1. Enzymes

2. 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

3. 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

4. Enzymes – General properties
Lactose
Sucrose

5. 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 compared to other catalysts; compared to uncatalyzed reactions A C
No enzyme A C E EA Ea
Free energy
Enzyme
E-S
E + S Ea
E + P
Progress of reaction

6. 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

7. 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)

8. 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

9. 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

10. Chemical kinetics S
1st Order Reaction
v = k [S]

11. 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

12. Enzymes – General properties
At low [S]
[E] and [S] determine rate
At high [S]
[E] determines rate

13. 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

14. 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

15. 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 1

16. 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

19. 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

20. 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

21. 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

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
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

23. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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)

33. 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

34. 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
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
Corn
Syrup
Glu isomerase
pH 7
50-60C
42% (HFCS) 52%

35. 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)

36. 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

37. 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?