Kuliah 4 Teknologi Enzim

FARMASI UHAMKA 2012

Priyo Wahyudi

Teknologi Enzim & Protein Sel Tunggal

a.

b.
c. d.

e.
f.

Definisi enzim Arti penting enzim Aplikasi teknologi enzim pada industri Sumber enzim Produksi enzim Karakterisasi dan aktivitas enzim

What is Enzyme ?

Most enzymes are Proteins (tertiary and quaternary structures) Act as Catalyst to accelerates a reaction Not permanently changed in the process

Enzim

Enzim = Biokatalisator yang mendorong terjadinya proses sintesa dan perombakan pada mahluk hidup Katalis = suatu zat yang meskipun dalam jumlah amat sedikit, mempunyai kemampuan unik untuk mempercepat berlangsungnya reaksi kimiawi tanpa terjadinya perubahan pada zat tersebut setelah reaksi selesai.

Skema reaksi enzimatis

Gambar skema fungsi katalisis enzim dalam sebuah reaksi kimia (S + E SE P + E)

Enzim dapat berupa:

Protein murni Gabungan protein dengan gugusan kimia lainnya Apoenzim, komponen protein dari sebuah enzim Koenzim, komponen molekul organik dengan BM rendah
+ Koenzim
Tidak aktif Molekul organik BM rendah Terdialisis

Enzim terdiri dari komponen:

Apoenzim
Tidak aktif Protein BM tinggi Tidak terdialisis

Holoenzim
Aktif

Beberapa enzim membutuhkan ion-ion logam untuk menjadikan teraktivasi, komponen tersebut disebut Kofaktor. Ion-ion logam yang dapat menjadi kofaktor a.l.: Mg2+, Mn2+, Fe2+, Zn2+ dll.

Enzim & energi aktivasi

Gambar skema pengaruh enzim dalam menurunkan energi aktivasi suatu reaksi kimia

Faktor-faktor yang mempengaruhi Aktivitas Enzim

1. Konsentrasi Substrat
Pengaruh konsentrasi substrat terhadap aktivitas enzim adalah bahwa pada tahap awal terjadi hubungan yang linear artinya pada suatu peningkatan konsentrasi substrat sampai batas tertentu terjadi peningkatan aktivitas enzim, namun setelah dicapai titik optimal maka adanya kenaikan konsentrasi substrat tidak berpengaruh lagi terhadap laju aktivitas enzim

Aktivitas enzim

Konsentrasi substrat

Faktor-faktor yang mempengaruhi Aktivitas Enzim

2. Konsentrasi Enzim Pengaruh konsentrasi enzim terhadap aktivitas enzim bersifat linear. Pada derajat kemurnian enzim tinggi dan dalam batas-batas tertentu maka semakin tinggi konsentrasi enzim semakin tinggi pula aktivitasnya

Aktivitas enzim

Konsentrasi enzim

Faktor-faktor yang mempengaruhi Aktivitas Enzim

3. pH Pengaruh pH pada aktivitas sebuah enzim sangat ditentukan oleh sifat / karakteristik enzim tersebut apakah acidofil, mesofil atau alkalifil. Aktivitas maksimum akan dicapai pada pH tertentu (pH optimal). Adanya penyimpangan dari nilai pH optimal akan menurunkan aktivitas enzim tersebut

Aktivitas enzim

3 4

9 10 11 12

pH

Faktor-faktor yang mempengaruhi Aktivitas Enzim

4. Suhu Pengaruh suhu terhadap aktivitas enzim adalah juga ditentukan oleh sifat / karakteristik dari enzim tersebut (mesofil atau termofil). Mulai suhu rendah sampai dicapai suhu optimal maka akan terjadi kenaikan aktivitas enzim, namun kenaikan suhu setelah melewati suhu optimal akan menyebabkan menurunnya laju aktivitas enzim

Aktivitas enzim
0 10 20 30 40 50 60 70 80 90

Suhu

Tipe enzim
1.

Enzim ektraseluler atau ektoenzim

Enzim disekresikan ke luar sel, berfungsi melangsungkan perubahan-perubahan seperlunya pada substrat di luar sel, sehingga memungkinkan substrat tersebut memasuki sel. Contoh: amilase menguraikan pati menjadi unit-unit gula yang lebih kecil

2.

