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Food Chemistry II > Enzymes I > Flashcards

Enzymes I Flashcards

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1
Q

Enzymes are _______

A

catalysts

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2
Q

why is it important to study minor components in foods?

A

they are major attributes to food
nutrition
defines the way we process foods

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3
Q

what is an enzyme?

A

o Most enzymes are proteins in nature with catalytic functions
o Their molecular weights and chains are usually smaller than other proteins such as collagen (100-400kDa), myosin (200kDa), etc. trypsin (20kDa), amylase (40-50kDa)

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4
Q

what are the functions of enzymes?

A

o Decrease the speed of biological reactions by reducing the activation energy (Ea) in organisms.
o Enzymes are biomolecules that enable biological reactions to proceed at perceptible rates in living organisms (animals, plants, microorganisms)
o All these organisms have the same functional classes of enzymes to enable them carry out the same kind of metabolic processes.

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5
Q

Why is it important to study enzymes in food chemistry?

A

o Desirable effect: processing

o Undesirable effect: rancidity, browning…

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6
Q

protein nature of enzymes

A

influence of H+ or OH-
influence of proteases
response to typical protein tests
evidence from X-ray crystallography

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7
Q

Protein nature of enzymes: influence of H+ or OH-

A

Enzymes can be hydrolyzed by dilute acid or alkali, just like proteins, to form free amino acids or low molecular weight peptides.

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8
Q

Protein nature of enzymes: influence of proteases

A

Enzymes can be broken down by proteases, just like proteins, to form AA or peptides with the participation of water.

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9
Q

Protein nature of enzymes: response to typical protein tests

A

Enzymes can respond to typical common protein tests, such as:

  • ninhydrin test,
  • biuret test,
  • Lowry test,
  • BCA test.
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10
Q

Protein nature of enzymes: evidence from X-ray crystallography

A

Enzymes are composed amino acids liked together by peptide bonds, just as it occurs in protein, by X-ray crystallography.

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11
Q

Holoenzyme

A

complete enzyme with functional activity

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12
Q

Proenzyme

A

not functioning, needs to be activated

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13
Q

Apoenzyme

A

protein part of the enzyme

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14
Q

Prosthetic group

A

essential non-protein part of the enzyme. Mainly metal ions such as Co, Fe, Zn..

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15
Q

Coenzyme

A

non-protein part, helps the functioning. Can be proteins or minerals.

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16
Q

Give examples of the enzymes that are composed only from holoenzyme.
Hint: these enzymes are just made up of proteins

A

trypsin, pepsin, chymotrypsin, renin

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17
Q

Some enzymes need a ____ in order to be able to function. These ions can be found on the active sites, they are bound to the protein very tightly.

A

prostethic group

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18
Q

Give examples of enzyme that cannot function without the prosthetic group

A

Polyphenol oxidase – Cu (in the active site or close to the active site)
Peroxidase – Fe (combines with the substrate)

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19
Q

What makes an enzyme different than other common proteins?

A

o They have catalysis function

o Enzymes have active sites

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20
Q

What are active sites in enzymes?

A

o Active site is a region where that substrates binds and transforms into products.
o Binds with substrates (S)
o Transform S to products (P)
o As a result of these 2 events, new bonds formed or broken to form the products

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21
Q

General features of active sites

A

o Small
o Crevices/Clefts
o 3D
o has two parts: binding site and the catalytic site where the catalytic reaction happens

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22
Q

Active site has two parts ____ and ____

A

binding site and the catalytic site where the catalytic reaction happens

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23
Q

Active sites have two functions

A

binding and transformation

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24
Q

What is the relation between binding and transformation of an enzyme?

A

o Binding of S precedes transformation
o Not all binding lead to transformation
o No transformation without binding

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25
Q

Enzyme specificity

A

o Different enzymes have different amino acid composition structure, molecular weight, unique active sites in terms of the shape, AA, co-factor (if any)
o Enzymes are specific
Enzymes select only limited amounts of compounds to act on

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26
Q

Enzyme specificity can be separated into 4 groups:

A

∇ Bond specificity
∇ Group specificity
∇ Absolute specificity
∇ Stereospecificity

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27
Q

Bond specificity

A

o Enzyme acts on the compound with similar bonds
o For example, lipase act on ester bonds in lipids/fats; protease act on peptide bonds in protein.
o It is also called relative, low specificity.

