2 Inside the chapter 1)Survey some of the applications of enzymes Sources of enzymeHydrolytic enzymes and applications2)Examine immobilized enzyme catalyst formulationswhich allow sustained, continuous use of the enzyme.
3 Sources of enzyme There are three major sources of enzyme a) animalb) plantc) or microbialAlthough all living cells produce enzymes, one of the three sources may be favored for a given enzyme or utilization
4 a) Animal sourcesSome enzymes may be available only from animal sources.Enzymes obtained from animals may be relatively expensive,e.g., rennin obtain from calf's stomach,the value depend on demand of lamb or beef,and their availability.
5 b) Plant sourcesWhile some plant enzymes are relatively easy to obtaine.g., papain from papaya, bromelain from pineapple, actinidin from kiwi fruittheir supply is also governed by food demands
6 c) Microbial enzymeMicrobial enzymes are produced by methods which can be scaled up easilyRecombinant DNA technology now provides the means to produce many different enzymes, including those not normally synthesized by microorganisms or permanent cell lines, in bacteria, yeast and cultured cells.
7 Due to the rapid doubling time of microbes compared with plants or animals microbial processes are attuned more easily to the current market demands for enzymes.On the other hand,for use in food or drug processes, only those microorganisms certified as safe may be exploited for enzyme production.
8 Although most of the enzymes used today are derived from living organisms, they are utilized in the absence of lifeExample –extracellular enzymes,secreted by cells in order to degrade polymeric nutrients into molecules small enough to permeate cell walls.Grinding, mashing, lysing, or otherwise killing and splittingintracellular enzymes,which are normally confined within individual cells.
9 Enzyme KineticThe enzyme kinetics study generally carried out with the purest possible enzyme preparations.Such research involvesthe fewest possible number of substrates (one if achievable)a controlled solution with known levels of activators (Ca2+, Mg2+,pH etc.),cofactors,and inhibitors.
10 Industrial enzymesMany useful industrial enzyme preparations are not highly purified.They contain a number of enzymes with different catalytic functions and are not used with either a pure substrate or a completely defined synthetic medium.Also, the simultaneous use of several different enzymes may be more efficient than sequential catalysis by a separated series of the enzymes.such enzyme preparations are kinetically more simple than the integrated living organisms from which they are produced
15 Hydrolytic enzymeEnzyme that catalyzes the hydrolysis of a chemical bondHydrolytic enzymes are normally associated with degradative reactions, (break down large molecules into small molecules) e.g.,conversion of starch to sugar,proteins to polypeptides and amino acids,and lipids to their constituent glycerols, fatty acids and phosphate bases
16 Hydrolytic enzymes 3 major group of hydrolytic enzyme Those involved in the hydrolysis ofEster (Esterase) – split ester into acid & alcoholGlycosidic (carbohydrase) – act on carbohydrateand various nitrogen bonds – act on proteins and polypetidesEnzymes are named according to the chemical reactions they catalyze, rather than according to their structure.
18 Since One-enzyme – one-reaction uniqueness does not generally exist, Enzymes from different plant or animal sources which catalyze a given reaction will not always have the same molecular structure or necessarily the same kinetics.Consequently,maximum reaction rate,Michaelis constant,pH of optimum stability or activity,and other properties –depend on the particular enzyme source used.
19 Application of hydrolytic enzymes In macroscopic degradations such asfood spoilagestarch thinning,and waste treatment,Also in the chemistry ofripening picked green fruitself-lysis of dead whole cells (autolysis),desirable aging of meat,curing cheeses,preventing beer haze,texturizing candies,treating wounds,and desizing textiles.
20 Application of Hydrolytic Enzymes In eucaryotes, hydrolases may be stored inside the cell in membrane-enclosed lysosome organelles, reside in the periplasm in microbes like yeast,or be secreted into the environment.Most hydrolytic enzymes used commercially are extracellular microbial products.
21 Hydrolysis of starch Carbohydrase Amylases - extensively applied enzymes- can hydrolyze the glycosidic bonds in starch and related glucose-containing compounds (eg. Cellulose).*(glycosidic bond – join carbohydrates toanother group)There are three major types of amylases-α-amylaseb-amylaseAmyloglucosidase/glucoamylase
22 Starch contains straight-chain glucose polymers called amylose and a branched component known as amylopectin.The branched structure is relative more soluble than the linear amylose and is also effective in rapidly raising the viscosity of starch solution.
