Lab Report How Lipase(enzyme) can be affected with Substrate Concentration

[Markee]

Yo! So, let me get down to business.

My Biology lab work or coursework will be using a factor - which mine will be Substrate Concentration - that affects an enzyme - which my teacher has given us Lipase to work on

Apparently, I am not that clear with what is what and how does that affect that and what can I use to experiment on... That is my current problem. (I know, shame on me for not listening to my teacher during class) I tried reading the book, but it explains how it reacts and it is rather irrelevant for the information I need to get this experiment going...

So, as of the moment, could anyone of you guys, explain that to me? Basically, How can my chosen factor affect the Lipase.

cheers!

[ShineeLikeMe]

Hiii. Ill write this real fast because i have a dentist appointment.

Lipase is located in your duodenum of your small intestine and acts as an enzyme that breaks down fats in your foods. However lipase is very small whereas their substrates are usually very large and so it needs the help of bile from the liver to break down the fats into smaller peices so that the fats can be broken down more easily. IT depends on what size your substrate is. Whether the fat is already emulsified or not. But normally as substrate concentration increases the enzymatic actvity also increases as there is a greater chance of collisions between enzyme and substrate. However it'll only reach a certain point before all active sites are filled so the graph will plane.

Sorry I wrote this in like 2 minutes. Let me know if you need more.

Edited March 20, 2011 by Mahuta ♥

[Markee]

Hey! Thanks for the facts! That cleared up my knowledge a bit even more, after I've asked my teacher about. So my substrate concentration will be the type of milk which would also be my independent variable. whilst Lipase would be my enzyme - obviously - and would be kept at a constant amount which would therefore be my dependent variable, yes?

Thus concludes me to make my research question "which will be How does the effect of substrate concentration affect the activity of lipase?"

Is that good so far?

[Keel]

You have to have a way to measure the rate of enzyme activity. After the talk we had I think the only way to do that would be to analyse the pH using a pH meter.

Independent Variable: Concentration of Substrate

Dependent Variable: Enzyme activity = Rate of fatty acids produced (measured by the decrease in pH over time)

RQ: How does the increase in concentration of substrate affect the rate at which lipase breaks down lipids? (Add specifics eg. milk)

The type of enzyme and its concentration are control variables.

Edited March 16, 2011 by Keel

[Markee]

Would using the Phenolphthalein be ok to measure the rate of enzyme activity?

Ok then. Thanks with that! So, Enzyme Activity will be the reaction between the enzyme and the types of milk that i will be using? Or have I gotten that wrong?

Edited March 16, 2011 by Mark10B

[Keel]

Would using the Phenolphthalein be ok to measure the rate of enzyme activity?

Ok then. Thanks with that! So, Enzyme Activity will be the reaction between the enzyme and the types of milk that i will be using? Or have I gotten that wrong?

If you vary the type or brand of milk that will count as an independent variable. You should be varying the concentration of one brand of milk, i.e. diluting the milk to give you different concs. eg. 20% milk, 40%...ect.(personally I don't think milk is a very good lipid to use, too weak)

Using an indicator would be a good idea but I don't think phenolphthalein is a good one to use as you are going to be starting off too alkali, maybe bromothymol blue or phenol red?

*Does anyone have a better way of measuring the enzyme activity?

[Markee]

Yeah, that's what I want to do.

Aww, seriously? =/ Well I'm sure raw meat won't do either. . .How about butter? But, it would just melt. . .That would be a bit difficult to collect data. . .

Hmm. . . Phenol Red would do. Since it is more used in cells biology. BTB, i'm not sure about it since after I've looked it up, it works only when CO₂ is added. . . But the problem would be, being able to get my hands on them. I have to ask my teacher if the school has a stock of them available.

Thanks for the reply in advance!

I hope no one has forgotten that I still need some assistance with this

Edited March 22, 2011 by Desy ♫

[Keel]

Why do you have to use lipase? Can't you use amalayse or catalase? The problem here is that you have no way of defining a cut off point where you can take the time and work out the rate. You could use the 'shake until there is not division between the solutions method', but aparently this only works with lipids that have short-chained fatty acids so you have to find a substrate with that property (apparently coconut oil has short/medium chains).

[Mahuta ♥]

Question..why are you going to use milk? There are so many other things you can use as a substrate for lipase, for example butter or margarine.

[Markee]

Why do you have to use lipase? Can't you use amalayse or catalase? The problem here is that you have no way of defining a cut off point where you can take the time and work out the rate. You could use the 'shake until there is not division between the solutions method', but aparently this only works with lipids that have short-chained fatty acids so you have to find a substrate with that property (apparently coconut oil has short/medium chains).

I could use it, yeah, I might be able to, I will just ask my teacher if I actually can.

What about the 2 indicators you mentioned?

Aight, lemme try to do some 30mins. research and see what I can find for a 6 carbon fatty acid.

Question..why are you going to use milk? There are so many other things you can use as a substrate for lipase, for example butter or margarine.

Because I like milk? lol.

Are those better than milk?

[Mahuta ♥]

You are investigating the effect of substrate concentration on LIPASE. An enzyme that digests lipids, it's going to be much much easier and more accurate for you if you use almost 100% lipid substance.

Milk contains:

• Lipids
  
• Proteins
  
• Carbohydrates
  

Same amounts of lipids and proteins and more of carbohydrates. Your investigation will be 80% inaccurate!

[Keel]

I could use it, yeah, I might be able to, I will just ask my teacher if I actually can.

What about the 2 indicators you mentioned?

Aight, lemme try to do some 30mins. research and see what I can find for a 6 carbon fatty acid.

