Food Chem 2- Enzymes
Card Set Information
Food Chem 2- Enzymes
What is a Holoenzyme?
Complete functional unit of an enzyme (protein part and non-protein part)
What is an apoenzyme?
Protein part of an enzye- some enzymes require only the protein party to function
What is a prosthetic group?
Essential non-protein part required by some enzymes for activity (eg metal ion)
What is an active site?
Region that binds and transforms substrates into products
Does all binding lead to transformation?
No, but you can not have transformation without binding
What is absolute specificity?
When an enzyme acts on only a single substrate (catalase)
What are racemases?
catalyze inversion of stereochemistry in biological molecules with only 1 assymetric carbon
What are epimerases?
Catalyze stereochemical inversion, but in molecules with 2 or more asymetric carbons
What are isomerases?
Catalyze inter-conversions between 2 isomers
What is group specificity?
Enzxyme will act on a group of closely related compounds
Ex. peroxidases break down all peroxides
What is stereospecificity?
Enzyme will act only on one stereo configuration
What are the different bases on which to purify an enzyme?
Based on size differences
Based on solubility differences
Based on charge differences
Based on selective adsorption
What is some ways to purify an enzyme based on size difference?
What is ultrafiltration?
You pressurize a compartment and only small molecules can get through the membrane
What are some ways to purify enzymes based on charge differences?
Ion exchange chromatography
What is isoelectric focusing?
Ampholyte are used to set up a pH gradient in the gel before samples are applied and subjected to electrophoresis
Molecules then migrate in the gels and stop at the pH zones corresponding the Isoelectic points of the protein/enzyme molecule
What are the different methods for purifying an enzyme based on solubility differences?
How does isoelectric precipitation work?
Proteins have different isoelectric points, by titration with acid or base, the different molecules can attain the isoelectric point value in which they are least soluble
Although, depending on how much of it is present, it will determine whether they precipitate or not
When present in a significant amount, the tendency is to precipitate
How does solvent precipitation work?
Hydrophilic vs Hydrophobic
Polar organized solvents such as acetone or ethanol
Behavior of the solvent causes the molecules to precipitate
It is important that solvents are at very cold temperatures (-20°C)
What is salt percipitation?
Put ammonium sulfate or sodium sulfate (neutral salt) into protein mixture
Salt will steal water from protein and cause protein to percipitate
Because different proteins have different surface groups, they wil percipitate at different concentrations of ammonium sulfate, so some fractionation can be achieved
What are the different methods for seperating an enzyme based on specific binding sites?
Hydrophobic interaction chromatography
How is affinity chromatography done?
Enzymes are bound in a gel. Substrates are titrated and bind to enzymes (can be the opposite). Bound molecules may be removed by changing the pH of the elution buffer
What is hydrophobic interaction chromatography?
You have a solution of soluble proteins in water
At high ionic strengths the protein will try to percipitate, so you keep it at an intermediate ionic strength
Use a hydrophobic surface to absorb the proteins out of solution
Can use for purification because different proteins have different inherent hydrophobicities
Use elution with lower ionic strength water to reverse the adsorption
HIC can be applied to the purification of most soluble proteins
What are the different ways for testing for enzyme purity?
Test for homogeneity
What is N-terminal analysis
N-terminus is the amino end of a peptide chain
Find out which amino acid is the end and you can tell the protein
React the unknwn peptide with a reagent which will selectively label the terminal amino acid with a color
Hydrolyze the protein
Compare to a standard to figure out what protein you have
What are the 6 types of enzyme classifications?
What do oxidoreductases do?
Catalyze oxidation/reduction reactions (eg. zanthine oxidase, glucose oxidase, ascorbic acid oxidase)
What do transferases do?
Catalyze the tansfer of groups between molecules
What are transferases used for in the food industry?
Used to improve the texture of soft tissue foods (eg alaskan pollock fillets into firm surimi-type products)
What do hydrolases do?