Enzim intraseluler atau endoenzim

Enzim diseksresikan di dalam sel, berfungsi mensintesis bahan seluler dan menguraikan nutrien untuk menghasilkan energi yang dibutuhkan oleh sel. Contoh: heksokinase mengkatalisis fosforilasi glukosa dan heksosa (senyawa-senyawa gula sederhana) di dalam sel.

Klasifikasi Enzim
1. 2. 3. 4. 5.

6.

Oksidoreduktase, Reaksi transfer elektron (pemindahan elektron atau atom Hidrogen) Transferase, Transfer gugus fungsional (cont: Fosfat, amino, metil, dll.) Hidrolase, Reaksi hidrolisis (penambahan molekul air untuk memecahkan ikatan kimiawi) Lyase, Penambahan ikatan ganda pada molekul dan juga pengusiran non hidrolitik gugus kimiawi Isomerase, Reaksi isomerisasi (pengubahan suatu senyawa menjadi isomer, misal. Suatu senyawa yang memiliki atomatom yang sama tetapi berbeda struktur molekulnya) Ligase, Pembentukan ikatan disertai pemecahan atau penambahan ATP

Naming conventions
The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers ; each enzyme is described by a sequence of four numbers preceded by "EC". The first number classifies the enzyme based on its mechanism.

The top-level classification is:

EC 1 Oxidoreductases: catalyze oxidation/ reduction reactions EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group). EC 3 Hydrolases: catalyze the hydrolysis of various bonds . EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation. EC 5 Isomerases: catalyze isomerization changes within a single molecule. EC 6 Ligases: join two molecules with covalent bonds.

Pemanfaatan Enzim

Sains & Teknologi

Enzim Endonuklease, Enzim Reverse Transcriptase

Protease mengempukkan daging Industri pulp, kertas, kain Industri sirup fruktosa Amilase Protease Lipase

Makanan

Industri

Kesehatan

Industrial Enzyme Classes

Commodity enzymes

High volume (tonnes p.a) Low purity (but not necessarily so) Low cost (e.g. $5-40 per kg) Low profit margins
Low volume (g kg) High purity High cost ($5 10,000 per g) High profit margins

Speciality enzymes

Enzymes in Industry

Distribution of enzymes by substrate

Protein hydrolysing
Carbohydrate hydrolysing

59%
28%

Lipid hydrolysing

3%

Speciality (analytical, pharma, research) 10%

Enzymes in Industry
Textile processing Grain processing Food processing Cleaning Feed enzymes Diagnostic/pharma Waste management

Process

% by value
Textile processing Grain processing Food processing Cleaning Feed enzymes Diagnostic/pharma Waste management Other

Other

10 12 18 44 4 4 4 4

Industrial enzymes: Market trends

Increasing 10-15% annually by volume Increasing 4-5% annually by value Decreased margins for commodity enzymes Increased use of speciality enzymes
Diagnostic enzymes Fine chemicals manufacture Chiral separation

Industrial enzymes

Food processing

Amylases in bread-making Lipases in flavour development Proteases in cheese making Pectinases in clarifying fruit juices Cellulases in treating denim to generate stone-washed texture/appearance

Textiles

Grain processing

Conversion of corn starch to high fructose syrups

Industrial enzymes

Feed enzymes

Enzymes to assist in the digestibility of animal feeds (cellulase, xylanase, phytase) Lipases as drain-cleaning agents
Reporter enzymes (alkaline phosphatase, glucose oxidase, bglucosidase) and diagnostic enzymes (DNA polymerase)

Waste management
Diagnostic enzymes

Industrial enzymes

Speciality

Biotransformations

Lipases, esterases and oxidoreductases for chiral separations Glucotransferases in synthesis of oligosaccharides Thermolysin in aspartame synthesis Nitrile hydratases in acrylamide and nicotinamide synthesis Proteases in peptide synthesis Penicillin acylase for manufacture of semisynthetic penicillins Aspartase in the manufacture of L-aspartate

Aplikasi Enzim pada Industri

Aplikasi Enzim

Stone-washing: Trichoderma Cotton: Gluconacetobacter Debleaching: Mushroom peroxidase Indigo: E. coli Plastic: Bacterial polyhydroxyalkanoate

