyme under their optimal conditions you might break it out this way: trypsin and pepsin are animal digestive enzymes while bromelain and papain are plant enzymes. So your hypothesis could be something like: Natural animal digestive enzymes break down gelatin better than plant-derived enzymes. I don’t know if this is true, but it would make a good hypothesis.

You did not give a source for pepsin. According to the wiki (http://en.wikipedia.org/wiki/Pepsin) it is isolated from the lining of hog stomachs. I don’t know if your butcher can get those for you. You might have to get them fresh from a slaughterhouse. Cow stomachs would not work as they are herbivores. Also, you probably need to get fresh stomachs because the enzyme is not very stable.

We use trypsin in the lab for digesting cellular proteins. We store it in the freezer at -20C and thaw it out at 37C just before use and then throw the remainder away. Proteases are proteins and they will digest themselves in time, so it is best to use your preparations as soon as possible or freeze them. Also, since trypsin and pepsin are most active at temperatures between 37 and 42C, you need to rig up a water bath to keep a constant temperature during the digestion. Test the bromelain and papain at the same temperature.

The digestion time is something you will have to determine by doing a preliminary experiment but I would guess an hour at 37C would digest quite a bit of gelatin. The one issue that bothers me is how you are going to quantitate how much of each enzyme you are using in the digestion solution. I’m hoping some of the other experts can help out here from their experience. It is simple when you are using purified single proteins because you can just weigh them and compare them gram for gram. But an extract of pancreas—now that’s a lot tougher!

If I were doing this in the lab I would measure the protein concentration using our protein assay kit and use equal amounts of protein for the digestion experiments. This would still not be ideal because your plant and animal extracts will contain many proteins, not just the protease, but at least it would be a starting point and you might be able to find a reference to how much trypsin, for example, on average there is per gram of total protein in pancreatic extract.

Oh, almost forgot. You asked about a control. Yes, you need to include a control which should be a piece of gelatin in the buffer solution you use for each enzyme but WITHOUT the enzyme—same temperature and time.

I hope these suggestions help you and I hope other experts will post their ideas about how best to do these experiments with limited resources.

Good luck!