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28
Q

Relative, low specificity is

A

bond specificity

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29
Q

Give an example for bond specificity

A

lipase act on ester bonds in lipids/fats; protease act on peptide bonds in protein.

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30
Q

Group specificity

A

o Enzyme acts on a group of closely related compounds
o Pepsin and trypsin cannot act on the same group of proteins. They can act on certain proteins that have certain amino acids on them.
o For example, pepsin can hydrolyze peptide bonds where here is an aromatic amino acid: phenylalanine, tryptophan, tyrosine. Whereas, trypsin can hydrolyze peptide bonds where there are basic amino acids: lysine, arginine, histidine.

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31
Q

Give an example for group specificity

A

pepsin can hydrolyze peptide bonds where here is an aromatic amino acid: phenylalanine, tryptophan, tyrosine.

Whereas, trypsin can hydrolyze peptide bonds where there are basic amino acids: lysine, arginine, histidine.

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32
Q

The group specificity is weaker/stronger than bond specificity

A

stronger

33
Q

Group specificity is also called the ____

A

structural or moderate specificity.

34
Q

Absolute Specificity

A

o Only acts on one single substrate
o Much higher than group and bond specificities
o Also called substrate specificity or high specificity

35
Q

Give examples for absolute specificity

A

o Lactase only acts on lactose
o Sucrase only acts on sucrose
o Maltase only acts on maltose

36
Q

Stereospecificity

A

o Acts on only one specific stereoisomer
o Enzyme acts on one isomer of a molecule
o It is also called high specificity
o Much higher than other groups

37
Q

Absolute specificity is also called the ________

A

substrate specificity or high specificity

38
Q

Give examples for Stereospecificity

A

For example, L-amino acid oxidase acts on only L-amino acids

39
Q

Rank the specificities form weak to strong

A

Bond Specificity < Group Specificity < Absolute Specificity < Stereospecificity

40
Q

Why is it necessary to know enzyme specificity fro food scientists?

A

we need to know how enzyme specificity works in order to improve the quality of our products

Example, starch (they have different, linear crossed ink polymers – a 14; and branched polymers – a 16) = AMYLASE –> Can we use amylase to solve the problem by debranching?

41
Q

Enzyme Classification

A

EC Numbers - 4 digits (EC a.b.c.d.)
• 1st No. ‘a’ denotes the type of reaction catalyzed.
• Based on this, 6 main types of Reactions are distinguished, numbered 1, 2, 3, 4, 5, & 6.

∇	Oxidoreductases
∇	Transferases
∇	Hydrolases
∇	Lyases
∇	Isomerases 
∇	Ligases
42
Q

Enzyme nomenclature is based on numbered system with 4 digits, E.C. X.X.X.X

Explain the X es

A

o The 1st digit of X is the group
o The 2nd digit of X is the subgroup
o The 3rd digit of X is the sub-sub group
o The 4th digit is the numbering in these groups

43
Q

E.C. 1 Oxidoreductase

A

o Catalyzes redox reactions

Examples:
∇	PPO (polyphenol oxidase)
∇	GOX (glucose oxidase)
∇	Peroxidase
∇	Lipoxygenase
44
Q

Give examples for oxidoreductases

A

∇ PPO (polyphenol oxidase)
∇ GOX (glucose oxidase)
∇ Peroxidase
∇ Lipoxygenase

45
Q

E.C. 2 Transferase

A

o Catalyze the transfer of various groups from one molecule (donor) to another molecule (acceptor). The donor will come first then the acceptor to the active site.
o Ex: transglutamniase (TG)

46
Q

Give an example for transferase

A

transglutamniase (TG)

47
Q

E.C. 3 Hydrolase

A

o Catalyzes the cleavage or hydrolysis of larger molecules into smaller molecules with H2O as the co-reactor.
o Ex: protease lipase, carbohydrase

