Transcript Enzymes - Food Science & Human Nutrition

Enzymes
Enzymes – General properties

All enzymes are proteins that function as biological
catalysts
◦ They are essential for reactions to occur in living (and dying) cells
◦ They can have a dramatic impact on the quality deterioration of
many foods
◦ They can be used to perform positive reactions in food and
beverage processing (also textile, leather and pharmaceutical
industry)
 Have specific ability to convert a particular substance into a particular
product
 Very rapid action
 Only need small amount
 Can easily control them by adjusting their environment, e.g. pH, T,
concentration…..
 They are all natural and non-toxic
Enzymes – General properties
Enzymes have one (or more) active sites in their structure that
have great specificity for certain substrates (bind only to these)
and catalyze their transformation into specific products
E
E-S
E-S#
E+P
Change in
environment
Enzymes – General properties
Lactose
Sucrose
Enzymes – General properties
The reason enzymes are able to speed up chemical reactions so much comes from
their ability to bind to their substrates with very high specificity and significantly
lower the activation energy (Ea) of the reaction converting a substrate to a product
 Speed up reactions by 103-1011 compared to other catalysts; 108-1020 compared to
uncatalyzed reactions

A
Free energy
No enzyme
Ea
A
C
E
Enzyme
E-S
E+S
C
Ea
E+P
Progress of reaction
EA
Enzymes – General properties

There are six main types/groups of enzymes classified based
on their chemical reaction mechanism
1. Oxidoreductases
 Catalyze oxidations or reductions of substrates
 Some important food reaction examples:
 Lipid oxidation – lipoxygenase (adds an oxygen on
fatty acids)
 Browning – polyphenol oxidase (oxidizes phenols in
food)
2. Transferases
 Catalyze a shift of a chemical group from a donor to
acceptor substrate
 Not so important in foods
Enzymes – General properties
3. Hydrolases
 Catalyze the hydrolysis (with help of water) of substrates
(i.e. breaking of bonds)
 By far the most important enzymes with respect to food
quality and use in food processing
 Some important food reaction examples:
 Texture, protein modification – proteases (cleave the
peptide bond)
 Texture, carbohydrate modification – e.g. amylases
(cleave glycosidic bonds) and pectinases (act on several
groups/bonds)
 Hydrolytic rancidity, fat crystallization modification –
lipases (cleave ester bonds)
Enzymes – General properties
4. Lyases
 Catalyze the removal or addition of chemical groups to
substrates
 Not so important in foods
5. Isomerases
 Catalyze intramolecular rearrangements
 An important food reaction example:
 Sweetness (Glu  Fru) – glucose isomerase (converts
aldose to ketose)
6. Ligases
 Catalyze combinations of substrates
 Not so important in foods
Enzymes – General properties
Factors affecting enzyme activity
1. Enzyme and substrate concentration
◦ When substrate concentration is kept constant the enzyme reaction
is proportional to the amount of enzyme (i.e. doubling enzyme will
double the speed of the reaction) up to a certain limit
Here you start to have more
enzyme than substrate and rate
starts to level off
There is no added benefit for a
food operation to have too much
enzyme ($$$).
That is why we have to study the
ideal concentration to work at
Chemical kinetics
120
100
C
h
a
n
g
e
S
80
1st Order Reaction
v = k [S]
60
40
20
0
-20
0
5
10
15
20
Time
25
30
35
40
Enzymes – General properties
o Increasing the substrate concentration under
fixed enzyme concentration leads to a non-linear
increase in reaction velocity that can be explained
by the formation of the Enzyme-Substrate
complex:
k1
o
k2
E + S ↔ E-S ↔E + P
k-1
o This reaction curve is shared by most enzyme and
gives us very useful information on the activity of
the enzyme and the affinity for its substrate
o Vmax gives us the maximum velocity that the
enzyme can produce (under the conditions
tested) – the higher the faster
o Km (determined as ½ Vmax) tells us the affinity
of the enzyme for its substrate
o Vmax/Km = catalytic efficiency (higher number
means more efficient)
Enzymes – General properties
At low [S]
[E] and [S] determine rate
A
B
A
At high [S]
[E] determines rate
Enzymes – General properties
2. Temperature
◦ Enzyme reactions increase with
temperature up to a point and
then activity declines as the
enzyme becomes denatured
◦ Different enzymes have different
temperature optima's (the point
when max activity is)
◦ Important to determine this to be
able to predict what type of
thermal treatment you need in
processing to inactivate
undesirable enzymes
Enzymes – General properties
3. pH
◦ All enzymes have a certain narrow range of pH where they perform
best
 Most active between 4.5-8
 Some active at very low (e.g. pepsin) or high pH
◦ Extremes of pH can affect the enzyme by denaturing it (remember it is
a protein) or affecting the charge of critical amino acids in its active site
(or charge on the substrate)
◦ For this reason pH control of foods with undesirable enzymes is
important
Pepsin
Trypsin
Activity
1
pH
12
Enzymes – General properties
4. Water activity
◦ Water can influence an enzyme in many ways
 It can be critical for the SP reaction (e.g. hydrolysis)
 It can be critical to solubilize the substrate and product
 It can be critical for the flexibility of the enzyme structure
◦ Water activity can be varied in foods to slow down enzymatic activity
Enzyme 1
Enzyme 2
Activity
0
aw
1
Enzymes – General properties
5. Inhibitors
◦ We can use chemical compounds to inhibit or slow down the
activity of enzymes
1. Competitive inhibitors
 Compete with the substrate for the active site
 Enzyme can only bind to either S (substrate) or I (inhibitor) at one time
2. Non-competitive inhibitors
 Bind to enzyme at another site than active site
 Enzyme can bind to both S and I at the same time
3. Un-competitive inhibitors
 Can only bind to the E-S complex (the intermediate state)
 Enzyme binds first to S and then can bind to I
◦ These can be reversible or irreversible
◦ Some food use for these but many have flavor, odor, color and
toxicity problems, plus can be very expensive
Enzymes – Important food enzymes
HYDROLASES
 They all have in common that they break bonds with the help of
water
1. Glycoside hydrolases
A) Enzymes that hydrolyze starch (glycosidic bonds)
◦ -amylase
 Hydrolyses -1-4 glycosidic bonds within starch
 Results in dextrins, maltose and maltotriose
◦ -amylase
 Hydrolyses -1-4 glycosidic bonds from the non-reducing end of starch
 Results in maltose
◦ Glucoamylase
 Hydrolyses -1-4 and -1-6 glycosidic bonds in starch
 Can hydrolyze all the way to glucose
◦ Pullulunase
 Hydrolyses -1-6 glycosidic bonds in starch

