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 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 Enzymes – General properties Lactose Sucrose 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 Free energy No enzyme Ea A C E Enzyme E-S E+S C Ea E+P Progress of reaction EA 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 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) 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 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 Chemical kinetics 120 100 C h a n g e S 80 1st Order Reaction v = k [S] 60 40 20 0 -20 0 5 10 15 20 Time 25 30 35 40 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: k1 o k2 E + S ↔ E-S ↔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 o Vmax/Km = catalytic efficiency (higher number means more efficient) Enzymes – General properties At low [S] [E] and [S] determine rate A B A 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) ◦ 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 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 Pepsin Trypsin Activity 1 pH 12 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 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 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 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 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 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 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) CO O C H CO O H 3 O CO O C H O O CO O C H O O CO O H 3 O PME 3 O O CO O C H O 3 O O O O O O O O O O O O CO O C H 3 CO O H O Ca C O O C H C O O H 3 O C O O C H O 3 O O O O O O O O C O O H 3 O O C O O C H O O 3 O O O 90C for 1 min O O O CO O C H 3 O O O CO O H O CO O C H 3 O O O CO O H Ca C O O C H CO O HO O O O O O CO O C H 3 OO O O O CO O C H 3 CO O H O O O O O Ca O CO O H O O O O O CO O C H 3 O O O O O Converts colloidal pectin to non-colloidal pectin We add this enzyme when clarity is desired (e.g. apple juice) 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 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 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 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 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 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 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) 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 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 Corn Syrup Glu isomerase pH 7 50-60C 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 42% (HFCS) 52% 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) 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 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?