lecture 4 teknologi enzim
DESCRIPTION
Kuliah 4 Teknologi EnzimFARMASI – UHAMKA 2012Priyo WahyudiTeknologi Enzim & Protein Sel Tunggala.b.c. d.e.f.Definisi enzim Arti penting enzim Aplikasi teknologi enzim pada industri Sumber enzim Produksi enzim Karakterisasi dan aktivitas enzimWhat is Enzyme ?Most enzymes are Proteins (tertiary and quaternary structures) Act as Catalyst to accelerates a reaction Not permanently changed in the processEnzimEnzim = Biokatalisator yang mendorong terjadinya prosesTRANSCRIPT
Kuliah 4 Teknologi Enzim
FARMASI – UHAMKA2012
Priyo Wahyudi
Teknologi Enzim & Protein Sel Tunggal
a. Definisi enzimb. Arti penting enzimc. Aplikasi teknologi enzim pada
industrid. Sumber enzime. Produksi enzimf. Karakterisasi dan aktivitas enzim
Most enzymes are Proteins (tertiary and quaternary structures)
Act as Catalyst to accelerates a reaction
Not permanently changed in the process
What is Enzyme ?
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)
Apoenzim + Koenzim Holoenzim
Tidak aktif Tidak aktif Aktif
Protein Molekul organikBM tinggi BM rendahTidak terdialisis Terdialisis 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 dapat berupa: Protein murni Gabungan protein dengan gugusan kimia
lainnya Enzim terdiri dari komponen:
Apoenzim, komponen protein dari sebuah enzim
Koenzim, komponen molekul organik dengan BM rendah
Enzim & energi aktivasi
Gambar skema pengaruh enzim dalam menurunkan energi aktivasi suatu reaksi
kimia
Faktor-faktor yang mempengaruhi Aktivitas
Enzim1. Konsentrasi
Substrat
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
Akti
vita
s en
zim
Faktor-faktor yang mempengaruhi Aktivitas
Enzim2. Konsentrasi
Enzim
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
Akti
vita
s en
zim
Faktor-faktor yang mempengaruhi Aktivitas
Enzim3. 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
Akti
vita
s en
zim
3 4 5 6 7 8 9 10 11 12
pH
Faktor-faktor yang mempengaruhi Aktivitas
Enzim4. 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
Akti
vita
s en
zim
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. Oksidoreduktase, Reaksi transfer elektron (pemindahan elektron atau atom Hidrogen)
2. Transferase, Transfer gugus fungsional (cont: Fosfat, amino, metil, dll.)
3. Hidrolase, Reaksi hidrolisis (penambahan molekul air untuk memecahkan ikatan kimiawi)
4. Lyase, Penambahan ikatan ganda pada molekul dan juga pengusiran non hidrolitik gugus kimiawi
5. Isomerase, Reaksi isomerisasi (pengubahan suatu senyawa menjadi isomer, misal. Suatu senyawa yang memiliki atom-atom yang sama tetapi berbeda struktur molekulnya)
6. 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 Makanan
Protease mengempukkan daging Industri
Industri pulp, kertas, kain Industri sirup fruktosa
Kesehatan Amilase Protease Lipase
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
Speciality enzymes Low volume (g – kg) High purity High cost ($5 – 10,000 per g) High profit margins
Enzymes in Industry
Distribution of enzymes by substrate
Protein hydrolysing 59%
Carbohydrate hydrolysing
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
Other
Textile processing 10Grain processing 12Food processing 18Cleaning 44Feed enzymes 4Diagnostic/pharma 4Waste management 4Other 4
Process % by value
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
Textiles
Grain 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
Conversion of corn starch to high fructose syrups
Industrial enzymes
Feed enzymes
Waste management
Diagnostic enzymes
Enzymes to assist in the digestibility of animal feeds (cellulase, xylanase, phytase)
Lipases as drain-cleaning agents
Reporter enzymes (alkaline phosphatase, glucose oxidase, b-glucosidase) and diagnostic enzymes (DNA polymerase)
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
Denim is faded by abrasive action of
pumice stones
Indigo dye adheresto denim surface
Cellulase enzymeremoves some of the dye
by partially hydrolyzing thecotton surface
• new looks• lower costs• shorter treatment times • less solid waste
• weakens the fabric
traditionalmethod
new method
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. • αKG, α-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 Therapeutic Use Basis Additional InformationProlactazyme Lactose Intolerance Prolactazyme is a proenzmye that
produces lactase in stomach.About 75% of the world’s 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 hereAglucerase Gaucher’s Disease type
IEnzyme replacement therapy This disease is characterized by the lack
of enzyme glucocerebrocidase.Streptokinase Heart Attacks (Myocar
dial Infarction)Used as “clot blusters” to dissolve clots in the arteries of heart wall. Plasminogen is converted to plasmin which is fibrinolytic.
Administered i.v. to patients as soon as possible after the onset of a heart attack
Asparaginase Acute Childhood Leukemia
Decreased level of serum asparagine and inhibition of aspargine dependent multiplication of tumor cells.