48
Q

Give a examples for hydrolases

A

protease lipase

carbohydrase

49
Q

E.C. 4 Lyase

A

o Remove groups from the substrate leaving behind product with low molecular weight (usually with unsaturated bonds)
o Ex: pectin lyase

50
Q

Give an example for lyase

A

pectin lyase

51
Q

E.C. 5 Isomerase

A

o Isomerase catalyzes the conversion of molecule into their isomers
- racemases
- epimerases
o Ex: glucose isomerase – transforms the glucose to fructose

52
Q

Give an example for isomerases

A

glucose isomerase

53
Q

Glucose isomerase

A

catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively.

The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS)

54
Q

E.C. 6 Ligase

A

o We don’t find them in food industry we find them in biology in DNA etc.
o Ligases catalyze the joining together of two or more molecules

55
Q

Racemases*

A

catalyze the stereochemical inversion around the asymmetric carbon atom in a substrate having only one center of asymmetry

56
Q

Epimerases*

A

catalyze the stereochemical inversion of the configuration about an asymmetric carbon atom in a substrate having more than one center of asymmetry, thus interconverting epimers

57
Q

E.C. 6 Ligase

A

o We don’t find them in food industry we find them in biology in DNA etc.
o Ligases catalyze the joining together of two or more molecules

58
Q

Which E.C. are most commonly used in the food industry

A

EC 1-2-3 are commonly used in the food industry

  • oxidoreductase
  • transferase
  • hydrolase
59
Q

Enzyme purification can be done based on

A

Based on size differences
Based on solubility differences
Based on charge differences
Based on selective adsorption

60
Q

Why is it necessary to purify enzymes?

A

To remove other undesirable components, form the source materials, such as toxin, other enzymes, and other biomolecules like DNA etc.…

61
Q

Extraction

A

= Purification

raw materials are blended or homogenized in buffer solution.

62
Q

Extraction –> filtration =

A

crude enzyme extract (salt, minerals, sugars, nucleic acids, RNA, DNA, other kinds of enzymes

63
Q

Enzyme purification techniques based on size difference

A

Dialysis
Ultrafiltration
Centrifugation
Gel filtration

64
Q

Explain Dialysis

A

is a technique used of enzyme purification based on size difference

∇ semi-permeable membrane (cellulose, sucrose) with different sizes of pores.
∇ Sample is placed in a dialysis
∇ The bag is places in a solvent of more dilute concentration than the sample solution
∇ Small molecules in the sample will travel through the pores to the outside of the dialysis bag, while the larger ones retain in the bag.
∇ When it gets to the equilibrium, small molecules can re-enter the bag.
∇ It is necessary to change the solvent several times to remove as much of the small molecules as possible.
∇ It is not very efficient; it is very difficult to remove all the small molecules with this technique.

65
Q

Explain Ultrafiltration

A

is a technique used of enzyme purification based on size difference

∇ Uses semi-permeable membrane
∇ Pressure in the form of gas (N2 or vacuum) is used to force the small molecules go through the pores in the membrane.

66
Q

Explain Centrifugation

A

is a technique used of enzyme purification based on size difference

∇ It uses the centrifugal force to hasten the sedimentation of different molecules in a suspension
∇ The larger/heavier molecules sediment at a faster rate then small/light molecules.

67
Q

Explain Gel Filtration Chromatography

A

is a technique used of enzyme purification based on size difference

∇ Resin is placed in column
∇ Apply the sample on the resin
∇ Sample migrate down to the column
∇ Big molecules will go down faster than big molecules
∇ The larger molecules pass through the space and elute faster.
∇ The small molecules enter the pores of resin and elute afterwards.