These enzymes are naturally present in the food or are found in
microorganisms added to the food
Enzymes – Important food enzymes

Food importance of the glycoside hydrolases
Corn syrup production
◦ Using a cocktail of enzymes
starch can be converted to a
glucose syrup (dextrose)
◦ Start with -amylase to break
amylose and amylopectin to
smaller units
◦ Then use glucoamylase to break
down to glucose
◦ If maltose is desired use amylase and pullulanase
Enzymes – Important food enzymes
Baking
◦ -amylases are important to “dextrinize” the disrupted starch granules
(rupture during milling) and the dextrins are then hydrolyzed to
maltose by -amylase  gives fermentable sugar for yeast to produce
CO2 (essential for rising of the bread)
◦ On baking there is further action of the amylases on the gelatinized
starch  plays an important role in the final texture and quality of
bread
◦ Amylases added to bakery products can minimize staling
◦ Need to add -amylases to some flours (what harvested in dry
climates)
Brewing
◦ High level of amylases in barley malt (no need to add more)
◦ During mashing (milled barley malt and water at 50C) amylases
hydrolyze starch to give maltose for yeast to utilize and produce CO2
and ethanol
Enzymes – Important food enzymes
B) Invertase
◦ An enzyme that hydrolyzes the glycosidic bond between
glucose and fructose in sucrose
 Results in invert sugar (free glu and fru)
 Popular in the confectionary industry because invert sugar
is sweeter than sucrose and has less tendency to
crystallize
 Popular in soft candy fillings
C) Lactase
◦ An enzyme that hydrolyses the glycosidic bond between
galactose and glucose in lactose
 Increases sweetness and solubility of the sugar
 Done in the dairy industry to minimize crystallinization in
ice cream and to produce lactose free products
Enzymes – Important food enzymes
2. Pectinases
 Occur widely in fruits and vegetables and are responsible
for the degradation of pectic substances
◦ Pectin methyl esterase
 Hydrolyze the methyl ester linkages of pectin
 Causes loss of cloud in citrus juice (big problem)
CO O C H
CO O H
3
O
CO O C H
O
O
CO O C H
O
O
CO O H
3
O
PME
3
O
O
CO O C H
O
3
O
O
O
O
O
O
O
O
O
O
O
O
CO O C H 3
CO O H
O
Ca
C O O C H
C O O H
3
O
C O O C H
O
3
O
O
O
O
O
O
O
O
C O O H
3
O
O
C O O C H
O
O
3
O
O
O
90C for 1 min
O
O
O CO O C H 3
O
O
O
CO O H
O
CO O C H 3
O
O
O CO O H
Ca
C O O C H
CO O HO
O
O
O
O
O
CO O C H 3
OO
O
O
O
CO O C H 3
CO O H
O
O
O
O
O
Ca
O
CO O H
O
O
O
O
O
CO O C H 3
O
O
O
O
O
 Converts colloidal pectin to non-colloidal pectin
 We add this enzyme when clarity is desired (e.g. apple juice)
Enzymes – Important food enzymes
3. Proteases
◦ Enzymes that hydrolyze peptide bonds in proteins
A) Papain
◦ Found in papaya
◦ Broad pH (3-11) and temperature stability
 For this reason very popular for a variety of food applications
1. Used as a meat tenderizer on inferior meat cuts (can also use slice of
pineapple on meat)
 The enzyme makes its way into the muscle and hydrolyzes
primarily connective tissue proteins (collagen etc.) and softens
muscle
 Have to use low amount to prevent liquefaction of muscle
 If you mix raw papaya into Jell-O it will not form a gel
 Other popular tenderizing enzymes are ficin (from figs),
bromelain (from pineapple) and microbial proteases
Enzymes – Important food enzymes
2. Papain can also be used to clear turbidity (chill haze) in beer
◦ When bottled or canned beer is kept below 10C (50F) a haze can form
 Interactions of proteins/polypeptides and tannins in beer
◦ This can be prevented using chill-proofing
 Protease (papain mostly used) added during post-fermentation
maturation to hydrolyze the proteins/polypeptides to prevent large
aggregates to form on cooling
Enzymes – Important food enzymes
B) Digestive proteases
◦ Trypsin & Chymotrypsin
 Found in animal pancreas
 Can cause quality problems in muscle foods if contamination
