Enzymes

Enzymes – General properties

 Allenzymes 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

Binding step E-S#

E+P Catalytic step

Change in
environment
Enzymes – General properties

Lactose Sucrose
Enzymes – General properties
 Activation energy (Ea) – the minimum energy that reacting molecules must acquire
before they will be converted to the reaction products
 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
◦ Allows biological reactions to occur in mild conditions
 Speed up reactions by 105-108 compared to chemical catalysts; 108-1020 compared
to uncatalyzed reactions

A C

No enzyme A C
Free energy

E EA
Ea
Enzyme
E-S

E+S Ea

E+P

Progress of reaction
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 functional group from a
donor to an 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, carbohydrate modification – e.g.
amylases (cleave glycosidic bonds) and
pectinases (act on several groups/bonds)
 Texture, protein modification – proteases
(cleave the peptide bond)
 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 joining of two molecules
 Not so important in foods
Enzymes – General properties
Factors affecting enzyme activity –
[substrate]
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 (substrate
limiting factor)

There is no added benefit for a

food operation to have too much
enzyme ($$$).

That is why we want to study the

ideal concentration to work at
Chemical kinetics

S 1st Order
Reaction
v = k [S]
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:
o E+S↔ k1 E-S →k2 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 Michaelis-Menten
o Vmax/Km = catalytic efficiency (higher equation
number means more efficient)
Enzymes – General properties

At low [S]
[E] and [S] determine
the rate
A B

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)
◦ It 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
 Usually most active between pH 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 Trypsin
Pepsin
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
E+S → ES + I → ESI
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
 Products are dextrins, maltose and maltotriose
◦ -amylase
 Hydrolyses -1-4 glycosidic bonds from the non-reducing end of
starch
 Product is maltose (if amylose), what about with amylopectin?
◦ Glucoamylase
 Hydrolyses -1-4 and -1-6 (slower) glycosidic bonds in starch
 Possible to hydrolyze all the way to glucose
◦ Pullulanase
 Hydrolyses -1-6 glycosidic bonds in starch (debranching
enzyme)
 These enzymes are naturally present in the food or are
found in microorganisms that are added to 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- glc)
◦ 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). Dextrins are then
hydrolyzed to maltose by -amylase  gives fermentable
sugar for yeast to produce CO2 (essential for rising of the
bread)
◦ On baking (first few minutes around 70 °C) 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 (wheat harvested in
dry climates) – doesn’t contain enough natural -amylase
Brewing
◦ High level of amylases in barley malt (no need to add more)
◦ During mashing (milled barley malt and water at ~ 50 °C)
amylases hydrolyze starch to give maltose for yeast to utilize
and produce CO2 and ethanol
Overview of the Brewing
Process

22
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
 Glucose has sweetness index value of 70, Fructose 170,
Sucrose 100, Lactose 16
 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 crystallization 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 (PME)
 Hydrolyze the methyl ester linkages of pectin
 Causes loss of cloud in citrus juice (big problem)

PME
C O O C H 3 C O O H C O O C H 3
O O O

O O O O O O O O

O O O
C O O CH 3 C O O H C O O CH 3 C O OH C O O C H3
O
O O O Ca
O
O O O C O OH O C O O C H3
O O O O O O O
O O O
O C O OH O C O O C H3 O O
O
C O O C H C O O H C O O C H Ca
3 3 O O O O
O
O O O C O OH C O O C H3

90C for 1 min

O O OO O O
C O OH C O O C H3
O O O O O
O O O O
Ca
O
C O OH C O O C H3
C O O C H 3 C O O H C O O C H 3 O O O
O O O
O O O O

O O O O

 Converts colloidal pectin to non-colloidal pectin

 We add this enzyme when clarity is desired (e.g. apple juice),
also has a minor effect on improving juice yield
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
processing 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 limit extent of proteolysis -
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
 Microbial proteases (A. oryzae, B. subtilis) preferentially
hydrolyze actin and myosin
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
◦ Prevention of this haze formation is called 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
 Produced in the pancreas, present in the intestines as
well
 Can cause quality problems in muscle foods if
contamination from intestines occurs (e.g. ground
products)  over-softening of the meat
 Often used to make protein hydrolysates for the food,
beverage, and pharmaceutical industry
 Most active at pH 7-9
 Can undergo autolysis at pH 8 during storage
◦ Pepsin
 Produced in the mucosal lining of the stomach
 Very acidic activity optima (pH 1- 3)
 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 (calf rennet)
 Very expensive and “inhumane” to process, now the enzyme
has been engineered to be produced by microorganisms
 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 and it aggregates leading
to a clot, the first step in cheese production

 Other proteases can initiate a milk clot like chymosin, but they
would continue casein hydrolysis producing bitter peptides and
eventually breakdown of 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 clots like
chymosin
 A protease cocktail from Aspergillus oryzae is used to
partially break down bread proteins (glutens) thus
reducing mixing time and making the dough more pliable
 Some are added to help with flavor and texture
development and speeding up fermentation in fermented
dairy products
 Fungal proteases can also be used to tenderize meat
◦ Bacterial proteases
 Subtilisin from Bacillus subtilisin is can be used in
combination with papain in beer chillproofing, and for the
production of protein hydrolyzates
Enzymes – Important food enzymes
4. Lipases
◦ Enzymes that hydrolyze bonds between the fatty acids
and the glycerol molecule
 Hydrolyze triglycerides at the water-oil interface in emulsions
◦ 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) Lipases 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 OIL in water but act on a lipid
substrate

The enzyme is located

inside the water
droplet of a water-in-oil
emulsion and acts on
the oil surrounding the
water droplet
Enzymes – Important food enzymes

ISOMERASES Corn
Syrup
 They all catalyze the isomeric
arrangement within a molecule
 Glucose isomerase
◦ The most important isomerase for the food
industry
◦ Catalyzes isomeric rearrangement of glucose Glu isomerase
to fructose (converts an aldose to a ketose) pH 7
 Gives a sweeter product than corn syrup 50-60 °C
 Sweetness glu = 70; fru = 170; sucrose = 100
 Can use less
 Product called high fructose corn syrup (HFCS)
 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
 Adding steps can get you to 55 % frc, 90 % frc
(HFCS)
50 % Glucose
42 % Fructose
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 (methylene
interrupted), 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 to get
stronger doughs
◦ 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 (β-carotene) can be bleached
 Lipoxygenases 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) such as
catechol, chlorogenic acid,
and caffeic acid in the
presence of O2 to give Polymerizes  Melanins
quinones which then react to Undesirable browning of apples,
form brown melanin bananas, mushrooms, shrimp, lobster,
pigments (desirable or human freckles?
undesirable) Up to 50% economic loss of tropical
fruit due to PPO activity
◦ Its activity can be inhibited
by: Desirable browning of tea, coffee,
 Removing O2 cocoa, raisins, prunes, tobacco,
human tan, freckles?
 pH < 4.5 (lemon juice)