Enzim Protease

1. Enzim Protease

2. Protease • Enzim protease adalah enzim yang memotong ikatan peptida pada polipeptida menjadi peptida-peptida sederhana atau asam-asam amino bebas. • Ikatan peptida adalah ikatan yang terjadi antara gugus amino dari asam amino pertama dengan gugu karboksil dari asam amino kedua.

3. Protease • Protease serin adalah enzim yang mempunyai residu serin pada sisi aktifnya, contohnya: tripsin, kimotripsin dan elastase. • Protease sulfhidril (protease thiol) adalah protese yang aktivitasnya tergantung adanya satu atau lebih residu sulfhidril pada sisi aktifnya.

4. Protease • Protease logamadalah protease yang aktivitasnyasangattergantungpadaadanya ion logampadasisiaktifnya. Enziminimembutuhkan Zn2+ untukaktivitasnya, danmembutuhkan Ca2+ untukmentabilkannya. • Protease asamadalah protease yang aktivitasnyadisebabkanadanyaduaguguskarboksilpadasisiaktifnya. Contohnya: pepsin, renin, danenzimdarikapang.

5. Digestive Proteses • Panceatic protease: trypsin, chymotrypsin, carboxypeptidase, aminopeptidase, elastase. • Gastric protease: pepsin, rennin.

6. Endopeptidase • Enzyme that catalyzes the cleavage of peptide bonds within a polypeptide or protein. • Example: pepsin, trypsin, chymotrypsin,

7. Exopeptidase • Enzyme that catalyzes the removal of an amino acid or dipeptidefrom the end of a polypeptide chain. • Example: carboxypeptidase, aminopeptidase.

8. Struktur primer protein Eksopeptidase Endopeptidase Eksopeptidase Endopeptidase Endopeptidase

9. Serine Proteses • The active site of serine proteases contains three critical amino acids: serine (S), histidine (H) and aspartate (D). • Included within this collection are three serine proteases: chymotrypsin, elastase and trypsin.

10. Metal-containing enzymes • Metalloenzymes are proteins which function as an enzyme and contain metals that are tightly bound and always isolated with the protein. • Hemoglobins and cytochromes contain Fe2+ or Fe3+, • Phosphotransferases, containing Mg2+; • Alcohol dehydrogenase, containing Zn2+; • Arginase, containing Mn2+; • Ferredoxin, containing Fe2+; • Cytochrome oxidase, containing Cu2+, • Zinc is the metal incorporated in carboxypeptidase.

11. Trypsin • Trypsinogen, the proenzyme (zymogen) form of trypsin, is produced in the acinar exocrine cells of the pancreas. • Enzyme Commission (EC) Number: 3.4.21.4 • Molecular Weight: 23.3 kDa. • Trypsin consists of a single chain polypeptide of 223 amino acid residues. • The pH optimum of trypsin is 7 – 9.

12. Trypsin • Isoelectric point: pH 10.5 • Optimum temperature for activity: 37.5oC. • Trypsin is stable as a dry powder at 5oC for years. • Trypsin will cleave peptides on the C-terminal side of lysine (K) and arginine (R) amino acid residues.

14. Chymotrypsin • Chymotrypsin is produced in the acinar cells of the pancreas as the inactive precursor, chymotrypsinogen. • Enzyme Comision E.C. 3.4.21.1 • Molecular Weight: 25 kDa • α-Chymotrypsin is the predominant form of active enzyme produced from it's zymogen.

15. Chymotrypsin • α-Chymotrypsin catalyzes the hydrolysis of peptide bonds on the C-terminal side of tyrosine (Y), phenylalanine (F), tryptophan (W), and leucine (L). • The molecule has three peptide chains: an A chain of 13 residues, a B chain of 131 residues, and a C chain of 97 residues. • α-Chymotrypsin is activated by trypsin. • α-Chymotrypsin is also completely inhibited by 10 mM Cu2+ and Hg2+.

16. Chymotrypsin • Optimum pH for activity: 7.8 • Optimum temperature: 50oC. • α-Chymotrypsin catalyzes the hydrolysis of peptide bonds on the C-terminal side of tyrosine, phenylalanine, tryptophan, and leucine.

18. Pepsin • Pepsin is the predominant digestive protease in the gastric juice of vertebrates. • Enzyme Commision E.C. 3.4.23.1. • Molecular Weight: 35 kDa. • Optimum pH for activity: 1-4 (several subst), 2-4 (synthetic subst). • Isoelectric point: pH 1.0 • Optimum temperature for activity: 37.5oC.

