Applied Enzyme Catalysis

1. Applied Enzyme Catalysis Pn Syazni Zainul Kamal School of Bioprocess Engineering ERT211 BIOCHEMICAL ENGINEERING

2. Inside the chapter 1)Survey some of the applications of enzymes • Sources of enzyme • Hydrolytic enzymes and applications 2)Examine immobilized enzyme catalyst formulations • which allow sustained, continuous use of the enzyme.

3. Sources of enzyme • There are three major sources of enzyme a) animal b) plant c) or microbial • Although all living cells produce enzymes, one of the three sources may be favored for a given enzyme or utilization

4. a) Animal sources • Some 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 sources • While some plant enzymes are relatively easy to obtain e.g., papain from papaya, bromelain from pineapple, actinidin from kiwi fruit • their supply is also governed by food demands

6. c) Microbial enzyme • Microbial enzymes are produced by methods which can be scaled up easily • Recombinant 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 life • Example – • 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 splitting • intracellular enzymes, • which are normally confined within individual cells.

9. Enzyme Kinetic • The enzyme kinetics study generally carried out with the purest possible enzyme preparations. Such research involves • the 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 enzymes • Many 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

11. Some industrially important enzymes

15. Hydrolytic enzyme • Enzyme that catalyzes the hydrolysis of a chemical bond • Hydrolytic 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 of • Ester (Esterase) – split ester into acid & alcohol • Glycosidic (carbohydrase) – act on carbohydrate • and various nitrogen bonds – act on proteins and polypetides • Enzymes are named according to the chemical reactions they catalyze, rather than according to their structure.

17. Classification of Hydrolytic Enzymes

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 as • food spoilage • starch thinning, • and waste treatment, • Also in the chemistry of • ripening picked green fruit • self-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 to another group) • There are three major types of amylases- • α-amylase • b-amylase • Amyloglucosidase/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.

23. Glucose Structure

24. a(1-4) glycosidic linkage between the C1 hydroxyl of one glucose and the C4 hydroxyl of a second glucose The b(1-4) glycosidic linkage is represented as a "zig-zag" line, but one glucose residue is actually flipped over relative to the other

25. Starch - polysaccharide of plant - 2 polysaccharide occur together in starch *amylose - α(1-4) glycosidic bond *amylopectin – β(1-6) glycosidic bond

26. Amylopectin Structure

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 bonds • α-amylase is often called the starch-liquefying enzymefor 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, plant e. 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)

34. Common Application of Amylase Preparation

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 depends • on the particular grain • and 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. Invertasehydrolyzes 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 sucrose • and a much higher sugar concentration before hardening.

38. Hydrolysis of Disaccharides • Maltose1. Maltose + H2O -*--> glucose + glucose * = enzyme; in this case maltase Enzymes end in -ase • Sucrose Sucrose + H2O -*-> glucose + fructose * = sucrase • Hydrolysis 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 units • Structural component of all plant cells from algae to tree

40. Hydrolysis of cellulose • cellulase – enzyme that hydrolyze cellulose • Trichoderma 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 products • Exo-b-1,4-cellobiohydrolase (CBH) • Endo-b-1,4-glucanase • b-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-glucanase 2)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

43. Hydrolysis of Cellulose

44. Cellulase sources • Many 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 biomass • Brewing • Waste treatment • Cereal processing • Pulp and paper industries

46. Proteolytic enzymes • Enzyme that catalyze the splitting of protein into smaller peptide fractions and amino acids by a process known as proteolysis • Eg. Proteinase + protein polypeptides polypeptidase + protein amino acids • Some can detach the terminal amino acids from the protein chain • i.e Exopeptidase – aminopeptidase, carboxypeptidase A • Others attack internal peptide bonds of a protein • i.e Endopeptidase – trypsin,pepsin,papain

47. Sources of proteolytic enzyme • Animal, plant, microb • Trypsin – animal pancrease • Papain – papaya • Protease – Bacillus sp., Aspergillus sp.

48. Application of proteolytic enzyme 1) Detergents • Enzyme used in laundry aid as early 1913 • Protease-contain a mixture of bacterial neutral-alkaline protease/lipase active at pH6-10 and 30-60°C • facilitate spot removal, so that it can be wash easily • Since 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 tenderization • eg. Bromelain and papain • breaking the peptide bonds between amino acids found in complex proteins • Meat is held together by a complex protein called collagen • Meat is often tenderized before cooking, to make it less tough and more suitable for consumption • If meat tenderizers are allowed to act for too long, the meat can become squishy and lose its special texture.

50. 3) Tanning • Making leather process from animal skin • Ground pancreases contain digestive proteases eg trypsins, lipases • Use for – dehairing animal hides & removal of noncollagen protein • Environmental friendly rather than using chemical