Enzyme Applications in Food Analysis Flashcards
The enzyme active site has definitive 3D structure caused by surrounding AA residues from various regions of polypeptide chain. This 3D structure must fit potential substrate structure, so that there can be multiple interaction points through different bonds, linkages and forces, but why no covalent bonds?
Becuase covalent bonds are very strong and they release of the substrate is needed
what are the 4 types of enzyme specificity and briefly describe them
- absolute specificity: the enzyme only acts on one specific substrate like ureases only cataylzes the breakdown of urea
- group specifity: the enzyme acts on a group of molecules with similar functional groups like hexokinase which works on several hexoses
- bond specificity: the enzyme can recognize specific type of bond like peptidases with peptide bonds
- stereochemical specificity: the enzyme differentiates between different stereoisomers of a compound so lactase can only act on specific beta-D-lactose
enzyme assays are used to? and what are the two types?
Determine quantiatively specific compounds which serve as substrates, activators, inhibotors of enzymes and also to look at kinetics. There is rate assay method and total change method
Define what infromation you get from the rate assay method
We can get enzyme kinects from Vmax, Kcat, Km etc.
Km= the substrate concentration at which v= 0.5 x Vmax
K cat= is the rate constants for the reaction
Vmax= max velocity
what is the rate assay method
The rate assay (kinetic assay) measures how fast an enzyme works by tracking changes in the concentration of a substrate or product over time. The reaction is set up with plenty of all necessary components, and small samples are taken at intervals. Each sample is stopped immediately using methods like changing pH or temperature. The amount of product formed or substrate used is then measured using chemical or physical methods. This helps determine the enzyme’s activity over time, often shown as a graph of increasing product concentration.
what are the advantages of the rate assay method?
- speed: it can be done in 1-5 minutes
- requires less enzyme
- determines [] of enzyme, activator, inhibitor or substrate, so many uses
which method is used for enzyme kinectics?
Rate Assay ONLY
enzyme assays may be designed to?
- determine amount of enzyme in sample
- determine the amount of substrate, activator, inhibitor
- determine isomeric configuration of molecules
explain the total change method?
The total change method measures the amount of product formed (or substrate used) after a fixed time period in an enzyme reaction. The enzyme and substrate are incubated together, then the reaction is stopped using acid, heat, or inhibitors. The final product concentration is measured using methods like spectrophotometry or chromatography. While this method is less affected by small changes in pH, temperature, or substrate levels, it is more sensitive to contamination by other enzymes that may alter the product and affect accuracy.
How can you determine L amino acids in the presesence of D-amino acids?
You use L amino acid oxidase, which will result in L-amino acid to be converted to a keto acid, ammonia, and hyrodgen peroxide and with the uptake of O2. You can then measure the amount of O2 consumption, or determination of ammonia/hydrogen perozide to measure how much L-amino acid.
what enzymes are used in the dairy industry?
- reninin (used in cheese manufacturing to cause caesin molecules to divide and re-coagulate into even larger clumps)
- lipases( in production of roqufort cheese to improve texture and motuhfeel and ehance ripening of blue cheese mold through the breakdown of fats)
- lactases
from measuring the decrease in the substrate concentraion or the increase in product concentration what can you measure?
- enzyme activity
- substrate []
- product []
- effect of inhibtors and activators
- effect of enzyme [], pH, temp on enzymatic activity
