BIOCHEMISTRY

LAB - ENZYMES

REAGENT/
TEST OBJECTIVE PRINCIPLE RESULTS
ENZYME/SUBSTRATE
FACTORS AFFECTING ENZYMATIC ACTIVITY
o To determine the o 1% starch o Optimum temp. o COLORLESS =
effect of solution, α- is the point in complete digestion
temperature on amylase, iodine which the o VIOLET/REDDISH =
enzyme activity enzymatic partial digestion
reactions are o DARK BLUE = no
Effect of most active digestion
Temperature o Most chemical
reactions
proceed at a
faster rate as
temp. increases

o To determine the o 1% starch o Enzymes should o COLORLESS =
effect of different solution, α- be kept at its complete digestion
pH buffer amylase, iodine optimum pH in o VIOLET/REDDISH =
Effect of pH
solutions in o buffer solutions order for it to partial digestion
enzyme activity (pH 4, pH 7, and function properly o DARK BLUE = no
pH 10) digestion
o To know the effect o Enzyme = α- o If there is o Blue-black (+)
of different amylase sufficient enzyme
enzyme o Substrate = 1% to bind with
concentrations on starch solution substrate then
the time it takes o Manipulated the reaction will
Effect of Enzyme
for digestion to variable = proceed fast and
Concentration
take place amount of α- if there are
amylase insufficient
enzymes present
then the reaction
will slow down
o To determine the o Enzyme = α- o Increasing o BRICK RED
effect of substrate amylase substrate PRECIPITATE =
concentration in o Substrate and concentration complete digestion
relation to the manipulated increases the (+)
Effect of Substrate enzyme activity variable = starch enzymatic o GREEN, YELLOW,
Concentration o Distilled water activity ORANGE = partial
o Benedict’s o The more starch digestion
reagent solution is added o BLUE = no
the least digestion (-)
digestion occurs
o To determine the o Enzyme = α- o Metal-ion o COLORLESS =
effect of metal-ion amylase poisons are complete digestion
poisons on o Substrate = known to be non- (+)
Effect of Metal-Ion enzyme activity. starch competitive o DARK BLUE = no
Poisons o Manipulated inhibitors of digestion (-)
variable = starch enzymes
solution o Enzymatic
activity depends

o Lead acetate on its tertiary
solution structure
o 1% silver nitrate
solution
o Iodine
PANCREATIC AND BILIARY DIGESTION
o To determine the o Enzymes = α- o When the BENEDICT’S
digestion of amylase, and pancreatic o Blue (-)
amylase and pancreatin amylase binds to o Green (+)
pancreatin & o Substrate = 1% the substrate, it o Brick red
sodium carbonate starch solution catalyzes the precipitate (+)
using the o Benedict’s breakdown of BARFOED’S
Pancreatic
Benedict's and reagent starch o Blue (-)
Amylase
Barfoed’s tests. o Barfoed’s (polysaccharide) o Brick red
reagent into maltose precipitate (+)
(disaccharide)
through
hydrolysis
reaction.
o To mimic digestion o Pepsin o Biuret test o Purple solution (+)
of protein to o Pancreatin o Blue solution (-)
amino acid in the o (Digestive)
Pancreatic
stomach Substrate = egg
Protease
white solution
o CuSO4
o HCl
o To determine the o Vegetable oil o Sodium choleate o pH 6, pH 7, pH 6
effect of lipase in o Pancreatin (bile salts)
Pancreatic Lipase digestion o Sodium choleate emulsifies fats
and Bile o Distilled water and help in
breakdown of
food

ADDITIONAL NOTES:
INTRODUCTION
o Enzymes are proteins that speed up catalytic reactions
o Enzymes are proteins that has the ability to catalyze these reactions. Catalysis is the acceleration of a chemical
reaction by a substance without undergoing permanent chemical change.
o Absolute specificity – only catalyze one reaction
o Group specificity – only on molecules that have a specific functional group
o Linkage specificity – particular type of bond regardless of the molecular structure
o The substrate binds to the active site of the enzyme to produce a product.
o Pepsin is only found in the stomach and digests protein
o Amylase is found in the mouth which digests starch