24 The b(1-4) glycosidic linkage is represented as a "zig-zag" line, but one glucose residue is actually flipped over relative to the othera(1-4) glycosidic linkage between the C1 hydroxyl of one glucose and the C4 hydroxyl of a second glucose
25 Starch - polysaccharide of plant - 2 polysaccharide occur together in starch*amylose - α(1-4) glycosidic bond*amylopectin – β(1-6) glycosidic bond
27 Hydrolysis of starch α-amylase The action of α-amylase reduces the solution viscosity by acting randomly along the glucose chain at α-1,4 glycosidic bondsα-amylase is often called the starch- liquefying enzyme for this reason.
28 Hydrolysis of starch β-amylase b-Amylase can attack starch a-1,4 glycosidic bond only on the nonreducing ends of the polymer and always produces maltose when a linear chain is hydrolyzed.Because of the characteristic production of the sugar maltose, b-amylase is also called a saccharifying enzyme.soluble mixture of starch and b-amylase yields maltose and a remainder of dextrins (starch remnants with 1,6- linkage on the end)
32 Hydrolysis of starch amyloglucosidase Another saccharifying enzyme, amyloglucosidase (also called glucoamylase) attacks primarily the nonreducing a-1,4 linkages at the ends of starch, glycogen, dextrins, and maltose. (a-1,6 linkages are cleaved by amyloglucosidase at much lower rates)Sequential treatment with a-amylase and glucoamylase or enzyme mixtures are utilized where pure glucose rather than maltose is desired,e.g., in distilleries and in the manufacture of glucose syrups (corn syrup) and crystalline glucose.
33 Sources of amylase The sources of amylases are very numerous Amylases are produced by – microb, plante. g., 1) amylase produced by Clostridium acetobutylicum which is clearly involved in the microbial conversion of polysaccharides to butanol and acetone.2) amylase produced by Aspergillus niger, Penicillium sp.3) amylase from Bacillus used in clothing and dishwasher detergent(amylase from microb, not suitable to be used in food industry)
35 Application of Amylase Commercial amylase preparations used in human foods are normally obtained from grains,e.g., barley, wheat, rye, oats, maize, sorghum, and rice.The ratio of saccharifying to liquefying enzyme activity dependson the particular grainand upon whether the grain is germinated.
36 Application of Amylase In the production of malt for brewing, the ungerminated seeds are exposed to a favorable temperature and humidity so that rapid germination occurs, with resulting large increase in a-amylase.The germinated barley is then kiln-dried slowly;this halts all enzyme activity without irreversible inactivation.The dried malt preparation is then ground, and its enormous liquefying and saccharifying power is utilized in the subsequent yeast fermentation.to convert starches to fermentable sugars.
37 Invertase hydrolyzes sucrose and polysaccharides containing a b-D-fructofuranosyl linkage. The hydrolyzed sucrose solution containing fructose and glucose rotates a polarized light beam in the direction opposite that of the original solution.The partially or completely hydrolyzed solution allows two properties desirable in syrup and candy manufacturing:a slightly sweeter taste than sucroseand a much higher sugar concentration before hardening.
38 Hydrolysis of Disaccharides Maltose 1. Maltose + H2O -*--> glucose + glucose * = enzyme; in this case maltase Enzymes end in -aseSucrose Sucrose + H2O -*-> glucose + fructose * = sucraseHydrolysis of Lactose Lactose + H2O -*-> galactose + glucose * = lactase
39 Hydrolysis of Cellulose Cellulose – polysaccharide consisting of a linear chain of several hundred to over ten thousand β-1,4 linked D-glucose unitsStructural component of all plant cells from algae to tree
40 Hydrolysis of cellulose cellulase – enzyme that hydrolyze celluloseTrichoderma fungi are commonly used at the present time.They are thoroughly developed and characterized at present.There are three major classes of enzymes for different substrates and productsExo-b-1,4-cellobiohydrolase (CBH)Endo-b-1,4-glucanaseb-glucosidase
41 Hydrolysis of cellulose 3 steps of reaction catalyze by cellulase :1)Breakage of non-covalent interactions present in the crystalline structure of cellulose by endo-β-1,4-glucanase2)Hydrolysis of individual cellulose fibers to break it into smaller sugars (cellobiose) by exo-β-1,4- cellobiohydrolase (CBH)3)Hydrolysis of dissacharides or tetrasaccharides into glucose by β-glucosidase