Catalyze hydrolysis (or breakdown) od larger molecules into smaller ones
Use H2O as a coreactant in the hydrolytic process
What do lyases do?
Remove groups from large molecules to result in smaller molecules (usually with unsaturated bonds)
What do isomerases do?
Catalyze interconversions between isomers
What do ligases do?
Put smaller molecules together to form larger molecules
What is an example of an isomerase frequently used in the food industry?
Glucose isomerase (glucose--> fructose)
What are the most important types of enzymes in the food industry?
Oxidoreductases, trasnsferases, hydrolases, and isomerases
What is THE most widely used group of enzymes in the food industry?
hydrolases (around 60% of total enzymes used)
How do enzymes facilitate reactions?
By reducing the energy barrier
What are the two methods for measuring reaction rate of enzymes?
Initial rate method
What is the initial rate method?
You measure the change as close as possible to time=0
Zero time is when the enzyme is added to the substrate in the initial rate method, measurements are taken as close to the zero time possible
What is end point method?
In the end point method, the E is added to the S and the reaction mixture is left to proceeed for a fixed time interval and then stopped, before measurements are taken
What are the advantages of initial rate method?
Enzymes are fully active to display its maximum potential
Not susceptible to substrate depletion
Not prone to inhibition by end product
What are the disadvantages of initial rate method?
Requires more skill to do
Requires sophisticated equipment
What are the advantages of end point method?
Simpler to do and does not require much skill
Inexpensive and simple instruments
What are the disadvantages of end point method?
Enzymes that are prone to inactivation, enzyme activity may decline with time, thus we can underestimate the catalytic capacity of the enzyme
Susceptible to errors due to the substrate depletion
Susceptible for end product inhibition (when the products inhibit the enzyme)
What are the 3 stages of enzyme catalysis?
Pre-steady state phase
Steady state phase
Post-steady state phase
What is pre-steady state phase?
When the rate of product formation is increasing with time
Very short duration
This occurs becasue the ES complex needs to get in equilibrium
What is steady state phase?
ES is forming and breaking down at the same rate (concentration of ES is almost constant)
What is post-steady state phase?
Substrate depleted, reaction slows down
What are the 3 types of reversible inhibition?
What is competitive inhibition?
'I' binds to active site of 'E' and prevents 'S' from binding
'I' resembles 'S' in structure, this competes for active site
eg. inhibition of succinate dehydrogenase by malonate
What is non-competitive inhibition?
'I' binds to a site other than the active site of 'E' and prevents transformation of 'S' to 'P'
Reduction of reaction rate by pH is an example of non-competitive inhibition- the 'I' being the H+ on the acid side of the optimum and the OH- on the alkaline side
What is uncompetitive inhibition?
'I' binds to site on 'E' molecule which becomes available only after 'S' has bound to the active site; ie. to the ES-Comples
eg. S inhibition which occurs at very high [S]. As in inhibition of invertase by high concentration of sucrose
How do we overcome enzyme inhibitors?
Establish type of inhibition by incubating E with I and assaying for residual enzyme activity at intervals
For irreversible inhibitors, E loses activity progressively with time
Irreversible inhibition cause by heavy metals, may be relieved by IEX
Reversible inhibition overcome by 1. increasing [S] 2. dialysis to dissociate I from E or 3. dilution out eh [S] or by increasing [E]
What do low temperatures do to enzyme activities?
Low temperatures slow down enzyme catalysis (used in foods to retard undesirable effects like texture softening, off-flavors, ripening)
What do high temperatures do to enzymes?
Higher tempertures enhance enzyme catalysis but may also deactivate enzymes
What are some factors affected by temperature?
Affinity to enzyme for substrte; inhibitors or activators
Rate of conversion of substrte to products
Changes in solubility of gases
pH of buffer
Ionization of prototropic groups
What does a graph of enzyme activity vs temperature look like?
Bell shaped curve
How does pH influence catalysis?