68
Q

Enzyme purification techniques based on Charge Differences

A
  • Ion exchange chromatography
  • Electrophoresis
  • Isoelectric focusing
69
Q

Explain Ion Exchange Chromatography

A

is a technique used of enzyme purification based on charge differences

∇ Uses resin as a purification material
∇ The resin is positively or negatively charged
∇ If it is positively charged, then it will bind to the anions who are negatively charged – ANION EXCHANGER
∇ If the resin is negatively charged, it will bind with the enzyme that is positively charged – CATION EXCHANGER
∇ Purify a (–) charged enzyme using IEX: by using a positively charged resin – apply the sample (mixture of different enzymes) and the negatively charged enzymes will bind with resin. The rest of the positively charged enzymes will elute first form the column. By using a buffer, we disrupt the binding of + and – charges. Bound enzyme is removed by the charge, the pH, or ion of the eluting buffer.

70
Q

Explain Electrophoresis

A

is a technique used of enzyme purification based on charge differences

∇ The proteins can move in the electrophoresis bc of their sizes and charges.
∇ Mobility x q/r2
∇ SDS – PAGE type of electrophoresis – the mobility is only related with the size of the enzyme

71
Q

Explain Isoelectric Focusing

A

is a technique used of enzyme purification based on charge differences

∇ pH. Difference is created by amino acids or peptides
∇ pH. Gradient gel generated by ampholyte.
∇ Electric current is applied
∇ Sample is a mixture of enzymes
∇ Positively charged enzyme will migrate to the cathode side and the enzyme that is negatively charged will move to the anode side of the plate.
∇ Under electric current enzymes with different charges migrate, up to a pH. Point in the gel where the overall charge of the enzymes is 0
∇ They will go up to a certain point and they stop.
∇ The pH. It reaches gives the isoelectric point of the enzyme/protein.
∇ Different enzymes have different isoelectric points. (PI)

72
Q

Explain Isoelectric Precipitation

A

∇ At the PI, enzyme has minimal solubility
∇ If we have an enzyme solution, we can add some acid or alkaline to change the pH. Of the solution. If the pH. Of the enzyme is equal to the pH. Of the medium then that enzyme will coagulate and precipitate at a pH. Value corresponding to its PI

73
Q

Explain Salt fractionation/salting out

A

The differential precipitation of proteins, or nucleic acids, from solution by the addition of neutral salts, often ammonium sulfate.

based on the electrolyte-non electrolyte interaction, in which the non-electrolyte could be less soluble at high salt concentrations.

It is used as a method of purification for proteins, as well as preventing protein denaturation due to excessively diluted samples during experiments.

74
Q

Solvent Precipitation

A

The sedimentation of a solid material (a ‘precipitate’) from a liquid solution caused by the addition of an additional different solvent.

75
Q

Enzyme purification techniques based on Specific Binding Sites

A
  • Affinity chromatography
  • Hydrophobic interaction chromatography (HIC)
  • Hydrophilic interaction chromatography (HILIC)
76
Q

Explain Affinity chromatography

A

∇ Substrate linked in resin have specific binding with particular enzyme. They can be: substrate, co-factor, inhibitor
∇ Unbound enzyme will elute from column at first, bound enzyme can be washed put of the column by changing the pH. Or ion strength of elution buffer.

77
Q

Explain Hydrophobic interaction chromatography (HIC)

A

Hydrophobic Interaction Chromatography is a separation technique that uses the properties of hydrophobicity to separate proteins from one another. In this type of chromatography, hydrophobic groups such as phenyl, octyl, or butyl, are attached to the stationary column. Proteins that pass through the column that have hydrophobic amino acid side chains on their surfaces are able to interact with and bind to the hydrophobic groups on the column.

78
Q

Hydrophilic interaction chromatography (HILIC)

A

HILIC uses hydrophilic stationary phases with reversed-phase type eluents.
Any polar chromatographic surface can be used for HILIC separations.
Separates by differences in polarity

cation exchange - retention of anionic groups
anion exchange - retention of cationic groups

now allow separation based more on the basic and/or neutral functional groups of these molecules.

79
Q

Enzyme purity can be tested with

A

o Electrophoresis and isoelectric focusing
o Activity testing
o Chromatographic behavior
o If the enzyme is pure you will get only one peak or one line on the test you have done.

Food Chemistry II (8 decks)

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