from intestines occurs (e.g. ground products)  over-softening of
the meat
 They are often used to make protein hydrolysates for the food,
beverage and the pharmaceutical industry
 Most active at pH 7-9
◦ Pepsin
 Very acidic activity optima (pH 1.8)
 Somewhat limits its use
 Used in cheese making, chillproofing and also in making protein
hydrolysates
 animal and fertilizer use primarily, some for food use
Enzymes – Important food enzymes
◦ Chymosin (rennin)
 Essential for the manufacture of good quality cheeses
 Found in the fourth stomach of suckling calf's
 Very expensive and “inhumane” to process now so it has been
engineered into a bacteria that mass produces it
 Has a very specific activity
 Hydrolyzes only one bond in к-casein, one of the many proteins that
make up the milk casein protein complex (к-, -, -casein)
 This breaks up the casein complex (micelle) and it aggregates leading
to a clot, the first step in cheese production
 Most other proteases can initiate a milk clot like chymosin but they
would continue the casein hydrolysis producing bitter peptides and
eventually breaking the clot
Enzymes – Important food enzymes
C) Microbial proteases
◦ Several fungal and bacterial proteases are used in the food industry
◦ Fungal proteases
 Some have almost equal ability to form cheese cloths like chymosin
 A protease cocktail from Aspergillus oryzae is used to partially break
down bread proteins (glutens) to reducing mixing time and making
them more extensible
 Some are added to help with flavor and texture development and
speeding up fermentation in fermented dairy products
 Fungal proteases are also used to tenderize meat
◦ Bacterial proteases
 Subtilisin from Bacillus subtilisin is popular and is used in combination
with papain in beer chillproofing
Enzymes – Important food enzymes
4. Lipases
◦ Enzymes that all hydrolyze ester bonds between fatty acids and a
glycerol molecule
 Work at the water-oil interface
◦ Two classes
a) 1,3-lipases: preferentially hydrolyze ester bonds at SN1 and SN3
b) 2-lipases preferentially hydrolyze ester bonds at SN2
Enzymes – Important food enzymes
◦ Lipases have a dramatic impact on the quality of food products
A) Lead to hydrolytic rancidity
 BAD when
 Free fatty acids released in muscle foods and react to proteins to
denature them and give a tough texture (happens on freezing
muscle)
 they are not inactivated in milk; release short chain fatty acids that
are very volatile and can also oxidize
 GOOD when
 Used in fermented products
 Extremely important in ripening of cheeses and dry-sausages
 Short chain fatty acids released from milk fat produces the
characteristic odor and flavor of these products (C:8 especially)
Enzymes – Important food enzymes
B) They can be used to modify the properties of lipids
 Very popular application in the margarine industry to modify lipid crystal
structure to give different textures and melting points
 Also used to produce mono and diglycerides for use as emulsifiers
 A very unique reaction system must be used for these enzymes since they are
soluble in water but act on a lipid substrate
The enzyme is located
in the water droplet of
a water-in-oil emulsion
and acts on the oil
surrounding the water
droplet
Enzymes – Important food enzymes
ISOMERASES
 They all catalyze the intramolecular
arrangement within a molecule
 Glucose isomerase
◦ The most important for the food industry
◦ Catalyzes isomeric rearrangement of glucose to
fructose (converts an aldose to a ketose)
 Gives a sweeter product than corn syrup
Corn
Syrup
Glu isomerase
pH 7
50-60C
 Sweetness glu = 70; fru = 170; sucrose = 100
 Product called high fructose corn syrup
 Made from corn syrup (which is made by amylase
digestion of starch)
 Enzymes are immobilized in large columns where
the reaction takes place – can reuse them
42% (HFCS) 52%
Enzymes – Important food enzymes
OXIDOREDUCTASES

