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Bioindustri Enzim

Bioindustri Enzim. Nur Hidayat Materi Kuliah Bioindustri. Jurusan Teknologi Industri Pertanian, Fak Teknologi Pertanian Universitas Brawijaya Malang http://nurhidayat.lecture.ub.ac.id http://ptp2007.wordpress.com. Enzim.

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Bioindustri Enzim

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  1. Bioindustri Enzim Nur Hidayat Materi Kuliah Bioindustri Jurusan Teknologi Industri Pertanian, Fak Teknologi Pertanian Universitas Brawijaya Malang http://nurhidayat.lecture.ub.ac.id http://ptp2007.wordpress.com

  2. Enzim • Enzim, dihasilkanoleh sistem hidup, merupakan protein yg memiliki sifat katalitik. • Sebagai katalis, enzim efisien dan sangat spesifik terkait keterlibatanya dalam reaksi kimia. • Cofactors terlibat dalam reaksi dimana molekul dioksidasi, reduksi, dioecah ataupun digabung.

  3. Biotechnology • Teknik yang melibatkan penggunaan oragnisme hidup atau produknya untukmembuat atau memodifikasi produk untuk tujuan komerial.

  4. Main Enzyme Classes ____________________________________________________ Enzyme class Catalyzed reaction ____________________________________________________ Oxidirectadases Oxidation-reduction reaction Transferases Transfer of functional group Hydrolases Hydrolytic reactions Lyases Group elimination (forming double bonds) Isomerases Isomerizaion reaction Ligases Bond formation coupled with a triphosphate cleavage ____________________________________________________

  5. Enzymes in Biotechnology • Enzymes in food and beverage production Dairy industry Beer industry Wine and juice industry Alcohol industry Protein industry Meat industry Baking industry Fat and Oil industry • Enzymes as industrial catalysts Starch processing industry Antibiotic industry Fine Chemicals industry

  6. Enzymes in Biotechnology •Enzymes as final products Detergent industry Cleaning agent industry Pharmaceutical industry Animal feed industry Analytical applications •Enzymes as processing aids Textile industry Leather industry Paper and pulp industry Sugar industry Coffee industry

  7. Faktor-faktor penting kenapa digunakan enzim • kemungkinan reaksi tidak dapat dilakukan secara kimia. • Reaksi spesifik • Mereduksi jumlah tahapan proses yang dibutuhkan. • Mengeliminasi kebutuhan pelarut organik dalam proses. • Enzim dapat digunakan ulang melalui imobilisasi. • Dapat dikombinasikan dengan proses lain. • Enzim dapat diperbaiki melalui rekayasa genetika.

  8. Industrial Enzyme Market Annual Sales: $ 1.6 billion Food and starch processing: 45 % Detergents: 34 % Textiles: 11 % Leather: 3 % Pulp and paper: 1.2 %

  9. Beberapa contoh enzim mikrobial • Protease: protease netral dari Aspergillus dan Alkali dari Bacillus • Deterjen biologi: subtilisin dari Bacillus licheniformis dan B. subtilis • Penjernihan wine • Pengolahan kulit • Pembuatan keju • Pengempukan daging dsb

  10. Lipase • Lipase terutama dari Bacillus, Aspergillus, Rhizopus, dan Rhodotorula • Deterjen biologis • Pengolahan kulit – penghilangan lemak • Produksi senyawa flavor • Pengolahan susu dan daging

  11. Alfa Amilase • Sumber: Aspergillus dan Bacillus • Untuk pengolahan pati menjadi sirup gula • Modifikasi tepung dalam pembuatan roti • Hidrolisis pati pada industri wine • Detergen biologis • Manufaktur tekstil

  12. Beta Amilase dan Amiloglukosidase • Bacillus polymyxa, Streptomyces, Rhizopus • Untuk produksi sirup maltosa • Industri beer: meningkatkan gula yg dapat difermentasi. • Amiloglukosidase: A. niger, R. niveus • Produksi sirup glukosa • Roti, • Beer, wine • Juice buah