19. Pepsin • Pepsin is a gastric proteinase, with substrate specificity on cleaving peptide bonds of N-terminal of phenylalanine (F), tyrosine (Y), leucine (L), dan methionine (M). • Pepsin has a broad range of substrates and demonstrates an esterase activity. • Conversion of pepsinogen to pepsin, which involves releasing a peptide, may be an autocatalytic process involving self-cleavage by the zymogen.

20. Carboxypeptidase • Carboxypeptidase A is pancreatic proteinase that catalyzes hydrolysis of the neutral or acid amino acids of C-terminal, position in polypeptides, except proline (P), glycine (G), arginine (R), and lysine (K). • Carboxypeptidase B is pancratic proteinase that catalizes hydrolysis of arginine (R) and lysine (K) of C-terminal position of polypeptides. • Molecular weight: 34,300 Da. • The enzyme contains 1 gram atom of zinc per mole.

21. Carboxypeptidase • Carboxypeptidase is activated by trypsin. • Carboxypeptidase is competitively inhibited by arginine, lysine, and ornithine. • Optimum pH for activity: 7-8. • Optimum temperature for activity: 37.5oC. • The enzyme is stable for 6-12 months when stored at -20oC. • Uses: digestive aids.

22. Aminopeptidase • Aminopeptidase is an intestinal mucosa and other animal tissues proteinase, that release an amino acid on N-terminal. • Optimum pH: 8 • Optimum temperature: 37.5oC. • Uses: digestive aids.

23. Elastase • Elastase is a serine protease that also hydrolyses amides and esters. • Elastase is produced in the pancreas as an inactive zymogen, proelastase, • Elastase is activated in the duodenum by trypsin. • Optimum pH: 8.5. • Elastase cleaved peptide bonds of C-terminal of alanine (A), leucine (L), Isoleucine (I), and valine (V) amino acid residues.

24. How are proteins degradated?

25. Asam-asam Amino

26. Protein sequences 1 msltrterti ilslwskist qadvigtetl erlfscypqa ktyfphfdlh sgsaqlrahg 61 skvvaavgda vksidnvtsa lsklselhay vlrvdpvnfk flshcllvtl ashfpadfta 121 dahaawdkfl sivsgvltek yr Karboksipeptidase Elastase Aminopeptidase Chymotrypsin Trypsin Pepsin 1 mddiykaave qlteeqknef kaafdifvlg aedgcistke lgkvmrmlgq nptpeelqem 61 idevdedgsg tvdfdeflvm mvrcmkddsk gkseeelsdl frmfdknadg yidleelkim 121 lqatgetite ddieelmkdg dknndgridy deflefmkgv e

28. Enzyme Lipase

29. Lipolitic Enzymes • Lipase, Esterase • Widely distributed in nature animal and plant tissues. • Hydrolyze fats and oils into glyceride and free fatty acids. • Economic significance in food industry. • Hydrolyze lipids, and produce undesirable rancid flavor in milk products. • Essential for production of desirable flavors in certain foods.

30. Lipase Tests and Assays • Potentiometric or pH-stat Method • Silica Gel Method • Survace Tension Reduction Method • Warburg Monomethric Technique • Turbidimetric Method • Photometric Methric

31. Factors Affecting Velocity and Kinetics of Lipase • Effect of pH and temperature • Most lipases have pH optimum on alkaline side (pH 8-9). It can sift down to acidic range, depends on substrate, presence of salt and kind of emulsifier. • Most lipases have wide temperature optimum range. Most studies assayed at 30-40oC. • Effects of various salts • Heavy metals inhibit lipase activity • NaCl is essential for porcine pancreatic lipases • Ca stimulates activity of lipases • Physical state of substrate • Substrate are plant or animal fat, oils, and synthetic glycerides • Emulsion form

32. Specificity • Hydrolyze natural fats and oils. • Hydrolyze synthetic mono, di, tri-glycerides. • Panceratic lipases hydrolyze ester of primary alcohol groups. • Unsaturated fatty acids were hydrolyzed from triglyceride more rapidly than saturated fatty acids. • Pancratic lipase is more specific on short chain that long chain of fatty acids.

33. Pancreatic Lipase • Produced by acinar cells of pancreas • Realased into doudenum • Lipase (MW about 38,000) was purified chromatographically. • Each mol of enzyme contains 6 disulfide bridges and 2 SH groups. • Rate of lipolysis is affected by emulsion form, length of chain of fatty acids, saturation of fatty acids.