EFFECT OF TEMPERATURE
o The optimum temperature of α-Amylase is 37 - 40°C
o Enzymes are globular proteins, at high temperatures the shape of the protein specifically the secondary and
tertiary structure is altered

EFFECT OF pH
o Optimum pH is the point where the enzyme is most active. It is considered as the highest rate of reaction
o The proteases (protein enzymes) in a human’s stomach works best in low pH (acidic).
o Extremely high or low pH levels generally result in complete loss of enzymatic activity.
o The enzyme amylase, which is present in saliva, has its maximum activity at pH 7. Enzymes are globular proteins
with a characteristic shape. This shape can alter with changing pH, the result being a decrease in activity.
EFFECT OF ENZYME CONCENTRATION
o Starch contains amylose. When iodine is added, amylose will form coils which will trap iodine and give of a
black-blue color.
o The more α-amylase present, the greater the degree of digestion.
o Amylase is an enzyme that cleaves the α(1,4) glyosidic links, leaving oligosaccharides: maltose and maltriose.
Therefore, the more α-amylase present, the more cleaved bonds which means the more starch is broken down.
o Iodine is not very soluble in water, therefore the iodine reagent is made by dissolving iodine in water in the
presence of potassium iodide.
o When there is an insufficient amount of enzyme present, the reaction will not progress as quick as it would
because the active sites present are occupied. The amount of enzyme concentration is directly proportional to the
rate of reaction.
o Enzymes act as catalysts that make digestion and all metabolic processes work; they are living proteins that direct
the life force into our basic biochemical and metabolic processes.
EFFECT OF SUBSTRATE CONCENTRATION
o So as there are larger amount of starch solution is added to each tube, there is more enzymatic activity thus as
there is lesser amount of substrate present, the faster the starch will be digested by the enzyme.
o Benedict’s reagent is used in the experiment to determine the presence of reducing sugars.
o Enediol is a powerful reducing agent which reduces cupric ions (Cu2+) to cuprous form (Cu+), which is responsible
for the change in color of the reaction mixture.
o Greatest digestion was observed in test tube 1 containing the least amount of starch solution because there are
plenty of active sites available to bind to the substrate following the principle that at low substrate concentration,
the reaction rate is proportional to the substrate concentration.
o Least digestion was observed in test tube 4 containing the greatest amount of starch solution because increasing
the substrate concentration also increases the rate of reaction forming more product which is maltose but once
the maximum rate or point of saturation is reached, any increase will have no effect on the reaction rate and the
enzyme is saturated with the substrate.
o Low amylase could be a factor in diseases that affect blood sugar and many forms of food sensitivities
EFFECT OF METAL-ION POISONS
o Metal ions are known to be examples of non competitive inhibitors. Metal ion poisons attack the -SH (sulfhydryl)
bond in the tertiary structure. The -SH bond is also responsible for the structure’s stability.
o Competitive inhibition – inhibitor binds to the active site of the enzyme and block the substrate’s access to it
o Uncompetitive inhibition – inhibitors only bind to ES complex
o Non-competitive inhibition – inhibitor binds to other location than the active site but it still leads to inhibition
o Irreversible inhibition – binding of an inhibitor to an enzyme which leads to permanent inactivation
o If the tertiary structure of an enzyme is disrupted, the substrate can no longer bind to the active site which leads
to the decrease in the enzymatic activity.
o The α- amylase breaks down starch by hydrolysis in order to yield maltose. The –SH bonds contribute to the
stability of the tertiary structure.