Yea, because its the same theory, the affect of changing substrate concentration on an enzyme. For amalyse you could test the time taken for starch with iodine solution to turn from blue-black to brick red or for starch with Benedict's solution to turn from blue to brick-red and hence find the rate. For catalase you could find the rate at which it decomposes hydrogen peroxide.

The main problem with this design is that its an enzyme design which means that the pH should be controlled with a buffer solution because pH is one of the variables that affects the effectiveness of an enzyme. So the indicator wouldn't work unless you were to ignore such a control variable which is not advised. As Mahuta stated, milk isn't a very good substrate to use, but if you are to use lipase as your enzyme be sure that the substrate has short chained fatty acids which can dissolve in water or ethanol solution. Otherwise the two substances will continue to be separated even though the enzyme has broken down the lipid.

[Markee]

You are investigating the effect of substrate concentration on LIPASE. An enzyme that digests lipids, it's going to be much much easier and more accurate for you if you use almost 100% lipid substance.

Milk contains:

• Lipids
  
• Proteins
  
• Carbohydrates
  

Same amounts of lipids and proteins and more of carbohydrates. Your investigation will be 80% inaccurate!

Ok. Thanks for that, Maha. Someday, I will pay you back for all of your help.

And I'm guessing those dairy products that you put in your bread as a filling are those kinds of Lipids?

I could use it, yeah, I might be able to, I will just ask my teacher if I actually can.

What about the 2 indicators you mentioned?

Aight, lemme try to do some 30mins. research and see what I can find for a 6 carbon fatty acid.

Yea, because its the same theory, the affect of changing substrate concentration on an enzyme. For amalyse you could test the time taken for starch with iodine solution to turn from blue-black to brick red or for starch with Benedict's solution to turn from blue to brick-red and hence find the rate. For catalase you could find the rate at which it decomposes hydrogen peroxide.

The main problem with this design is that its an enzyme design which means that the pH should be controlled with a buffer solution because pH is one of the variables that affects the effectiveness of an enzyme. So the indicator wouldn't work unless you were to ignore such a control variable which is not advised. As Mahuta stated, milk isn't a very good substrate to use, but if you are to use lipase as your enzyme be sure that the substrate has short chained fatty acids which can dissolve in water or ethanol solution. Otherwise the two substances will continue to be separated even though the enzyme has broken down the lipid.

Oh wow, it sounds like I'm doing a Chemistry coursework. Haha! Ok, I'll bear that in mind. And it does seem easier to use. So that means, if I could use those two enzymes, I could use the Universal indicator then?

aaaagh! I wish our Biology teacher could have gone into more detail with this experiment. Anyway, So, at best, I should use either Amalayse or Catalase if I don't want to give myself such a troublesome matter?

[Keel]

No I'm saying that because you need a buffer solution to keep the pH constant, testing a change in pH is not an option. You no indicators will be use for any of the experiments. So for amalase its time taken for a colour change, for catalase, its time taken to produce x volume of gas or volume of gas produced in x seconds.

[Mahuta ♥]

What do you mean those kind of lipids? You could simply use butter, it's about 81% butter.

Butter nutritional data

[Markee]

So a Buffer Solution would work best with the experiment I'm doing then? Regardless of what enzyme and substrate I'm going to use?

[Keel]

No No. OK, you are trying to investigate a variable that will affect the activity of an enzyme. Here is a list of variables that will affect enzyme activity: concentration of substrate, concentration of enzyme, concentration of inhibitors, concentration of heavy metals, pH, temperature. You are investigating the affect the concentration of substrate has on the activity of an enzyme, thus all the other variables need to be controlled. As you can see, the pH needs to be controlled, how do you control pH? with a buffer solution. So the pH is part of your control variables and the buffer is how you are going to control the pH. Therefore, using the change in pH as a method of testing the activity of the enzyme will not work as it is a constant controlled by the buffer. If you were to measure the activity you would need to find another method which is your major problem now for lipase.

However, if you were to use amalase, your method for determining activity would be a colour change. This is not affected by controlling any of the control variables. For catalase, it would be the volume of gas given off, again not affected by the control of control variables.

[Markee]

What do you mean those kind of lipids? You could simply use butter, it's about 81% butter.

Butter nutritional data

Oh wow. . .I feel like an utter slacker now. Haha. Ok then. Butter it shall be. I just need to get my head around the enzyme and how to collect the data for the reaction. . .But knowing that. . .I have to wait till tomorrow or my next Biology class since I have to ask my teacher >__< aaagh! And yet another coursework has been postponed. . .

No No. OK, you are trying to investigate a variable that will affect the activity of an enzyme. Here is a list of variables that will affect enzyme activity: concentration of substrate, concentration of enzyme, concentration of inhibitors, concentration of heavy metals, pH, temperature. You are investigating the affect the concentration of substrate has on the activity of an enzyme, thus all the other variables need to be controlled. As you can see, the pH needs to be controlled, how do you control pH? with a buffer solution. So the pH is part of your control variables and the buffer is how you are going to control the pH. Therefore, using the change in pH as a method of testing the activity of the enzyme will not work as it is a constant controlled by the buffer. If you were to measure the activity you would need to find another method which is your major problem now for lipase.

However, if you were to use amalase, your method for determining activity would be a colour change. This is not affected by controlling any of the control variables. For catalase, it would be the volume of gas given off, again not affected by the control of control variables.

Ahh, I see now. I really should ask my teacher now if I can use Amalase now. . .Arigato Sensei!

[Mahuta ♥]

Whats wrong with the enzyme? As in what is it that you cant get your head around?

[Markee]

Nothing at all Maha. heh heh heh. It is just because I am not thinking properly today. That's all. . .

Keel, you know when you mentioned Amylase? And that it breaks down starch with iodine solution. Then that would mean, I would not use a lipid(butter) as my substrate concentration?