34. Milk Lipase • Milk contains esterolytic enzymes. • Chromatographically purified from milk, skim milk. • Highly unstable enzyme by light, oxygen, and heavy metals (copper, nickel, mercury and cobalt). • Labile to heat, inactive lower than normal pasteurisation.

35. Milk Lipase • Antibiotic will be a competitive inhibitor of the enzyme. • Sulfhydryl reagents irreversibly inhibit the enzyme. • Hydrolyze fats, oils glycerides in an emulsion form. • Hydrolyze physiological substrate (milk fat), and natural fats and oils. • Hydrolyze simple triglycerides. • Hydrolyze short chain faster than long chain of fatty acids.

36. Microbial Lipases • Medical and industrial applications, including in food industry. • Lipase of contaminant microorganisms may cause undesirable rancid flavors. • Produced by Candida and Torulopsis yeasts; Rhizopus, Pinicillium, Aspergillus, Geotrichium and Muccor molds; and Pseudomonas, Achromobacter, and Staphylococcus bacteria.

37. Temperature and pH optima of various Microbial Lipases

38. Microbial Lipases • Hydrolyze natural oils and fats as well as synthetic glycerides. • Low concentration of Ca, Na, K and Mg salts activate lipolysis. • Heavy metal salts strongly inhibit the most microbial lipases. • Most microbial lipases have the same positional specificity as pancreative and milk lipases.

39. Oxidoreductase • Enzymes catalyzing electron transfers. • Intracellular enzymes. • Deteriorative changes in foods due to the enzymes. • Enzymatic browning (polyphenyl oxidase), bleaching (lipoxidase), destruction of ascorbic acid (ascorbic acid oxidase), oxidative flavor deterioration (peroxidase).

40. Oxidoreductase • Glucose oxidase for protection of foods against oxidative deterioration and maillard browning • Catalase for elimination of residual hydrogen peroxide after low temperature pasteurization of milk.

41. Glucose Oxidase • Microbial enzymes in food processing and analytical uses. • Produced by Penicillium sp. and Aspergillus niger. • pH activity 4.5-7. • Temperature optimum 30-60oC. • Oxidixes  form of glucose more rapidly than  form. • Inhibited by copper ions.

42. Galactose Oxidase • Fungal enzymes • Isolated from Dactylium dendoides. • Activated by Ca2+, active on wide variety of substrates. • Oxidizes galactose to galactohexodialdose, and produces peroxide. • More active on galactosides than on galactose.

43. Lipoxygenase/Lipoxidase • Carotene-destroying enzyme • Found in alfalfa, various legums • Active in low temperature • Oxidation of PUFA (linoleic, linolenic, arachidonic) • Catalytic reaction results a organic peroxide; one of the bonds shift to a conjugated position, cis trans isomer.

44. Ascorbic Acid Oxidase • Isolated from mature oranges. • Inhibited by diethyldithio carbamate, potassium ferrocyanide, flouride, and histidine. • Labile to heat. • Specificity on L-ascorbic acid • pH optimum 6.6 • Has role in development of citrus flavor.

45. Xanthine Oxidase • Found in cow milk, calf liver • Oxidizes hypoxanthine and xanthine to uric acid • Milk xanthine oxidase has MW of 275,000. • Inhibited irreversibly by metal ions and flavin. • pH optimum 8.3 • Formation of oxidized flavor in milk.

46. Catalase • Oxidizes hydrogen peroxide to oxygen, and water. • Important in food applications • Found in bovine liver, fungal (Aspergillus niger), bacteria (Micrococcus lysodeikticus) • Inhibited by cyanide, phenol, alkali and urea, • pH optimum 5.3-8.0

47. Glucose Oxidase in Food Processing • To form hydrogen peroxidase • To form gluconic acid • To remove glucose • To remove oxygen

48. Formation of Hydrogen Peroxidase • To form nascent oxygen in presence of catalase. • In conjunction with a secondary system for the peroxide (i.e. peroxidase and chromogen) as a test for glucose. • In the treatment of flour, by forming peroxide catalase-free glucose axidase. • In biological chlorination.

49. Formation of Gluconic Acid • To obtain higher purity of salt.

50. Removal of Glucose • In the analysis of mixed sugars by determining the reducing power before and after removing glucose. • Galactose tolerance test. • To prevent Maillard browning in egg, dried meat and potatoes.