Enzymology for food and biorefinery
Introduction
What are enzymes & protein structure
Primary: amino acid sequence, glycosylation, peptide bonds
Secondary: alpha-helices and beta-sheets
Tertiary: 3D structure (folding), stabilised by
o Non-covalent interactions (H-bridges, electrostatic interactions, van der Waals
forces, hydrophobic interactions)
o Covalent interaction: S-S bridges
Quaternary: formation of subunits
Disulphide bridges (S-S): stabilize all protein conformations
General classification & EC numbering
Each enzyme:
- systematic name (substrate, enzyme class)
- trivial name (papain; amylase)
- enzyme commission number (EC) + 4 digits
(1) enzyme class, (2-4): type of reaction and specificity of the reaction
Carbohydrates: CAZy classification, not EC
First digit: enzyme class
1. Oxidoreductases oxidation-reduction
2. Transferases remove & transfer of functional groups
3. Hydrolases cleave of bonds with introduction of water
4. Lyases cleave of bonds (no water), creation double bond
5. Isomerase rearrangements
6. Ligase formation of a linkage, with the use of ATP, UTP or CTP
Substrate or reaction specificity
Specificity: arises from the 3D structure of the enzyme active site (tertiary structure)
- Substrate specificity: enzyme is specific for a structural characteristic of the substrate
- Reaction specificity: only one type of reaction takes place for a certain enzyme
Michaelis-Menten parameters describe a typical enzyme reaction, curve: substrate vs. velocity
Km: a measure for the affinity of the enzyme for its substrate (substrate binding); substrate
concentration at which Vmax/2, lower Km: reaction faster with low [substrate], high affinity!
Vmax: maximal reaction rate (substrate conversion), saturation
kcat: amount of product formed per enzyme molecule, when all the enzyme has bound to the
substrate
,Higher [substrate], more product formed with same enzyme concentration, when all enzymes are
full, increasing [substrate] will slow down the reaction rate, Slope: reaction rate or velocity (V)
Endogenous/exogeneous enzymes, endo/exo-activity
Endogeneous: already present in food - Quality control (ripening, fruit, meat, cheese), product
formation (dough, beer)
Exogeneous: added enzymes - Production (cheese), quality (fruit juices)
Endo-activity: enzyme cleave internal bonds
Exo-activity: enzyme cleave at the outside of the chain
Effect of pH or T for enzymes
Effect of pH
- Activation through protonation: activity determined by the degree of protonation of
residues in active site
- Irreversible inactivation (loss of active conformation)
Effect of temperature
- Increase of temperature: reaction rate increases until optimum temperature
- Above the inactivation temperature: denaturation. Mainly the 2nd and 3rd
(and if present also the 4th) structure lost (loss of interaction), 1 st maintained
Temperature optimum a result of enzyme catalysis and enzyme inactivation
Q10 value: factor that gives the increase of reaction rate at a temperature increase of 10 degrees
D value: time needed to heat an enzyme at a certain temperature in order to maintain 10% of its
original activity; rate of inactivation at a certain temperature
Lower D: less time needed to inactivate the enzyme
Z value: increase of temperature needed to decrease the D-value from 100% to 10%
thermostability of an enzyme at increasing temperature
Higher Z: higher temperature needed to inactivate the enzyme
Other parameters affecting temperature stability: ion-strength, water activity, stabilisers (e.g.
immobilization, other proteins, Ca2+)
Enzyme inhibition: competitive inhibition, non-competitive inhibition, substrate inhibition
,Carbohydrate degrading enzymes
Classification of carbohydrate active enzymes & glycoside
hydrolases
Classification of CHO active enzymes
CAZy: carbohydrate active-enzyme database, based on:
- Homology based on amino-acid sequences & secondary structure motives (helix, strand,
turn)
- Classification has a highly predictive value (catalytic amino acids, homology modelling)
CAZy database: families of structurally-related catalytic and carbohydrate-binding modules (or
functional domains) of enzymes that degrade, modify or create glycosidic bonds
Enzyme classes
- Glycoside Hydrolases (GH) hydrolysis of glycosidic bonds (amylase, alpha-galactosidase)
- Glycosyl Transferases (GT) formation of glycosidic bonds (use activated sugar
phosphates as glycosyl donors, and catalyze glycosylgroup transfer to a nucleophilic group)
- Polysaccharide Lyases (PL) non-hydrolytic cleavage of glycosidic bonds (pectin lyase)
- Carbohydrate esterases (CE) hydrolysis of carbohydrate esters (pectin methyl esterase)
- Auxiliary activities (AA) redox enzymes that act in conjunction with CAZymes (LPMO)
CBM: carbohydrate binding module
Use of CAZy classification: optimized enzyme mixtures (enzyme selection increasingly important),
improvement of enzymes within reach – predictive value enables a better understanding of
structure-function relationship; attribute functions to unknown genes in unknown genomes by
alignment to families
Question: What is the difference with the EC classification? EC system is based on substrate
specificity and occasionally on the molecular mechanism, in CAZy enzymes the grouping in one
family reflects similar structural features of the enzymes, which helps to gain mechanistic
information of the enzyme (structure-function relationship).