  13. Production of High Fructose Corn Syrups from Starch Corn Starch Slurry (30-35% DS, pH 6.0-6.5, Ca2+ 50 ppm) Liquefaction Thermostable a-Amylase Gelatinization (105°C, 5 min) Dextrinization (95°C, 2h) Liquefied Starch DE 10-15 Saccharification Glucoamylase (60°C, pH 4.0-4.5, 24-72 h) Glucose Syrups DE 95-96 Isomerization Glucose isomerase (pH 7.5-8.0, 55-60°C, 5 mM Mg2+) High Fructose Corn Syrups (42% fructose)

  14. Production of Glucose from Starch _______________________________________________________________ Liquefaction Saccharification DE Glucose _______________________________________________________________ Acid Acid 92 85 Acid Glucoamylase 95 91 Acid/α-amylase Glucoamylase 96 92 α-Amylase/High pressure Glucoamylase 97 93 cooking/ α-amylase α-Amylase (thermostable) Glucoamylase 97 94 α-Amylase (thermostable) Glucoamylase 97-98.5 95-97.5 _______________________________________________________________

  15. Conversion of Glucose to Fructose HO HO OH OH glucose isomerase O O OH HO OH HO OH OH

  16. Enzim mikrobial komersial • Enzim detergent • Enzim dalam pengolahan Pati dan karbohidrat • Enzim dalam produksi keju • Enzim dalam produksi juice • Enzim dalam Manufaktur tekstil • Enzim dalam manufaktur kulit • Enzim dalam penanganan pulp kayu • Enzim dalam sintesis bahan organik

  17. 6-Aminopenicillanic Acid (6-APA) Penicillin: First discovered by Fleming in 1932 19% of worldwide antibiotic market. Superior inhibitory action on bacterial cell wall synthesis Broad spectrum of antibacterial activity Low toxicity Outstanding efficacy against various bacterial strains Excessive use has led to development of resistant pathogens 6-APA:Raw material for production of new semisynthetic penicillins (amoxycillin and ampicillin) Fewer side effects Diminished toxicity Greater selectivity against pathogens Broader antimicrobial range Improved pharmacological properties

  18. Chemical and Enzymatic Deacylation of Penicillins to 6-APA H R C N S S CH3 CH3 NH2 Penicillin acylase CH3 CH3 O Alkaline N N COOH COOH [Enzymatic] O O Penicillin V or G (6-APA) [R=Ph or PhO] PCl5 ROH H2O [Chemical] Pyridine Me3SiCl H R C N S CH3 CH3 O N COOSiMe3 O

  19. 6-Aminopenicillanic Acid (6-APA) Chemical method: Use of hazardous chemicals - pyridine, phosphorous pentachloride, nitrosyl chloride Enzymatic method: Regio- and stereo-specific Mild reaction conditions (pH 7.5, 37 oC) Enzymatatic process is cheaper by 10% Enzymes: Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium, Streptomyces lavendulae Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium, Fusarium sp., Pseudomonas acidovorans Immobilized Enzyme: Life, 500-2880 hours

  20. Enzymatic Modification of Penicillins to 6-APA and Semisynthetic Penicillins H R C N S S CH3 CH3 NH2 Penicillin acylase CH3 CH3 O Alkaline N N COOH COOH [Deacylation] O O Penicillin V or G (6-APA) Penicillin acylase [Acylation] Acidic Semisynthetic Penicillins

  21. Synthesis of Acrylamide • Monomeric raw material for the manufacture of polymers • and synthetic polymers • Obtained by hydration of the cyanide function of • acrylonitrile • World market, 200,000 tpa • Chemical Process: • Reaction of acrylonitrile with water in the presence of H2SO4 (90 oC) or a metal catalyst (80-140 oC) • Formation of toxic waste (HCN) • The reaction must be stopped to prevent the acrylamide itself being converted to acrylic acid Enzymatic Process: 99.9% yield Kg quantity product/g cells Acrylic acid is not produced • Fewer process steps are involved • Much more environmental friendly • Nitto Chemical Industry: 6,000 tons annually

