DEWI SUSAN B (20509040) ISTHY EKA R (20509044) Cellobiose dehydrogenase DEWI SUSAN B (20509040) ISTHY EKA R (20509044)

AGENDA PENDAHULUAN APLIKASI KESIMPULAN 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" AGENDA PENDAHULUAN ISOLASI, PEMURNIAN, DAN KARAKTERISASI APLIKASI KESIMPULAN

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" 12/04/2017 Cellobiose dehydrogenase... ??? Cellobiose dehydrogenase merupakan enzim yang mengkatalisis reaksi oksidasi-reduksi Pada umumnya terdapat pada white-rot ,soft-rot ,brown-rot fungi (Co: Termitomyces clypeatus, Sclerotium rolfsii, Ceriporiopsis subvermispora) Berperan dalam transfer elektron dari cellobiose dan cello-oligomers ke berbagai akseptor (co : ion logam & senyawa organik) yang dimediasi oleh gugus heme dan flavin pada enzim

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" Tatanama 1 1 99 18 Kelas 1: Oxidoreductases Enzim yang mengkatalisis reaksi oksidasi-reduksi Sub-kelas 1.1 Acting on the CH-OH group of donors EC 1.1.99 With other acceptors Contoh : 2,6 dichloroindophenol ;phenol blue; cytochrome c EC 1.1.99.18 cellobiose dehydrogenase

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" Nama enzim Nama lain cellobiose dehydrogenase; cellobiose oxidoreductase; Phanerochaete chrysosporium cellobiose oxidoreductase; CBOR; cellobiose oxidase; cellobiose:oxygen 1-oxidoreductase; CDH; cellobiose:(acceptor) 1-oxidoreductase Nama Sistematis cellobiose:acceptor 1-oxidoreductase

13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" FUNGSI ENZIM Mengoksidasi cellobiose menjadi cellobionic acid dan lactobionic acid Berperan dalam hidrolisis selulosa, hemiselulosa atau lignin biosensor, bioremediation,biocatalysis

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" STRUKTUR ENZIM Cellobiose dehydrogenase flavoprotein fragment in complex with cellobionolactam Ligand Interaction fo the CDH active site

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" STRUKTUR ENZIM Ligand Interaction fo the CDH active site

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" STRUKTUR ENZIM Hasil X-Ray CDH

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" SUBSTRAT ENZIM Karbohidrat Co: Selobiosa,laktosa,maltosa dll. Akseptor elektron Co: 2,6 dichloroindophenol; phenol blue; cytochrome c

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" CELLOBIOSA Disakarida dengan ikatan β(1→4) glikosidik Rumus Molekul : [HOCH2CHO(CHOH)3]2O Dihasilkan secara enzimatis dari hidrolisis selulosa

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" KOFAKTOR ENZIM FAD Heme

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" Mekanisme Reaksi

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" MEKANISME

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" Produk cellobiono-1,5-lactone reduced acceptor

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" 12/04/2017 METODOLOGI Cultivation Pemurnian Karakterisasi Deglikosilasi SDS-Page & Isoelectric focusing Uji Aktivitas Protein determination Parameter Kinetik

Cultivation Standard cultivation Bioreactor cultivation Medium Medium 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" Cultivation Standard cultivation Bioreactor cultivation Medium (30 g /L yeast extract, 30 g/L cellulose, 1 g/L MgSO4.7H2O, 0,3 ml/L trace element solution) Medium (30 g /L yeast extract, 30 g/L cellulose, 1 g/L MgSO4.7H2O, 0,3 ml/L trace element solution) pH 5,4; Autoclaves pH 5,4; Autoclave Inokulasi Inokulasi Shaking flask cultures (300 ml medium, 30°C, 110 rpm, 30 days) Shaking flask cultures (30°C, 150 rpm, 30 days)

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase"

Pemurnian Enzim Supernatan Mono Q column Sentrifuga Filtrasi 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" Pemurnian Enzim Supernatan Mono Q column Sentrifuga (6000 g/ 15 menit) Filtrasi (Polyethersulfone hollow fiber cross-flow module) Supernatan enzim PHE Sepharose Filtrasi (Polyethersulfone hollow fiber cross-flow module) DEAE-Sepharose