o Heavy metal ions such as silver and lead can react with sulfhydryl group on cysteine, replacing the hydrogen
atom with the heavy metal ion.
o The solution that has water in it yielded a positive (full digestion) result and became colorless.
o Lead acetate bonded with the -SH group thus causing it to denature the enzyme and having a negative (dark
blue) result.
o Silver nitrate yielded a negative result as well because the silver also bonded with the -SH group thus altering its
structure and inhibiting the reaction of enzyme with the substrate.
o The rate of enzyme reaction decreases with increasing concentration of metal-ion poisons.
PANCREATIC AMYLASE
o Benedict’s test is used to detect reducing sugars (carbohydrates having free aldehyde or ketone functional
group).
o Barfoed’s test is used to distinguish monosaccharides from reducing disaccharides.
o The purpose of the sodium carbonate solution in the pancreatic amylase experiment is to stop an enzymatic
reaction, to neutralize the high acidity of the stomach during digestion. It neutralizes the action of digestive and
pancreatic juices which also contains digestive enzymes that contributes in the digestive process in the stomach.
o Salivary amylase is used to act on the raw form of carbohydrates, starch. It is produced in the salivary glands.
o Partial digestion of carbohydrates is already ongoing in the mouth even before food goes into the stomach and
small intestines.
o Pancreatic amylases are produced in the pancreas wherein there are powerful enzymes that help dissolve
carbohydrates in the stomach and small intestines; act more on complex carbohydrates which take longer to
dissolve.
o Sodium carbonate can be used to stop an enzymatic activity
o Starch can be broken down into simple sugars through hydrolysis which involves the addition of a water
molecule.
o If there was a change in color in the Benedict’s test, this implies that there was no digestion that occurred due to
the presence of sugar in the solution.
o In Barfoed’s test, if there was an implication of brick red precipitate, this also means that the starch was not
digested by the α- amylase.
o The negative result may be due to the presence of sodium carbonate solution
PANCREATIC PROTEASE
o HCl or Hydrochloric acid is used to make the solution acidic.
o Pepsin is an enzyme that begins the digestion of proteins. It cuts peptide bonds breaking them down to amino
acids.
o Pancreatin contains the enzyme trypsin which is normally found in the small intestine. Pancreatin is used to
demonstrate that proteins can be digested by trypsin.
o The biuret test is used to detect the presence of peptide bonds.
o In the biuret test, copper forms a violet colored complex in the presence of peptide bonds. This is due to the
formation of a copper (II) ion that complexes in an alkaline solution.
o The peptide bonds were cut via hydrolysis therefore complexation cannot occur.
o Pepsin is a protease produced in the gastric mucosa and secreted into the stomach as a zymogen, pepsinogen.
o HCl is necessary for the activation of pepsinogen to pepsin.
o Pancreatin is a combination of several digestive enzymes produced by the exocrine cells of the pancreas and is
secreted into the lumen of the small intestine.
o The two major pancreatic proteases are trypsin and chymotrypsin. They are synthesized and packaged into
secretory vesicles as the inactive proenzymes trypsinogen and chymotrypsinogen.

o Pancreatic juice is the clear alkaline digestive fluid secreted by the pancreas.
o Pancreatic proteases are better at digesting proteins because pepsin can only digest proteins found in food.
PANCREATIC LIPASE/BILE
o Sodium choleate is a bile salt. This is usually used as a digestive aid in dietary supplements. Bile salts are
produced in the liver and cholecystokinin or CCK triggers its release.
o Pancreatin contains amylase, lipase, and protease that helps in digestion.
o These enzymes produced by the pancreas important for digesting fats, proteins, and sugars.
o Lipase is the enzyme that catalyzes the hydrolysis of into fatty acids and glycerol.
o Temperature affects the reaction rate of enzymes.
o As the temperature increases the rate of reaction increases
o At optimum temperature, the highest rate of reaction occurs because the enzymes are maximally active but the
enzyme activity decreases once it has reached beyond the optimum temperature because at high temperatures,
enzymes and denatured the activity stops. At optimum pH, enzymes work at their maximum capacity.
o It is necessary to break down fats in the digestive system to make it easier to absorb through the membranes of
the intestine and also for it to be it soluble enough to transport in the blood.