44 Cellulase sourcesMany other microorganisms including the molds bacteria produce cellulases with distinctive activities and properties. e.g.-Fusarium solani,Aspergillus niger,Penicillium funicolsum,Sporotrichum pulverulentum,Cellulomonas species,Clostridium thermocellum,and Clostridium thermosaccharolyticum
45 Applications of cellulase Alcohol fermentation from biomassBrewingWaste treatmentCereal processingPulp and paper industries
46 Proteolytic enzymesEnzyme that catalyze the splitting of protein into smaller peptide fractions and amino acids by a process known as proteolysisEg. Proteinase + protein polypeptidespolypeptidase + protein amino acidsSome can detach the terminal amino acids from the protein chaini.e Exopeptidase – aminopeptidase, carboxypeptidase AOthers attack internal peptide bonds of a proteini.e Endopeptidase – trypsin,pepsin,papain
48 Application of proteolytic enzyme 1) DetergentsEnzyme used in laundry aid as early 1913Protease-contain a mixture of bacterial neutral-alkaline protease/lipase active at pH6-10 and 30-60°Cfacilitate spot removal, so that it can be wash easilySince one enzyme molecule can act on many substrate (i.e., soil) molecules, a small amount of enzyme added to a laundry detergent can provide a big cleaning benefit to the consumer
49 2) Meat tenderizationeg. Bromelain and papainbreaking the peptide bonds between amino acids found in complex proteinsMeat is held together by a complex protein called collagenMeat is often tenderized before cooking, to make it less tough and more suitable for consumptionIf meat tenderizers are allowed to act for too long, the meat can become squishy and lose its special texture.
50 3) TanningMaking leather process from animal skinGround pancreases contain digestive proteases eg trypsins, lipasesUse for – dehairing animal hides & removal of noncollagen proteinEnvironmental friendly rather than using chemical
51 4) Dairy industries- Coagulation of milkRennin remove glucopeptide from soluble calcium casein to yield paracaseinateParacaseinate precipitate to form curdCurd further process to make cheese
55 lipase Most important enzyme in esterase group Hydrolyze triglycerides into diglycerides, monoglycerides, fatty acids and glycerolsApplications in food, detergent, pharmaceuticals, leather, textile, cosmetic
56 Applications of lipase 1)Dairy industriesLipase used for hydrolysis of milk fatCurrent applications ; flavour enhancement of cheese, acceleration of cheese ripening (lipase degrade protein, fat, lactose)Addition of lipase to cow’s milk, generate flavour similar to that of ewe’s 0r goat’s milk
57 2)DetergentsLipase add to detergentsRemove fat and oils based stains3) Lipase in oleochemical industryBefore this used organic solvent and emulsifier in oleochemical industryNow using immobilized lipase from Candida cylindracea in production of soapResulted in high productivity & continuous running of the processReduce cost for expensive equipment & thermal energy
58 4) Meat processing industry to produce fat free meatsPartial fat hydrolysis of the meat cut using lipase5) Pulp & paper industryDeinking – removal of ink process from surface of paperConventional method – used chemical to remove ink ; cause water pollution & high costDeinking by enzyme – Lipase used to remove oil based ink
59 Enzyme mixtures Mixture of enzymes may contain : a) same general type = α- & β- amylase and amyloglucosidaseb) Different type = found in pancreas extract (trypsin, lipase, amylase)are often used more successfully than single enzyme preparationseg. Blend of diff. amylase yields large amount of saccharified starch suitable for yeast fermentation
60 Other applications of enzyme in solution Hydrolysis enzymes applications dominate past and present enzyme technologyOther enzyme processes currently serve important function in food, pharmaceutical and biochemical industries
61 Medical applications of enzymes Recently free or extracellular enzymes were used in medicine1)Lysozyme(in nasal mucus, saliva, tears)It hydrolyze mucopolysaccharides of bacterial cell walls.Used as an antibacterial agent, treatment of ulcer, skin disease
62 2) AsparaginaseUsed as anticancer agentEnzyme that catalyze hydrolysis of asparagine to aspartic acidSome cancer cells require asparagine (nurient), their growth can be inhibited using asparaginaseCan be given to patient as intramuscular, subcutaneous or intravenous injection (differ from other chemo agent, no tissue irritation)
63 3)Penicillinase1st isolated from gram negative bacteria E.colihuman beings do not produce penicillinaseRemove allergenic form of penicillin from allergic individualsConvert the drug into nonallergic form