Enzymes : techniques

Question 1

The enzymes naturally present in the foods are __

Answer 1

endogenous

Question 2

The enzymes added to foods are ____

Answer 2

exogenous

Question 3

name 4 evidences we know enzymes are proteins

Answer 3

1. inlfuenced by the acid or base medium
2. influenced by proteases
3. Response to typical protein tests
4. evidence from x-ray crystallography

Question 4

what are the two enzymes seen in lass that need copper as a co-factor?

Answer 4

xanthine oxidase

polyphenol oxidase

Question 5

what is the difference between apoenzyme and holoenzyme

Answer 5

apoenzymeis the catalytically-inactive, protein component of the enzyme whereasholoenzymeis the catalytically-active form of the enzyme, consisting of theapoenzymeand the cofactor. A cofactor can be either a metal ion or a small organic molecule

Question 6

What is the differnece between a co-factor and a prosthetic group?

Answer 6

Prosthetic groupsarecofactorsthat bind tightly to proteins or enzymes, They are permentaly bound to the enzyme and required for its function.
Acofactoris a non-proteinchemical compoundormetallic ionthat is required for anenzyme’s activity(it is not bound tightly to the enzyme like it is the case for the prosthetic group).

Question 7

what are the features of the active site

Answer 7

small
3D entities
Crevices
Region where the substrate binds and is transformed into a product

Question 8

T or F : binding at the active site will result in a transformation of the substrate into product

Answer 8

binding is necessary, but not all the binding at the active site will result in the transformation.

Question 9

What are the different types of enzyme specificity ?

Answer 9

1. absolute specificity
2. Group specificity
3. Stereospecificity
4. Racemases
5. Epimerases

Question 10

Give examples given in class of enzymes having a absolute specificity

Answer 10

1. glucose kinase (adding p on glucose)
2. catalase (breakdown of hydrogen peroxide)
3. glucose oxidase (oxidize only glucose).

Question 11

What is group specificity

Answer 11

enzymes will only act on closely related molecules like hexoses or lipase

Question 12

Lipase is in the category of ____ specificity

Answer 12

group

Question 13

Hexokinase is in the category of ____ specificity

Answer 13

group

Question 14

What are stereospecific/stereoselective enzymes

Answer 14

A number of enzymes have the ability to discriminate between enantiomeric substrates or products; such enzymes are referred to as stereospecific/stereoselective enzymes

Question 15

What are the techniques used to purify enzymes based on size differences

Answer 15

1. dialysis
2. ultrafiltration
3. high speed centrifugation
4. gel filtration
5. Electrophoresis (SDS-page)

Question 16

Explain dialysis

Answer 16

it is the osmosis of a sample placed inside a bag. The molecules will diffuse from the higher concentration (interior of the bag) to the lower concentration (surrounding of the bag).

Question 17

What is gel filtration

Answer 17

a technique used to separate molecules based on their size. Inside a column, there is gel beads of selected size. only the enzymes of smaller size than the beads selected will be able to pass through the column

Question 18

What are the techniques used to purify enzymes based on their charge differences

Answer 18

1. Ion exchange chromatography
2. Electrophoresis
3. Isoelectric focusing

Question 19

explain ion exchange chromatography

Answer 19

beads that can bind anion (-) or cations (+) are place inside a column. When a sample of enzymes of mixed charged is passed through the column. The enzymes of similar charge to the beads will pass through the column and the enzymes of inverse charge to the beads will stay inside the column and can be later washed.

Question 20

Electrophoresis will separate a molecule based on ____

Answer 20

charge and size

Question 21

Explain electrophoresis

Answer 21

Electrophoresis (PAGE) is a technique used to separate proteins according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation and charge of the molecule. They will migrate on a gel with a current applied on each side.

Question 22

Explain isoelectric focusing

Answer 22

) is an electrophoresis technique that separates proteins based on theirisoelectricpoint (pI). The proteins migrate on the gel that has a pH gradient. They will strop at the pH corresponding to their isoelectric point (no net charge means no effect of the charge differences applied in the gel).

Question 23

What are the techniques used to purify enzymes based on their solubility differences

Answer 23

1. isoelectric precipitation
2. salt fractionation
3. solvent precipitation

Question 24

explain isoelectric precipitation

Answer 24

at the isoelectric point, the molecule is less soluble but is not insoluble. Alkali or acid may be used to adjust the pH (upwards or downwards to attain the PI to result in precipitation to separate proteins or enzymes

Question 25

At the pi : the net charge on the enzyme is ___
Above the PI : the net charge on the protein or enzyme molecule is ____
Below PI : the net charge is ___

Answer 25

+

Question 26

explain solvent precipitation / fractionation

Answer 26

The solvation layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface and binds it in hydration layers around the organic solvent molecules. With smaller hydration layers, the proteins can aggregate by attractive electrostatic and dipole forces.

Question 27

what are the technique used to separate proteins based on specific binding sites?

Answer 27

1. affinity chromatography

2. Hydrophobic interaction chromatography

Question 28

explain the affinity chromatography

Answer 28

example would be to have a columm with trypsin inhibitor as the ligand and trypsin will go in the colum and be attracted by the ligand and stay in the column. The ligand in the column can be anything that has an affinity with the enzyme (cofactor), coenzyme, inhibitor, substrate

Question 29

complete : Hydrophobic interaction chromatography(HIC) separates molecules based on their_____

Answer 29

hydrophobicity

Question 30

What are the techniques used to verify the enzyme purity

Answer 30

1. test for homogeneity (bands in electrophoresis)
2. chromatogrpahic behavior
3. activity testing
4. isoelectric focusing

Question 31

How can you test for activity of trypsin?

Answer 31

trpysin will be color yellow a sample when you do the BAPNA test (benzoylarginine p-nitroanilide.) because is will separate the p-nitroanilide from the benzoylarginine p-nitroanilide. p-nitroanilide is yellow so the sample will be colored yellow.

Question 32

How can you test for activity of lipase?

Answer 32

Lipases will degrade the p-nitrophenyl palmitate (PNPP) into p-nitrophenyl and palmitic acid. P-nitrophenyl is yellow.

Question 33

In chromatography, you measure the absorbance of your sample. symmetrical peaks will denotes more purity. ____ and ____ in the chromatography graph means the sample is not pure.

Answer 33

Shoulders or tailing