Enzymes Flashcards
MM Kinetics
Lower Km = greater affinity for the substrate
What affects enzymatic rate?
Enzyme concentration
Substrate concentration
Temp
pH
Activators
Inhibitors
Enzymatic methods
Any physical or chemical property that can relate to substrate or product concentration
Enzymatic method categories
Viscosity
Titration
Chromatography
Absorbance spec
Fluorimetry
Monometric
Coupled reactions
Two or more enzymatic reactions
Indicating and measuring
S1 → P1
P1 → P2
Sulfites
Enhance flavor
Preserve freshness
Slow bacterial growth
Prevent oxidation and browning
Beer, wine, fruit juices
Sulfite enzymatic test
Sulfite oxidase converts to sulfate
NADH peroxidase converts H2O2 to Nad+
Amount of sulfite is proportional to amount of NADH oxidized
Glucose enzymatic test
Glucose oxidase
Converts glucose to lactone
Peroxidase
Converts H2O2 to oxidized dye
Oxidized dye is a colored product
Starch and dextrins enzymatic test
Amyloglucosidase cleaves alpha 1,4 and 1,6 bonds
Starch, glycogen, dextrins
Liberates glucose
Lactose enzymatic test
Hydrolyzed to glucose and galactose
NADH is the end product
Fast, selective, accurate
Malic acid enzymatic test
Only L malic is natural
Mixture of D and L in synthetic
Measure NADH via change in absorbance
Peroxidase test
Treatment that destroys peroxidase is adequate to destroy other enzymes and microbes
Shows how well food is processed
Peroxidase enzyme
Heat stable
Produces brown pigments
Destroyed by blanching
Peroxidase reaction
Add guaiacol and H2O2 to form colored product
High enzyme activity = oxidation
ALP
Alkaline phosphatase
Heat stable enzyme in milk
Thermal stability > non spore forming pathogens
ALP test
Determine if pasteurization was performed correctly
ALP: Scharer variation
ALP cleaves phosphate of reagent
Creates indophenol
Blue color
ALP: Rutgers variation
Phenolphthalein dye as a reagent
End with a pink color if there is enzyme activity
Immunoassays
Antibody antigen interaction
Detect allergens or GMOs
High specificity for target antigen
Epitope
Specific region bound by a single antibody
ELISA
Enzyme linked immunosorbent assay
Binding of soluble antigen or antibody to a solid support
Bound and unbound molecules separated via washing
Enzyme label converts colorless substrate to colored product
Sandwich method
Antibody attached to surface
Add sample
Antibody recognizes and captures the substance/antigen
Wash
Add a second antibody w/ enyzme
Wash
Add the substrate to the enzyme
ELISA applications
Pesticide residue
Detect foodborne pathogens
Detect toxins
Meat and fish species identification
Food allergens
GMOs
Meat species identification kits
Cooked and uncooked product
Qualitative
Sensitive and specific
Lateral flow immunoassay
pros
Sensitive and specific
Low sample volume
No wash steps
Short time
Low cost
Simple
Lateral flow immunoassay
Test strip
Add sample
Moves in certain direction
Test and control lines
Antibodies embedded in the strip capture the antigens
Positive or negative result
ELISA enzyme tags
Horseradish peroxidase
ALP