Enzymes that catalyze the oxidation or
reduction of substrates
A) Lipoxygenase
◦ Found in a wide variety of plants
(primarily legumes) and have also been
identified in animal tissue (e.g. in the
skin of fish)
◦ Specific for the oxidation of fatty acids
that have a cis, cis penta-1,4-diene unit,
so there are three naturally occurring
fatty acids that can be substrates
 Linoleic acid (2 double bonds)
 Linolenic acid (3 double bonds)
 Arachidonic acid (4 double bonds)
Enzymes – Important food enzymes

Importance of lipoxygenases in foods
◦ Desirable
 The enzyme plays a role in bleaching of wheat and soybean flours
 It contributes to the formation of S-S bonds in gluten in dough, thus one
does not have to add chemical oxidizers
◦ Undesirable
 Lipid oxidation and reactions of its products
 Breakdown products of hydroperoxides give off-flavors and odors
 Oxidation products (the free radicals or hydroperoxide) can bind and/or
oxidize proteins to lead to textural problems
 Lipid oxidation also leads to nutritional loss of essential polyunsaturated fatty
acids
 Vitamins may also be oxidized by the oxidation products
 Chlorophylls and carotenes can be bleached
 Its action can be effectively delayed by using antioxidants
Enzymes – Important food enzymes
B) Polyphenol oxidase (PPO)
◦ Found in plants (fruits and
vegetables), animals (including
humans), insects and microbes
◦ Catalyzes the oxidation of phenolic
compounds (mono and/or
diphenols) in the presence of O2 to
give quinones which polymerize into
melanin pigments (desirable or
undesirable)
◦ Its activity can be inhibited by:
 Removing O2
 pH < 4.5 (lemon juice)
 Ascorbic acid (vit-C) (again,
lemon juice)
 Bi-sulfites
 EDTA
Polymerizes  Melanins
Undesirable browning of apples,
bananas, mushrooms, shrimp, lobster,
human freckles?
Up to 50% economic loss of tropical
fruit due to PPO activity
Desirable browning of tea, coffee,
cocoa, raisins, prunes, tobacco,
human tan, freckles?