Stone washing denim

Indigo dye adheres to denim surface
traditional method new method

Denim is faded by abrasive action of pumice stones

Cellulase enzyme removes some of the dye by partially hydrolyzing the cotton surface

weakens the fabric

new looks lower costs shorter treatment times less solid waste

Aplikasi Enzim

Protease pada penyamakan kulit

Dehairing Bathing

Aplikasi Enzim

Enzim Protease pada detergen

Penghilang noda pada kain

Aplikasi Enzim

Enzim Protease pada detergen

Penghilang noda pada kain

Enzyme prodrug therapy (EPT)

Nature Reviews Drug Discovery 10, 671-684 (September 2011)

Targeting metabolic enzymes as a strategy to block biosynthesis or induce energy stress

The pathways of central carbon metabolism are presented. Some of the metabolic enzymes that are currently being considered as therapeutic targets for cancer are marked with a target (shown as a pink circle ). Five drugs that influence metabolism and have been tested in humans are shown in pink boxes. This figure illustrates how these enzyme targets are involved in the synthesis of important macromolecules (shown in brown boxes) that are needed for cell growth.
KG, -ketoglutarate; DCA, dichloroacetate; DNP, 2,4-dinitrophenol; F-2,6-BP, fructose-2,6-bisphosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; G6P, glucose-6-phosphate; GLS, glutaminase; GLUT1, glucose transporter type 1; HK2, hexokinase 2; I, complex I; IDH, isocitrate dehydrogenase; III, complex III; IV, complex IV; LDH, lactate dehydrogenase; MCT4, monocarboxylate transporter 4; OAA, oxaloacetate; PDH, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; PEP, phosphoenolpyruvate; PFK1, phosphofructokinase 1; PFK2, phosphofructokinase 2; PKM2, pyruvate kinase M2 isoform; V, complex V.

Enzymes Prolactazyme

Additional Information About 75% of the worlds population is intolerant to lactose in adulthood. It occurs due to lack of lactase in digestive system. Beta-Lactamase Penicillin Allergy Penicillin is converted to penicillioate Learn more about penicillin allergy here Aglucerase Gauchers Disease type Enzyme replacement therapy This disease is characterized by the lack I of enzyme glucocerebrocidase. Streptokinase Heart Attacks Used as clot blusters to dissolve Administered i.v. to patients as soon as (Myocardial Infarction) clots in the arteries of heart wall. possible after the onset of a heart Plasminogen is converted to plasmin attack which is fibrinolytic. Asparaginase Acute Childhood Decreased level of serum asparagine Tumor cells cannot synthesize LLeukemia and inhibition of aspargine asparagine due to lack of aspartatedependent multiplication of tumor ammonia ligase. cells. Collagenase Skin ulcers Causes collagen hydrolysis Break up and remove dead skin and tissue DNAse Cystic Fibrosis (CF) DNAse hydrolyses extracellular DNA DNA present in the mucous, which responsible for Cystic Fibrosis. arises from dead WBCs and bacterial cells, serves to cross link the mucous, changing it from a fluid gel to a semisolid. Lysozyme Antibiotic Therapy Causes Bacterial cell wall hydrolysis Ribonuclease Antiviral Therapy Causes RNA hydrolysis Trypsin Inflammation Causes Protein hydrolysis Uricase Gout Converts Urate to allantoin Enzyme To increase the efficacy Against resistant bacterisa Example: Beta lactamase inhibitor like inhibitors of drugs clavulanic acid along with amoxicillin.

Therapeutic Use Lactose Intolerance

Basis Prolactazyme is a proenzmye that produces lactase in stomach.