Question: Are enzymes in one family (in CAZy) always having the same substrate/linkage specificity?
No, in one family the specificity may differ, or different families may cleave the same bonds
Glycoside hydrolases
Catalytic mechanisms of GH
Inverting GH one-step mechanism, inverting stereochemistry (alpha-beta; beta-alpha)
Single displacement reaction, large active site (6.6-9.5 A), H 2O and substrate can enter
Retaining GH two-step mechanism, retention of stereochemistry (alpha-alpha, beta-beta)
Smaller active site (5.5A), first substrate binds, H 2O acts as an acceptor in the second step
Retaining GH & Transferase activity with a high substrate concentration, the substrate
can act as an acceptor, resulting in transglycosylation
Recap: monomeric CHO equilibrium linear/ring form, alpha: opposite face to the CH2OH- group at
C4 position, beta: same face
, What is endo/ exo-activity and processivity
Endo-activity: enzyme cleave internal bonds
Exo-activity: enzyme cleave at the outside of the chain
Processivity: the enzyme’s ability to repetitively continue its catalytic function without releasing the
substrate - Consequence for product profile
Analytical methods of enzyme activity
Hydrolysis - reducing end group analysis
During hydrolysis, more and more reducing end groups become
available, CHO or sugar residue ring that can open is called the
reducing end, polysaccharides: only one reducing ends
DE glucose: 100, maltose: 50, starch ~0, expectation: higher DE with
an exo-active enzyme, since it leads to mono and di-saccharides instead of oligomers; DE as a
characteristic of the enzyme: analyse Reducing Ends and Total Sugar, calculate DE in time
Reaction: copper, hydroxide (blue) reacts with reducing ends to copperoxide and water (red)
Spectrophotometric assay: quantify the amount or number of reducing end groups, calibration curve
Hydrolysis – HPSEC high performance size exclusion chromatography
Separation on the hydronamic volume in solution, RI/UV detector
Application: shift in molecular weight distribution
Big molecules cannot enter the pores in between the particles: shorter retention time
Introduction
What are enzymes & protein structure
Primary: amino acid sequence, glycosylation, peptide bonds
Secondary: alpha-helices and beta-sheets
Tertiary: 3D structure (folding), stabilised by
o Non-covalent interactions (H-bridges, electrostatic interactions, van der Waals
forces, hydrophobic interactions)
o Covalent interaction: S-S bridges
Quaternary: formation of subunits
Disulphide bridges (S-S): stabilize all protein conformations
General classification & EC numbering
Each enzyme:
- systematic name (substrate, enzyme class)
- trivial name (papain; amylase)
- enzyme commission number (EC) + 4 digits
(1) enzyme class, (2-4): type of reaction and specificity of the reaction
Carbohydrates: CAZy classification, not EC
First digit: enzyme class
1. Oxidoreductases oxidation-reduction
2. Transferases remove & transfer of functional groups
3. Hydrolases cleave of bonds with introduction of water
4. Lyases cleave of bonds (no water), creation double bond
5. Isomerase rearrangements
6. Ligase formation of a linkage, with the use of ATP, UTP or CTP
Substrate or reaction specificity
Specificity: arises from the 3D structure of the enzyme active site (tertiary structure)
- Substrate specificity: enzyme is specific for a structural characteristic of the substrate
- Reaction specificity: only one type of reaction takes place for a certain enzyme
Michaelis-Menten parameters describe a typical enzyme reaction, curve: substrate vs. velocity
Km: a measure for the affinity of the enzyme for its substrate (substrate binding); substrate
concentration at which Vmax/2, lower Km: reaction faster with low [substrate], high affinity!
