Enzymes

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 Binding step E-S# E + P Catalytic step Change in environment

4. Enzymes – General properties Lactose Sucrose

5. 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 A C No enzyme E EA Ea Free energy Enzyme E-S Ea E + S 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 functional group from a donor to an 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, 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)

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 joining of two molecules • Not so important in foods

9. 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

10. Chemical kinetics P 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 • Vmaxgives 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 Michaelis-Menten equation

12. Enzymes – General properties At low [S] [E] and [S] determine the 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) • It 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 • 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 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 0 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

17. E+S → ES + I → ESI

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 • 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

20.  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 • Food importance of the glycoside hydrolases

21. 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

22. Overview of the Brewing Process

23. 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

24. 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) • 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

26. 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

27. 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

28. 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

29. 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

30. 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 Aspergillusoryzae 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

31. 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

32. 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)

34. 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 OIL The enzyme is located inside the water droplet of a water-in-oil emulsion and acts on the oil surrounding the water droplet

35. Enzymes – Important food enzymes Corn Syrup ISOMERASES • They all catalyze the isomeric arrangement within a molecule • Glucose 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 Gluisomerase pH 7 50-60 °C (HFCS) 50 % Glucose 42 % Fructose

36. 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)

37. 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

38. Enzymes – Important food enzymes • B) Polyphenoloxidase (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 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?