Sumber Enzim

Virus :

Enzim reverse transcriptase

Enzim DNA endonuclease, DNA polimerase Enzim protease, lipase, amilase Enzim selulase Enzim protease, lipase, amilase Enzim protease Enzim selulase

Bakteri :

Fungi

Tanaman

Hewan

Sumber Enzim

Tanaman

Papain

Bromelin

Sumber Enzim

No fibre digesting enzymes or phytase

Produksi Enzim

Mikroorganisme yang direkayasa Sistem fermentasi Pemisahan produk enzim Purifikasi Immobilisasi

Produksi Enzim

Diagram Alir Preparasi Enzim

Enzim-Enzim Komersial yang Diperoleh dari Hewan

Enzima Katalase EC numberb 1.11.1.6 Sumber Hati Intra/extra -selulerc I Skala produksid Penggunaan Industri Makanan

Khimotripsin
Lipasee Rennetf Tripsin

3.4.21.1
3.1.1.3 3.4.23.4 3.4.21.4

Pankreas
Pankreas Abomasum Pankreas

E
E E E

+ -

Kulit
Makanan Keju Kulit

Enzim-Enzim Komersial yang Diperoleh dari Tanaman

Enzima Aktinidin a-Amilase b-Amilase EC numberb 3.4.22.14 3.2.1.1 3.2.1.2 Sumber Buah Kiwi Malted barley Malted barley Intra/extra -selulerc E E E Skala produksid +++ +++ Penggunaan Industri Makanan Brewing Brewing

Bromelain
b-Glukanaseg Fisin Lipoksigenase

3.4.22.4
3.2.1.6 3.4.22.3 1.13.11.12

Getah nanas
Malted barley Fig latex Kedelai

E
E E I

++ -

Brewing
Brewing Makanan Makanan

Papain

3.4.22.2

Getah pepaya

++

Daging

Enzim-Enzim Komersial yang Diperoleh dari Jamur

Enzima a-Amilase Aminoasilase Glukoamilasek Katalase Selulase EC numberb 3.2.1.1 3.5.1.14 3.2.1.3 1.11.1.6 3.2.1.4 Sumber Aspergillus Aspergillus Aspergillus Aspergillus Trichoderma Intra/extra -selulerc E I E I E Skala produksid ++ +++ Penggunaan Industri Roti Farmasi Pati Makanan Pengolahan limbah

Dekstranase

3.2.1.11

Penicillium

Makanan

Enzim-Enzim Komersial yang Diperoleh dari Jamur

Enzima Glukosa oksidase Laktasel Lipasee Rennetm Pektinasen Pektin liase Proteasem Raffinaseo EC numberb 1.1.3.4 3.2.1.23 3.1.1.3 3.4.23.6 3.2.1.15 4.2.2.10 3.4.23.6 3.2.1.22 Sumber Aspergillus Aspergillus Rhizopus Mucor miehei Aspergillus Aspergillus Aspergillus Mortierella Intra/extr a -selulerc I E E E E E E I Skala produksid ++ ++ + Penggunaan Industri Makanan Susu Makanan Keju Minuman Minuman Roti Makanan

Enzim-Enzim Komersial yang Diperoleh dari Khamir

Enzima Invertasep Laktasel Lipasee Raffinaseo EC numberb 3.2.1.26 3.2.1.23 3.1.1.3 3.2.1.22 Sumber Saccharomyces Kluyveromyces Candida Saccharomyces Intra/extra -selulerc I/E I/E E I Skala produksid Penggunaan Industri Gula-gula Susu Makanan Makanan

Enzim-Enzim Komersial yang Diperoleh dari Bakteri

Enzima a-Amiase b-Amilase Asparaginase Glukosa isomeraseh Penisilin amidase Proteasei EC numberb 3.2.1.1 3.2.1.2 3.5.1.1 5.3.1.5 3.5.1.11 3.4.21.14 Sumber Intra/extra -selulerc E E I I I E Skala produksid +++ + ++ +++ Penggunaan Industri Pati Pati Kesehatan

Bacillus Bacillus Escherichia coli Bacillus Bacillus Bacillus

Sirup Fruktosa
Farmasi Detergen

Pululanasej

3.2.1.41

Klebsiella

Pati

Regulasi Enzim

Competitive inhibitors

Many drugs (like Cipro and anti-HIV drugs) are enzyme inhibitors Two major kinds of inhibitors: competitive and noncompetitive. Competitive inhibitors bind to the active site of the enzyme. Alter Km but not Vmax.

Noncompetitive inhibitors

Noncompetitive inhibitors bind somewhere besides the active site. They alter the behavior of the enzyme in a manner analogous to allosteric regulation