Vmax: maximal reaction rate (substrate conversion), saturation
kcat: amount of product formed per enzyme molecule, when all the enzyme has bound to the
substrate
,Higher [substrate], more product formed with same enzyme concentration, when all enzymes are
full, increasing [substrate] will slow down the reaction rate, Slope: reaction rate or velocity (V)
Endogenous/exogeneous enzymes, endo/exo-activity
Endogeneous: already present in food - Quality control (ripening, fruit, meat, cheese), product
formation (dough, beer)
Exogeneous: added enzymes - Production (cheese), quality (fruit juices)
Endo-activity: enzyme cleave internal bonds
Exo-activity: enzyme cleave at the outside of the chain
Effect of pH or T for enzymes
Effect of pH
- Activation through protonation: activity determined by the degree of protonation of
residues in active site
- Irreversible inactivation (loss of active conformation)
Effect of temperature
- Increase of temperature: reaction rate increases until optimum temperature
- Above the inactivation temperature: denaturation. Mainly the 2nd and 3rd
(and if present also the 4th) structure lost (loss of interaction), 1 st maintained
Temperature optimum a result of enzyme catalysis and enzyme inactivation
Q10 value: factor that gives the increase of reaction rate at a temperature increase of 10 degrees
D value: time needed to heat an enzyme at a certain temperature in order to maintain 10% of its
original activity; rate of inactivation at a certain temperature
Lower D: less time needed to inactivate the enzyme
Z value: increase of temperature needed to decrease the D-value from 100% to 10%
thermostability of an enzyme at increasing temperature
Higher Z: higher temperature needed to inactivate the enzyme
Other parameters affecting temperature stability: ion-strength, water activity, stabilisers (e.g.
immobilization, other proteins, Ca2+)
Enzyme inhibition: competitive inhibition, non-competitive inhibition, substrate inhibition
,Carbohydrate degrading enzymes
Classification of carbohydrate active enzymes & glycoside
hydrolases
Classification of CHO active enzymes
CAZy: carbohydrate active-enzyme database, based on:
- Homology based on amino-acid sequences & secondary structure motives (helix, strand,
turn)
- Classification has a highly predictive value (catalytic amino acids, homology modelling)
CAZy database: families of structurally-related catalytic and carbohydrate-binding modules (or
functional domains) of enzymes that degrade, modify or create glycosidic bonds
Enzyme classes
- Glycoside Hydrolases (GH) hydrolysis of glycosidic bonds (amylase, alpha-galactosidase)
- Glycosyl Transferases (GT) formation of glycosidic bonds (use activated sugar
phosphates as glycosyl donors, and catalyze glycosylgroup transfer to a nucleophilic group)
- Polysaccharide Lyases (PL) non-hydrolytic cleavage of glycosidic bonds (pectin lyase)
- Carbohydrate esterases (CE) hydrolysis of carbohydrate esters (pectin methyl esterase)
- Auxiliary activities (AA) redox enzymes that act in conjunction with CAZymes (LPMO)
CBM: carbohydrate binding module
Use of CAZy classification: optimized enzyme mixtures (enzyme selection increasingly important),
improvement of enzymes within reach – predictive value enables a better understanding of
structure-function relationship; attribute functions to unknown genes in unknown genomes by
alignment to families
Question: What is the difference with the EC classification? EC system is based on substrate
specificity and occasionally on the molecular mechanism, in CAZy enzymes the grouping in one
family reflects similar structural features of the enzymes, which helps to gain mechanistic
information of the enzyme (structure-function relationship).
Question: Are enzymes in one family (in CAZy) always having the same substrate/linkage specificity?
No, in one family the specificity may differ, or different families may cleave the same bonds
Glycoside hydrolases
Catalytic mechanisms of GH
Inverting GH one-step mechanism, inverting stereochemistry (alpha-beta; beta-alpha)
Single displacement reaction, large active site (6.6-9.5 A), H 2O and substrate can enter
Retaining GH two-step mechanism, retention of stereochemistry (alpha-alpha, beta-beta)
Smaller active site (5.5A), first substrate binds, H 2O acts as an acceptor in the second step
Retaining GH & Transferase activity with a high substrate concentration, the substrate
can act as an acceptor, resulting in transglycosylation
Recap: monomeric CHO equilibrium linear/ring form, alpha: opposite face to the CH2OH- group at
C4 position, beta: same face
, What is endo/ exo-activity and processivity
Endo-activity: enzyme cleave internal bonds
Exo-activity: enzyme cleave at the outside of the chain
Processivity: the enzyme’s ability to repetitively continue its catalytic function without releasing the
substrate - Consequence for product profile
Analytical methods of enzyme activity
Hydrolysis - reducing end group analysis
During hydrolysis, more and more reducing end groups become
available, CHO or sugar residue ring that can open is called the
reducing end, polysaccharides: only one reducing ends
DE glucose: 100, maltose: 50, starch ~0, expectation: higher DE with
an exo-active enzyme, since it leads to mono and di-saccharides instead of oligomers; DE as a
characteristic of the enzyme: analyse Reducing Ends and Total Sugar, calculate DE in time
Reaction: copper, hydroxide (blue) reacts with reducing ends to copperoxide and water (red)
Spectrophotometric assay: quantify the amount or number of reducing end groups, calibration curve
Hydrolysis – HPSEC high performance size exclusion chromatography
Separation on the hydronamic volume in solution, RI/UV detector
Application: shift in molecular weight distribution
Big molecules cannot enter the pores in between the particles: shorter retention time