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase"

SDS Page & Isoelectric Focusing 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" SDS Page & Isoelectric Focusing

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" SPEKTRUM

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" 12/04/2017 PARAMETER KINETIK Berdasarkan deglikosilasi -Cellobiose memiliki nilai Km yang rendah dan efisiensi katalitik yang tinggi -Lactose memiliki nilai Km 30 kali lebih tinggi dan tidak terdapat substrate inhibition, serta nilai Kcat yang sedikit lebih tinggi dari selobiosa -Maltosa menghasilkan nilai Km yang sangat tinggi dan efisiensi katalitik yang sangat rendah -Glukosa juga menghasilkan nilai yang hampir sama dengan maltosa yang berbeda jauh dengan selobiosa -Nilai parameter yang hampir sama didapatkan dari electron acceptors DCIP and cyt c -Pada cellobiose terjadi perbedaan nilai KM and KI values observed when using cyt c.

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" 12/04/2017 PARAMETER KINETIK The almost-twofold-higher kcat values of the one-electron acceptors over the two-electron acceptors indicate that the rate-limiting step is either the binding or oxidation of the carbohydrate and not the binding of the electron acceptor or subsequent electron transfer. The affinity of CDH for electron acceptors is generally high, resulting in low KM values, especially for ferricenium and cyt c. These values yield catalytic efficiencies between 1.7 106 and 1.8 108 M1 s1, revealing a very efficient and almost diffusionlimited reoxidation process.

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" 12/04/2017 AKTIVITAS VS SUHU The interdomain electron transfer and the “temperature stability” of this transfer were investigated using the standard DCIP and cyt c assays at different temperatures, which were run for 5 min and the average activity calculated (Fig. 5). The activity of the flavin domain (DCIP activity) increased with temperature until reaching a temperature maximum at 60°C. The activation energy was 18.7 kJ mol1, which is low in comparison to other values published for CDHs, and was calculated from initial rates in the range of 21 to 50°C. For the heme domain-dependent reaction (cyt c activity), the activity increment with higher temperatures was much lower, leveled off at 30°C, and decreased rapidly above 40°C. From the linear increase of the absorbance in cyt c assays up to 50°C, which indicates a stable steady-state turnover, it can be concluded that denaturation of the heme domain is not the cause of the decrease in activity, but there is a less efficient interdomain electron transfer between both domains at higher temperatures.

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" aplikasi Sebagai elektroda pada amperometri biosensor untuk mengukur selobiosa dan laktosa Pengukuran selobiosa secara kolorimetri Biosensor untuk mendeteksi laktosa pada makanan (penderita laktosa intoleran) Mengukur aktivitas selulosa dan mengontrol kadar laktosa pada pembuatan keju Bleaching pulp Degrading and modifying poisonous aromatic waste products

13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" 27 Kesimpulan Cellobiose dehydrogenase (CDH) merupakan enzim ekstraseuler yang dihasilkan oleh wood-degrading fungi Cellobiose dehydrogenase berperan dalam mengkatalisis reaksi oksidasi-reduksi pada karbohidrat seperti selobiosa CDH banyak digunakan sebagai biosensor 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase"

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase" DAFTAR PUSTAKA Harreither W., Sygmund C., Dünhofen E., Vicuna R., Haltrich D., Ludwig R., 2009. Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete Ceriporiopsis subvermispora . Appl. Environ. Microbiol. 75:9, 2750–2757 Hallberg B. , Henriksson G., Pettersson G., Vasella., Divne C., 2003. Mechanism of the Reductive Half-reaction in Cellobiose Dehydrogenase. The journal of biological chemistry. 278:9, 7160-7166 Henriksson G., Pettersson G., Johansson G., 2000. A critical review of cellobiose dehydrogenases. Journal of Biotechnology.78, 93–113

Presentasi Enzimologi " Cellobiose Dehydrogenase" 13/11/2009 Presentasi Enzimologi " Cellobiose Dehydrogenase"