Lesson 10 : EXTRACTION AND EFFECTS OF PH AND TEMPERATURE Flashcards
protein polymers that catalyze chemical reactions to maintain operating rate to sustain life.
Enzymes
a substance the enzymes act on to produce a final product
Substrate
Factors affecting enzymes
substrate and enzyme concentration, pH, temperature, and presence of inhibitor.
proposes a specific substrate for a specific enzyme shape.
Lock & Key Model
proposes that in order to achieve optimal binding upon conformational shape changes of the enzyme and substrate.
Induced Fit Model
optimal temperature for peak enzyme function is at _______; average: _______. If temperature exceeds, enzymes become _________.
35 - 40C
37C
denatured
optimal pH for peak enzyme function is at ___________.
neutral pH: 7
EXTRACTION OF INVERTASE FROM YEAST
● Weigh _____ of ________. Dissolve in ___________
● Let the solution stand for ________ at ____________
● Decant the supernatant if _________ occurs/ Dilute the supernatant to _________ using distilled water
● The resultant supernatant will be used as __________ in the succeeding experiments.
10g of baker’s yeast
30mL of 0.1M NaHCO3
24 hours
room temperature
sedimentation
1:100
enzyme solution
What is the objective of extraction of invertase from yeast
To isolate invertase from yeast
__________ is the enzyme produced by ___________ that hydrolyzes the glycosidic bond of sucrose producing ___________ and ________.
Invertase
Saccharomyces cerevisiae
glucose and fructose
PREPARATION OF DENATURED INVERTASE
● Incubate ______ of __________ in ______ for ___________.
● Let the solution cool
● Collect only ___________ if __________ occurs. Resultant supernatant is used as _________________ in succeeding experiments.
25 mL of enzyme stock solution
boiling water bath for 10 minutes.
supernatants
frothing
denatured enzyme stock solution
EFFECTS OF TEMPERATURE
● Set up __,__,__,__,__,__ and __ water baths
● Prepare six test tubes with __, __, and __ of __, __ . Incubate test tubes separately for _____ in each water bath
● Add ____ to the tubes, incubate test tubes for another ______
● Add ___ of ____. Immerse test tubes in a ___ water bath for ____ to develop a characteristic _____ color.
● Cool test tubes in an ice water bath. Add ____ of ___ to each tube. Mix well.
● Prepare another test tube for the reagent blank containing ____, ___, ___, and ___, ____. Incubate for _____ at ________.
● Repeat steps 4-5
● Measure the absorbance at _____
● Determine the amount of invert sugar produced in each test tube reaction using the invert sugar standard curve constructed in the first experiment.
● Plot the temperature against the amount of the invert sugar
20°C, 30°C, 40°C, 50°C, 60°C,70°C, and 90°C
1mL 0.03M sucrose, 1.4mL distilled water, and 0.5 mL of 0.05M acetate buffer, pH 4.7
5 minutes
0.1mL enzyme solution
5 minutes
2mL of DNS reagent
95°C
10 minutes
red-brown
5mL of distilled water
0.1mL of denatured enzyme, 1mL of the 0.03M sucrose, 1.4mL of distilled water, and 0.5mL of the 0.05M acetate buffer solution, pH 4.7
5 minutes
room temperature
540 nm
PROCEDURE: EFFECTS OF PH
● Prepare ___ numbered test tubes. Add ___ of appropriate ________ as described. Label accordingly.
● To each test tube, add _______ and ________. Mix thoroughly. Incubate all tubes in ___________ for _______
● Add ____ of _______ and incubate the reaction mixture in a ________ for ________
● Add _____ of ______. Immerse the test tube in a __________ for __________ to develop a characteristic __________ color
● Cool test tubes in an ice water bath. Add _____ of ______ to each tube. Mix well.
● Prepare another set of six test tubes for the reagent blank, each containing _____ of _____, ____, and ________. Mix thoroughly. Incubate tubes in ______ for _______
● Repeat steps 3-5
● Measure the absorbance at _________
● Determine the amount of invert sugar produced in each test tube reaction using the invert sugar standard curve constructed in the first experiment.
● Plot the pH against the amount of the invert sugar
six
0.5 mL
0.1M buffer solution
0.10mL enzyme solution
1.4mL of distilled water
60°C water bath
5 minutes
1mL
0.03M sucrose
60 °C water bath
5 minutes
2mL
DNS reagent
boiling water bath
10 minutes
red-brown
5mL of distilled water
0.10mL of denatured enzymes, 1.4mL distilled water, and 0.5mL of buffer.
60°C water bath
5 minutes
540 nm
EFFECTS OF PH & TEMPERATURE ON INVERTASE OBJECTIVE
To determine invertase activity in varying pH and temperature using spectroscopy.
study of absorption and emission of light.
Spectroscopy
an instrument measuring the amount of light that a sample absorbs.
Spectrophotometer
Spectrophotometer measures a substance using the principle of ________ which measures ___________ by its _____, ________ and ___________.
Beer-Lambert Law
absorbance of the substance
light absorptivity, concentration and optical path length
reacts with a reducing sugar from the hydrolysis of invertase
DNS reagent with heat
______, a product of DNS and a reducing sugar = ________ color detected at _____
ANSA
red-brow
540 nm
amount and activity of reducing sugar directly proportional to activity of invertase
Absorbance of light
Most enzymes are __________ that speed up chemical reactions. Some enzymes are RNA catalysts known as __________. Enzymes work by ____________
protein-based catalysts
Ribozymes
lowering the activation energy.
Enzymes have an ________ with a unique __________ that is ______for the _________ that it binds with.
active site
3D shape
specific
substrate
Factors that affect enzyme activity include
pH, temperature, concentration, inhibitors & activators
○ _________- include inorganic metal ions.
○ __________ -include organic molecules like vitamins.
Cofactors
Coenzymes
pH, temperature, concentration, inhibitors &activators. bind to the active site and block the substrate.
Competitive Inhibitors
___________ also called as ____________. It binds into the _________ of the enzyme or somewhere away from the _______ and changes the enzyme’s _______.
Non-competitive Inhibitors
allosteric inhibitors
allosteric site
active site
shape
breaks down proteins or polypeptides into amino acids.
Protease
breaks down fats or lipids like triglycerides into glycerol and fatty acids.
Lipase
catalyzes rearrangement reactions.
Isomerase
catalyzes transfer of functional groups from one molecule to another.
Transferase
transfers phosphate group (particularly from ATP) to another.
Kinase
removes hydrogen atoms from a molecule.
Dehydrogenase
breaks down starch into simple sugars.
Amylase
catalyzes the transfer of electrons
Oxidoreductase
catalyzes hydrolysis reactions.
Hydrolase