Tumor cells cannot synthesize L-asparagine due to lack of aspartate-ammonia ligase.
Collagenase Skin ulcers Causes collagen hydrolysis Break up and remove dead skin and tissue
DNAse Cystic Fibrosis (CF) DNAse hydrolyses extracellular DNA responsible for Cystic Fibrosis.
DNA present in the mucous, whicharises from dead WBCs and bacterial cells, serves to cross link the mucous, changing it from a fluid gel to a semi-solid.
Lysozyme Antibiotic Therapy Causes Bacterial cell wall hydrolysisRibonuclease Antiviral Therapy Causes RNA hydrolysisTrypsin Inflammation Causes Protein hydrolysisUricase Gout Converts Urate to allantoinEnzyme inhibitors
To increase the efficacy of drugs
Against resistant bacterisa Example: Beta lactamase inhibitor like clavulanic acid along with amoxicillin.
Sumber Enzim
Virus : Enzim reverse transcriptase
Bakteri : Enzim DNA endonuclease, DNA polimerase Enzim protease, lipase, amilase
Fungi Enzim selulase Enzim protease, lipase, amilase
Tanaman Enzim protease
Hewan Enzim selulase
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
Enzima EC numberb Sumber
Intra/extra
-selulerc
Skala produksid
Penggunaan
Industri
Katalase 1.11.1.6 Hati I - Makanan
Khimotripsin 3.4.21.1 Pankreas E - Kulit
Lipasee 3.1.1.3 Pankreas E - Makanan
Rennetf 3.4.23.4 Abomasum E + Keju
Tripsin 3.4.21.4 Pankreas E - Kulit
Enzim-Enzim Komersial yang Diperoleh dari
Hewan
Enzima EC numberb Sumber
Intra/extra
-selulerc
Skala produksid
Penggunaan
Industri
Aktinidin 3.4.22.14 Buah Kiwi E - Makanan
a-Amilase 3.2.1.1 Malted barley E +++ Brewing
b-Amilase 3.2.1.2 Malted barley E +++ Brewing
Bromelain 3.4.22.4 Getah nanas E - Brewing
b-Glukanaseg 3.2.1.6 Malted
barley E ++ Brewing
Fisin 3.4.22.3 Fig latex E - Makanan
Lipoksigenase
1.13.11.12 Kedelai I - Makanan
Papain 3.4.22.2 Getah pepaya E ++ Daging
Enzim-Enzim Komersial yang Diperoleh dari
Tanaman
Enzima
EC number
b
SumberIntra/extra
-selulerc
Skala produksi
d
Penggunaan
Industri
a-Amilase 3.2.1.1 Aspergillus E ++ Roti
Aminoasilase
3.5.1.14
Aspergillus I - Farmasi
Glukoamilasek 3.2.1.3 Aspergillu
s E +++ Pati
Katalase 1.11.1.6
Aspergillus I - Makanan
Selulase 3.2.1.4 Trichoderma E - Pengolah
an limbah
Dekstranase
3.2.1.11
Penicillium E - Makanan
Enzim-Enzim Komersial yang Diperoleh dari
Jamur
Enzima
EC number
b
SumberIntra/extra
-selulerc
Skala produksi
d
Penggunaan
Industri
Glukosa oksidase 1.1.3.4 Aspergillu
s I - Makanan
Laktasel 3.2.1.23
Aspergillus E - Susu
Lipasee 3.1.1.3 Rhizopus E - Makanan
Rennetm 3.4.23.6
Mucor miehei E ++ Keju
Pektinasen 3.2.1.15
Aspergillus E ++ Minuman
Pektin liase 4.2.2.10
Aspergillus E - Minuman
Proteasem 3.4.23.6
Aspergillus E + Roti
Raffinaseo 3.2.1.22
Mortierella I - Makanan
Enzim-Enzim Komersial yang Diperoleh dari
Jamur
Enzima
EC number
b
SumberIntra/extra
-selulerc
Skala produksid
Penggunaan
Industri
Invertasep 3.2.1.26 Saccharomyces I/E - Gula-
gula
Laktasel 3.2.1.23 Kluyveromyces I/E - Susu
Lipasee 3.1.1.3 Candida E - Makanan
Raffinaseo 3.2.1.22 Saccharomyces I - Makanan
Enzim-Enzim Komersial yang Diperoleh dari
Khamir
Enzima
EC number
b
SumberIntra/extra
-selulerc
Skala produksid
Penggunaan
Industri
a-Amiase 3.2.1.1 Bacillus E +++ Pati
b-Amilase 3.2.1.2 Bacillus E + Pati
Asparaginase 3.5.1.1 Escherichia coli I - Kesehata
n
Glukosa isomeraseh 5.3.1.5 Bacillus I ++ Sirup
Fruktosa
Penisilin amidase
3.5.1.11 Bacillus I - Farmasi
Proteasei 3.4.21.14 Bacillus E +++ Detergen
Pululanasej 3.2.1.41 Klebsiella E - Pati
Enzim-Enzim Komersial yang Diperoleh dari
Bakteri
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