  22. Synthesis of Acrylamide Copper-catalysed process Microbial process Nitrile hyratase and amidase reactions

  23. Aspartame (L-Asp-L-Phe-Methyl Ester) • Aspartame is dipeptide sweetener formed by linking the • methyl ester of phenylalanine with aspartic acid • Extensively used in food and beverages • 200 times as sweet as sucrose • Annual sale: 200 million Ibs, $ 850 million • Nutrasweet Corp. retains 75% of the US market • Chemical method: • The amino group of aspartic acid needs to be protected to prevent its • reacting with another molecule of aspartic acid to give unwanted • by-products • The correct single enantiomer of each of the reactants must be used • to give the required stereochemistry of aspartame (beta-aspartame is • bitter tasting) • Enzymatic method: • Thermolysin promotes reaction only at the alpha-functionality • Mild condition, pH 6-8, 40 oC • Cbz, benzyloxycarbonyl

  24. Biocatalytic Production of Aspartame HO2C Ph + thermolysin CO2H CO2Me PhCH2OCNH H2N O H2O N-Cbz-aspartic acid D,L-phenylalanine Methyl ester HO2C Ph CO2Me CNH PhCH2OCNH O O Cbz-aspartame Cbz, benzyloxycarbonyl

  25. L-Carnitine Thyroid inhibitor Slimming agent Dietary supplement for athletes Only one enantiomer of the compound is used Two biocatalytic routes are available to make L-carnithine. Saccharomyces cerevisiae Rhizobiaceae

  26. Synthesis of L-Carnitine O O O reductase HO H Cl Cl OC8H17 OC8H17 g-chloroacetoacetic acid octyl ester (R)-g-chloro-b-hydroxybutanoic acid octyl ester O HO H Me3N OH L-carnitine O O hydroxylase HO H Me3N Me3N OH OH g-butyrobetaine L-carnitine

  27. Synthesis of Naproxene CO2H * biocatalysts CH3O CH3O CO2H CO2H * * resolution multistep (D/L) CH3O CH3O CH3O • Tartaric acid • Br2 • 3) Hydrolysis O CO2H C CH2CH3 * CH3O CH3O

  28. Synthesis of Calcium – Antagonist Drug Diltiazem OMe OMe O O (R,R) esterase MeO2C HO2C racemate OMe S (S, S) O Diltiazem N O O H

  29. Synthesis of L-malic Acid and L-Aspartic Acid from Fumaric Acid HO2C HO2C fumarase H H2O CO2H CO2H HO fumaric acid L-malic acid HO2C HO2C aspartase H NH3 CO2H CO2H HO fumaric acid L-aspartic acid

  30. Environmentally Compatible Synthesis of Catechol from Glucose acetone hydroquinone a b HO c benzene cumene phenol CO2H CO2H OH HO OH OH d O d d catechol OH HO OH O HO OH OH OH D-glucose 3-dehydroshikimic acid protocatechuic acid (a) propylene, solid H3PO4 catalyst, 200-260°C, 400-600 psi. (b) O2, 80-130°C then SO2, 60-100°C. (c) 70% H2O2, EDTA, Fe2+ or Co2, 70-80°C. Draths and Frost, 1995 (d) E. coli AB2834/pKD136/pKD9.069A, 37°C.

  31. Debittering of Protein Hydrolyzates • Treatment with activated carbon • Extraction with alcohol • Isoelectric precipitation • Chromatographic separation • Masking of bitter taste • Enzymatic hydrolysis of bitter peptides • with aminopeptidase • with alkaline/neutral protease with carboxypeptidase • Condensation reactions using protease

  32. Mill Scale Xylanase-aided Bleaching Trials ____________________________________________________ Sequence after Pulp Total active chlorine Enzyme treatment consumption decrease (%) ____________________________________________________ (CD)EDED Softwood kraft 21 (CD)EoDED Pine kraft 18.4 (CD)EpDEpD Birch kraft 18 (CD)EopDEpD Pine kraft 12 DEopDED Softwood kraft 15 ____________________________________________________ C, elemental chlorine (Cl2), D, chlorine dioxide (ClO3), E, alkaline extraction (NaOH), Eo/Ep, oxygen/hydrogen peroxide reinforced alkaline extraction

