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Bioindustri Enzim Nur Hidayat Materi Kuliah Bioindustri Jurusan Teknologi Industri Pertanian, Fak Teknologi Pertanian Universitas Brawijaya Malang

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Presentasi berjudul: "Bioindustri Enzim Nur Hidayat Materi Kuliah Bioindustri Jurusan Teknologi Industri Pertanian, Fak Teknologi Pertanian Universitas Brawijaya Malang"— Transcript presentasi:

1 Bioindustri Enzim Nur Hidayat Materi Kuliah Bioindustri Jurusan Teknologi Industri Pertanian, Fak Teknologi Pertanian Universitas Brawijaya Malang

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 Corn Starch Slurry (30-35% DS, pH , Ca ppm) Production of High Fructose Corn Syrups from Starch Liquefaction Thermostable  -Amylase Gelatinization (105°C, 5 min) Dextrinization (95°C, 2h) Liquefied Starch DE Saccharification Glucoamylase (60°C, pH , h) Glucose Syrups DE Isomerization Glucose isomerase (pH , 55-60°C, 5 mM Mg 2+ ) High Fructose Corn Syrups (42% fructose)

14 Production of Glucose from Starch _______________________________________________________________ Liquefaction Saccharification DE Glucose _______________________________________________________________ Acid Acid92 85 Acid Glucoamylase Acid/α-amylase Glucoamylase α-Amylase/High pressure Glucoamylase cooking/ α-amylase α-Amylase (thermostable) Glucoamylase α-Amylase (thermostable) Glucoamylase _______________________________________________________________

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

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 % 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 CH 3 RCN H O N O SS O N COOH Penicillin V or G NH 2 CH 3 COOH (6-APA) Penicillin acylase Alkaline [Enzymatic] CH 3 RCN H O N O S COOSiMe 3 [Chemical] [R=Ph or PhO] Pyridine Me 3 SiCl PCl 5 ROH H 2 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 o C) 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, hours

20 Enzymatic Modification of Penicillins to 6-APA and Semisynthetic Penicillins CH 3 RCN H O N O SS O N COOH Penicillin V or G NH 2 CH 3 COOH (6-APA) Penicillin acylase [Acylation] Acidic Semisynthetic Penicillins Penicillin acylase Alkaline [Deacylation]

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 H 2 SO 4 (90 o C) or a metal catalyst ( o C) 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 o C Cbz, benzyloxycarbonyl

24 Biocatalytic Production of Aspartame HO 2 C CO 2 H H2OH2O thermolysin D,L-phenylalanine Methyl ester Cbz-aspartame PhCH 2 OCNH O + Ph CO 2 Me H2NH2N HO 2 C CNH PhCH 2 OCNH O CO 2 Me Ph O N-Cbz-aspartic acid 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 Cl reductase O Cl OC 8 H 17 HO H O OC 8 H 17  -chloroacetoacetic acid octyl ester (R)-  -chloro-  -hydroxybutanoic acid octyl ester HO H O Me 3 N OH L-carnitine hydroxylase O Me 3 N OH Me 3 N HO H O OH L-carnitine  -butyrobetaine

27 Synthesis of Naproxene CH 3 O CO 2 H * CH 3 O CO 2 H * (D/L) CH 3 O CO 2 H * CH 3 O O C CH 2 CH 3 CH 3 O CO 2 H * biocatalysts multistep resolution 1)Tartaric acid 2)Br 2 3) Hydrolysis

28 Synthesis of Calcium – Antagonist Drug Diltiazem OMe O MeO 2 C esterase racemate O HO 2 C OMe (R,R) (S, S) Diltiazem N H O O O OMe S

29 Synthesis of L-malic Acid and L-Aspartic Acid from Fumaric Acid HO HO 2 C CO 2 H H2OH2O fumarase HO 2 C CO 2 H H fumaric acidL-malic acid HO 2 C CO 2 H HO 2 C H CO 2 H HO fumaric acidL-aspartic acid NH 3 aspartase

30 Environmentally Compatible Synthesis of Catechol from Glucose benzene a cumenephenol catechol protocatechuic acid 3-dehydroshikimic acid D-glucose acetone b c hydroquinone dd d HO OH HO OH O CO 2 H OH HO O CO 2 H (a) propylene, solid H 3 PO 4 catalyst, °C, psi. (b) O 2, °C then SO 2, °C. (c) 70% H 2 O 2, EDTA, Fe 2+ or Co 2, 70-80°C. (d) E. coli AB2834/pKD136/pKD9.069A, 37°C. Draths and Frost, 1995

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)E o DED Pine kraft 18.4 (CD)E p DE p D Birch kraft 18 (CD)E op DEpD Pine kraft 12 DE op DED Softwood kraft 15 ____________________________________________________ C, elemental chlorine (Cl 2 ), D, chlorine dioxide (ClO 3 ), E, alkaline extraction (NaOH), E o /E p, 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 o C In alkaline solutions, it is a powerful sequestrant of metallic ions It is about half as sweet as sucrose

35 Hydrogenation of D -Fructose H 2, catalyst D -Fructose D -Sorbitol D -Mannitol H 2 C OH C O HO CH HC OH H 2 C OH HC OH HO CH HC OH H 2 C OH HO CH HC OH H 2 C OH +

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

37 Mannitol Production from Fructose in pH-Controlled Batch Fermentation Fructose (g/L) At 37 o C, 130 rpm, Initial pH 6.5, pH controlled at 5.0, 500 ml fleaker with 300 ml medium. Time (h) Mannitol (g/g) Lactic Acid (g/g) Acetic Acid (g/g)  ± ± ± ± ± ± ± ± ± ±0.00

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

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

40 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 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 necessary to avoid catalyst inactivation Catalytic Hydrogenation

41 Enzymatic Conversion of Fructose to Mannitol Mannitol 2-Dehydrogenase D -fructoseMannitol CH 2 OH O HO H CH 2 OH HO H CH 2 OH NAD(P)HNAD(P)

42 Cofactor Regeneration Chemical Photochemical Electrochemical Biological Enzymatic

43 Mannitol Dehydrogenase Na-Formate NADHNAD D-Fructose CO 2 + H 2 0 Mannitol Formate Dehydrogenase Enzymatic Conversion of Fructose to Mannitol with Simultaneous Cofactor Regeneration

44 Mannitol Dehydrogenase Glucose + H 2 0 NADHNAD + D-Fructose Gluconic acid Mannitol Glucose Dehydrogenase Enzymatic Conversion of Fructose to Mannitol with Simultaneous Cofactor Regeneration

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