1. Enzim Protease
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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.
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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 logam adalah protease yang aktivitasnya sangat tergantung pada adanya ion logam pada sisi aktifnya. Enzim ini membutuhkan Zn2+ untuk aktivitasnya, dan membutuhkan Ca2+ untuk mentabilkannya.
Protease asam adalah protease yang aktivitasnya disebabkan adanya dua gugus karboksil pada sisi aktifnya. Contohnya: pepsin, renin, dan enzim dari kapang.

5. Digestive Proteses
Panceatic protease: trypsin, chymotrypsin, carboxypeptidase, aminopeptidase, elastase.
Gastric protease: pepsin, rennin.
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6. Endopeptidase
Enzyme that catalyzes the cleavage of peptide bonds within a polypeptide or protein.
Example: pepsin, trypsin, chymotrypsin,
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7. Exopeptidase
Enzyme that catalyzes the removal of an amino acid or dipeptide from the end of a polypeptide chain.
Example: carboxypeptidase, aminopeptidase.
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8. Struktur primer protein
Eksopeptidase
Endopeptidase
Eksopeptidase
Endopeptidase
Endopeptidase
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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.
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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.
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11. Trypsin
Trypsinogen, the proenzyme (zymogen) form of trypsin, is produced in the acinar exocrine cells of the pancreas.
Enzyme Commission (EC) Number:
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.
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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.
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13. 平成31年8月21日

14. Chymotrypsin
Chymotrypsin is produced in the acinar cells of the pancreas as the inactive precursor, chymotrypsinogen.
Enzyme Comision E.C
Molecular Weight: 25 kDa
α-Chymotrypsin is the predominant form of active enzyme produced from it's zymogen.
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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+.
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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.
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17. 平成31年8月21日

18. Pepsin
Pepsin is the predominant digestive protease in the gastric juice of vertebrates.
Enzyme Commision E.C
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.
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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.
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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.
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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.
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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.
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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.
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24. How are proteins degradated?
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25. Asam-asam Amino Amino Acids Alanine ala A Leucine leu L Arginine arg R
Lysine
lys K
Asparagine
asn N
Methionine
met M
Aspartic Acid
asp D
Phenylalanine
phe F
Cysteine
cys C
Proline
pro P
Glutamine
gln Q
Serine
ser S
Glutamic Acid
glu E
Threonine
thr T
Glycine
gly G
Tryptophan
trp W
Histidine
his H
Tyrosine
tyr Y
Isoleucine
ile I
Valine
val V
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26. Protein sequences 平成31年8月21日
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
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27. 平成31年8月21日

28. Enzyme Lipase
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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.
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30. Lipase Tests and Assays
Potentiometric or pH-stat Method
Silica Gel Method
Survace Tension Reduction Method
Warburg Monomethric Technique
Turbidimetric Method
Photometric Methric
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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
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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.
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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.
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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.
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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.
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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.
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37. Temperature and pH optima of various Microbial Lipases
Source of lipase
pH optimum
Temp. Optimum (oC)
Penicillium chrysogenum 37
Pseudomonas fragi 32
Rhizopus delemar
5.6 35
Aspergillus niger 25
Penicillium roqueforti
8.0
Staphylococcus aureus
8.5 45
Geotrichium candisum
8.2
Achromobacter lipolyticum
7.0
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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.
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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).
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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.
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41. Glucose Oxidase
Microbial enzymes in food processing and analytical uses.
Produced by Penicillium sp. and Aspergillus niger.
pH activity
Temperature optimum 30-60oC.
Oxidixes  form of glucose more rapidly than  form.
Inhibited by copper ions.
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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.
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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.
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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.
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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.
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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
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47. Glucose Oxidase in Food Processing
To form hydrogen peroxidase
To form gluconic acid
To remove glucose
To remove oxygen
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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.
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49. Formation of Gluconic Acid
To obtain higher purity of salt.
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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.
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51. Removal of Oxygen
To stabilize against the deteriorative effect of light .
To costabilize ascorbic acid and cyanocobalamin (B12) in vitamin preparations.
To determine glucose in closed system by measuring residual oxygen polarographically.
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52. Removal of Oxygen
To remove occluded air from oil in water emulsions to prevent the development of rancidity.
To prevent oxidation of beer.
To prevent wine from going to vinegar.
To prevent corrosion of can.
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53. Maillard Reaction
Major use of glucose oxidase is prevention of Maillard reaction.
Maillard reaction occurs between an aldehyde group and an amino group.
Maillard reaction: disappearance of glucose and free amino acids, loss of solubility, change in flavor and odor, and development of a brown color.
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54. Preventing Maillard Reaction
Acidification to pH 5.5.
Drying to a maximum moisture 0f 2%.
Refrigeration.
Removing or inactivating one of the reactants.
Converting glucose to gluconic acid with glucose oxidase-catalase.
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55. Applications of Glucose Oxidase
Desugaring eggs,
Modification of fructose-glucose ratio,
Curdling milk,
Protecting animal fats against oxidation,
Protection of water and oil emulsions.
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56. Kuiz 25 April 2017
Buatlah grafik Linewaver-Burk Plot dari inhibitor yang mempunyai aktivitas competitive inhibitor, dan jelaskan makna grafik tersebut.
Apakah yng dimaksud dengan stabilitas enzim.
Jelaskan dengan gambar 3 macam metode immobilisasi enzim.
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