Enzymes

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Enzymes
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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

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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
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Enzymes General properties
Sucrose
Lactose
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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
C
No enzyme
E
EA
Ea
Free energy
Enzyme
E-S
Ea
E S
E P
Progress of reaction
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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

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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)

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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

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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
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Chemical kinetics
S
1st Order Reaction v k S
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Enzymes General properties

• 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
• E S ? E-S ?E P
• 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
• Vmax gives us the maximum velocity that the
  enzyme can produce (under the conditions tested)
  the higher the faster
• Km (determined as ½ Vmax) tells us the affinity
  of the enzyme for its substrate
• Vmax/Km catalytic efficiency (higher number
  means more efficient)

k1
k2
k-1
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Enzymes General properties
At low S E and S determine rate
At high S E determines rate
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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

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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

Trypsin
Pepsin
Activity
pH
1
12
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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
aw
0
1
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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

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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

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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

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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

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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

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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)
• Converts colloidal pectin to non-colloidal pectin
• We add this enzyme when clarity is desired (e.g.
  apple juice)

PME
90C for 1 min
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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

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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

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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

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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 ?-casein, one of the
  many proteins that make up the milk casein
  protein complex (?-, ?-, ?-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

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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

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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

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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 (C8 especially)

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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
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Enzymes Important food enzymes
Corn Syrup

• 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
• 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

Glu isomerase pH 7 50-60C
42 (HFCS) 52
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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)

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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

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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 lt 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?
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