  33. Mannitol • Food additive • Reduces the crystallization tendency of sugars and is used as such to increase the shelf-life of foodstuffs • Used in chewing gum • Pharmaceutical formulation of chewable tablets and granulated powders • Prevents moisture adsorption from the air, exhibits excellent mechanical compressing properties, does not interact with the active components, and its sweet cool taste masks the unpleasant taste of many drugs

  34. Mannitol • Mannitol hexanitrate is a well-known vasodilator, used in the treatment of hypertension • The complex of boric acid with mannitol is used in the production of dry electrolytic capacitors • It is an extensively used polyol for the production of resins and surfactants • It has low solubility in water of only 18% (w/w) at 25 oC • In alkaline solutions, it is a powerful sequestrant of metallic ions • It is about half as sweet as sucrose

  35. H2C OH C O HO CH HC OH HC OH H2C OH H2C OH HC OH HO CH HC OH HC OH H2C OH H2C OH HO CH HO CH HC OH HC OH H2C OH Hydrogenation of D-Fructose H2, catalyst + D-Fructose D-Sorbitol D-Mannitol

  36. Heterofermentative Conversion Pathway of Fructose into Mannitol Fructose 2 Fructose ATP ADP Fructose – 6-P Glucose – 6-P NADP+ NADPH + H+ 6 - Phosphogluconate NADP+ CO2 NADPH + H+ Ribulose – 5-P Xylulose – 5-P Acetyl - P Glyceraldehyde - 3-P 2 ADP NAD+ ADP NADH + H+ 2 ATP Pyruvate NADH + H+ ATP NAD+ Acetate 2 Mannitol Lactate

  37. Fructose (g/L) Time (h) Mannitol (g/g) Lactic Acid (g/g) Acetic Acid (g/g) Mannitol Production from Fructose in pH-Controlled Batch Fermentation 150 200 250 300 15 40 64 136 0.720.00 0.69±0.03 0.70±0.02 0.66±0.03 0.17±0.00 0.17±0.00 0.16±0.00 0.15±0.01 0.12±0.00 0.13±0.00 0.12±0.00 0.11±0.00 At 37oC, 130 rpm, Initial pH 6.5, pH controlled at 5.0, 500 ml fleaker with 300 ml medium.

  38. Fructose and Glucose (2:1) Co-Utilization and Mannitol Production 100 Fructose ) Mannitol L / g ( t O 37 C c u pH 5.0 d o r P 50 r o e t Lactic acid a r t s Acetic acid b u S Glucose 0 12 36 0 48 24 Time (h)

  39. Mannitol Production in pH-Controlled Fed-Batch Fermentation O 37 C 200 ) pH 5.0 L / g ( t c u 150 Mannitol d o r P r o 100 Fructose e t a r t Fructose used: 300 g/L (final concentration) s Lactic b 50 u acid S Acetic acid 0 48 24 96 0 72 Time (h)

  40. Catalytic Hydrogenation Only half of fructose converted to mannitol Co-product: sorbitol in large excess (3) Highly pure hydrogen gas necessary Nickel catalyst essential Ion exchanger for nickel ions removal Highly pure substrates necessaryto avoid catalyst inactivation Fermentation All fructose converted to mannitol Co-product: lactic acid and acetic acid one half of mannitol Glucose is hydrogen source in hydrogenation Nitrogen source essential for growth Electrodialysis for removing organic acids Use of less pure substrates poses no problem

  41. CH2OH O HO H H OH H OH CH2OH CH2OH HO H HO H H OH H OH CH2OH Enzymatic Conversion of Fructose to Mannitol Mannitol 2-Dehydrogenase NAD(P)H NAD(P) D-fructose Mannitol

  42. Cofactor Regeneration • Chemical • Photochemical • Electrochemical • Biological • Enzymatic

  43. Enzymatic Conversion of Fructose to Mannitol with Simultaneous Cofactor Regeneration Mannitol Dehydrogenase Mannitol D-Fructose NADH NAD Na-Formate CO2 + H20 Formate Dehydrogenase

  44. Enzymatic Conversion of Fructose to Mannitol with Simultaneous Cofactor Regeneration Mannitol Dehydrogenase Mannitol D-Fructose NADH NAD+ Gluconic acid Glucose + H20 Glucose Dehydrogenase

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