E's active within narrow pH rang
pH optimum of enzymes- duration of reaction, nature of S and [S], ionic strength of medium, purity of E, presence/absence of an inhibitor or activator
How do we determine the pH stability of an enzyme?
Incubate enzyme at various pH values
Apply enzyme to the substrate
Measure relative activity
How do we determing the pH optimum for an enzyme?
Prepare substrates at different pHs
Add enzyme, and then measure activity
What are some methods used to inactivate enzymes in food?
What happens if cheese is not properly pasteurized?
Incomplete inactivation of the enyme after the curd is formed could lead to continued proteolysis to: decrease yield, softer texture, form bitter peptides
How is cheese curd formed?
k-casein , under proteolytic enzymes becomes para-k-casein and macro-casein
Para-k-casein is insoluble and leads to the curd formation
Macro-casein is a glycopeptide, soluble, responsible for a couple of good health effects (digestion efficiency, inhibition of gastric pathogens)
Why are lipases necessary in cheese making?
Lipases break down fats in the milk to form free fatty acids which impart the flavors in the cheese
How are enzymes used in alcoholic beverages?
Starches are degraded by amylases to produce fermentable sugars
These sugars are degraded by zymases to form ethanol and CO2
Proteases break down protein molecules in the raw material to form low molecular weight peptites and free amino acids --> flavor change
Whern you reduce the temperature the small molecules stay in solution and the heavier ones percipitate
How are enzymes used in non-alcoholic beverages?
Amylases improve flavor
Pectinases increase juice yield and facilitate juice clarification
Naringinases reduce bitterness in the juice (especially the biterness from naringin compound in grapefruit juice)
What do alpha-amylases do?
Act randomly on alpha-1,4 bonds within the molecule to form a mixture of oligosaccharides
What do beta-amylases do?
Remove 2 glucose units sequentially from the end of the molecule by cleaving alpha-1,4 bonds
Action be beta-emylase stops at the branch point because it does not act on alpha-1,6 bonds
What enzyme acts on beta-1,4 linkages?
What are pectins?
Carbohydrates made up of anhydro-galacturonic acid units
Why is pectin important in juices such as tomato?
to stabilize the 'cloud'
How are enzymes used in baking?
Amylases break down starches again and again into fermentable sugars
Lipoxidase/lipoxygenase- used for whitening flour (bleaching) by breaking down carotenoid pigments
Proteases may be added to flour to break down proteins and improve texture
Transglutaminases gets the texture firmer from crosslinking between proteins (creamier yogurt, firmer noodles, firmer meat)
Where do proteases come from?
Mostly derived from plants and are quite heat stable
Proteases used to produce protein hydrolysates derived mostly from meat scraps, bones and fish frames for use as flavorants
What are the different ways a protease can hydrolyze a protein?
What are exoproteases?
Remove amino acids stepwise from the termini of the proteins
Useful in flavor modifications
2 kinds- aminopeptidases/another one
What are endoproteases?
Act randomly on peptide within the protein molecules to form peptides
Useful in preparation of hydrolysates
What are the 2 major carb chains found in food?
Starch (alpha-1,4 linkage)
Cellulose (beta-1,4 linkage)
Which is more resistant to enzymatic hydrolysis, cellulose or starch?
What is the most imporant use of lipases in the food industry?
Hydrolysis of acyl glycerols
Triglycerides+ nH2O --> Glycerol + nFFA
Why is the action of lipases usually undesirable in food?
FFAs formed are less stable than the acyl glycerols, and are responsible for undesirable rancid odors
What are some industrial applications of proteases?
Removal of bitterness
Modification of milk an whey proteins for incorporation into dietetic products
Hydrolysis of wheat gluten for use as flavorants
Modification of colagen and gelatin
Recovery of scrap protein from offal, bones, and blood
What are some lesser known applications of enzymes?
Removal of dental plaque with toothpaste containing dextranases
Elimination of hair with keratinase
Solubilization of cold tea solids with tannases
Synthesis of protein-like molecules