enzymes. enzymes – general properties all enzymes are proteins that function as biological...
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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
Change in environment
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
Enzymes – General properties
E E-S
E-S#E + P
Binding step
Catalytic step
SucroseLactose
Enzymes – General properties
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
Fre
e e
nerg
y
Progress of reaction
No enzyme
Enzyme
E + S
E-S
E + P
Ea
Ea
A C
A C
E EA
Enzymes – General properties
There are six main types/groups of enzymes classified based on their chemical reaction mechanism1. 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
-20
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35 40
Change
Time
S 1st Order Reactionv = k [S]
P
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 ↔ E-S →E + P
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 substrateo 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)
k1 k2
k-1
Vo = Vmax[ S] oKm + [S ]o
Michaelis-Menten equation
A
A B
Enzymes – General properties
At low [S][E] and [S] determine the rate
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
pH1 12
Activity
PepsinTrypsin
Enzymes – General properties
4. Water activity◦ Water can influence an enzyme in many ways
It can be critical for the SP 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
aw
Activity
0 1
Enzyme 2Enzyme 1
Enzymes – General properties
5. Inhibitors◦ We can use chemical compounds to inhibit or slow
down the activity of enzymes1. Competitive inhibitors
Compete with the substrate for the active site Enzyme can only bind to either S (substrate) or I (inhibitor) at
one time2. 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 water1. 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. PectinasesOccur 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)
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
PME
90C for 1 min
O OO
O O OO
C O O C H 3 C O O H C O O C H 3
O OO
O O OO
C O O C H 3 C O O H C O O C H 3
O OO
O O OO
C O O C H 3 C O O H C O O C H 3
O OO
O O OO
C O O C H 3 C O O H C O O C H 3
O OO
O O OO
C O OH C O OCH3
O
O OO
O O OO
C O OH C O OCH3O
Ca
O OO
O O OO
C O OH C O OCH3
O
O OO
O O OO
C O OH C O OCH3O
Ca
O OO
O O OO
C O OH C O OCH3
O
O OO
O O OO
C O OH C O OCH3O
Ca
O OO
O O OO
C O OH C O OCH3
O
O OO
O O OO
C O OH C O OCH3O
Ca
Enzymes – Important food enzymes3. Proteases
◦ Enzymes that hydrolyze peptide bonds in proteinsA) 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 classesa) 1,3-lipases: preferentially hydrolyze ester bonds at SN1 and
SN3b) 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 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
OIL
Enzymes – Important food enzymes
ISOMERASESThey all catalyze the isomeric
arrangement within a moleculeGlucose isomerase
◦ The most important isomerase for the food industry
◦ Catalyzes isomeric rearrangement of glucose to fructose (converts an aldose to a ketose) Gives a sweeter product than corn syrup
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
Glu isomerasepH 750-60 °C
CornSyrup
(HFCS)50 % Glucose42 % 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 quinones which then react to form brown 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
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?
Polymerizes Melanins