ISOLASI DAN KARAKTERISASI

EKSTRAK KASAR PROTEASE

Kelompok 6

Chofifah Fitrotul Hasanah

Moch. Akbar Reno Marsasi
Titis Rahmawati

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 Alat dan Bahan
Akuades
Shaker Biakan murni Pseudomonas sp. MD 24
Lampu spiritus Nutrient broth
Tabung reaksi Pepton
Stopwatch Glukosa
Pipet ukur Kalium dihidrofosfat
Pipet volume Magnesium sulfat heptahidrat
Pipet mikro Besi(ii) sulfat heptahidrat
Kertas saring Larutan TCA 20%
Larutan buffer fosfat 0,1 M pH 8
Sentrifuge
Larutan asam klorida 0,1 M
Seperangkat spektrofotometer UV-Vis
Larutan natrium hidroksida 0,5 M
Autoklaf
Larutan kasein 2% (dalam larutan buffer fosfat 0,1 M
Tusuk gigi steril / kawat ose steril ph 8),
Gelas kimia Larutan tripsin 0,4% (dalam larutan buffer fosfat 0,1
Rak tabung reaksi M ph 8)
Penangas air Reagen Biuret
Reagen Folin-Ciocalteu
Batang pengaduk
Larutan BSA 200 μg/ml.
 Metode (Pembuatan Media Starter)
0,16 g nutrient broth
• dilarutkan dalam 20 mL akuades • Nutrient broth difungsikan sebagai
sumber nutrien bagi mikroba
• diaduk • Untuk menghomogenkan campuran
• diautoklaf pada suhu 121°C, teka • Untuk mensterilisasi campuran dari
nan 15 psi selama 15 menit mikroba kontaminan

Media starter
 Metode (Pembuatan Media Produksi)

0,38 g pepton; 0,25 g glukosa; Modern

0,25 g kalium dihidrofo
PowerPoint
Presentation
sfat; 0,25 g magnesium sulfat heptahidrat; 0,005 g
besi(II) sulfat heptahidrat

• dilarutkan dalam akuades • Pepton berperan sebagai sumber protein,

glukosa sumber karbohidrat, Mg dan Fe
sebagai sumber mikronutrien dan KH2PO4
sebagai buffer sel
• diaduk
• Untuk menghomogenkan campuran
• diatur pHnya hingga 7 • Untuk menyesuaikan pH optimum mikroba
• diautoklaf pada suhu 121°C, tekanan • Untuk mensterilisasi campuran dari mikroba
15 psi selama 15 menit kontaminan

Media produksi
 Metode (Pembuatan Starter)

Isolat MD 24

• diambil sebanyak 2 ose

• diinokulasikan dalam 20 mL
media starter
• diinkubasi pada shaker dengan
penggojogan sekitar 100 rpm
selama semalam pada suhu 37°C
Kultur starter
• Untuk memindahkan mikroba
• Untuk menghasilkan kondisi optimum
pertumbuhan mikroba dan memberi-
nya waktu untuk berkembang biak
serta beradaptasi
 Metode (Produksi Ekstrak Kasar Protease)
Kultur starter berumur 20 jam

• diambil sebanyak 2 mL
• diinokulasikan dalam 50 mL media
produksi
• diinkubasi pada shaker dengan penggojog
an sekitar 100 rpm selama 2-5 hari pada
suhu 37°C
• disentrifugasi pada 2500 rpm selama 5
menit
• Untuk memindahkan mikroba
• Untuk menghasilkan kondisi optimum
pertumbuhan mikroba dan memberinya
waktu untuk menghasilkan protease
secara maksimal
• Untuk memisahkan ekstrak kasar
protease dengan sel

Residu
Ekstrak kasar protease
 Metode (Penentuan Aktivitas Enzim)
2 mL buffer fosfat 0,1 M pH 8; 1 mL tripsin 0,4%
• dicampurkan dalam tabung reaksi AT0
• ditambah 3 mL TCA 20%
• divortek
• diinkubasi selama 30 menit dalam
penangas air 35°C
• ditambahkan 4 mL kasein 2%
• divortek
• direndam dalam air es selama 15 menit
• disentrifuge 5 menit
Supernatan AT0 Residu
• Buffer untuk mengatur pH optimum, tripsin
sebagai kontrol positif aktivitas protease
• Untuk mendeaktivasi enzim
• Untuk menghomogenkan
• Untuk menghasilkan kondisi optimum aktivitas
enzim
• Kasein sebagai protein target untuk aktivitas
enzim
• Untuk menghomogenkan
• Untuk mengendapkan protein
• Untuk memisahkan peptida pendek larut air dan
protein
 Metode (Penentuan Aktivitas Enzim)

2 mL buffer fosfat 0,1 M pH 8; 1 mL ekstrak

kasar protease • Buffer untuk mengatur pH optimum

• dicampurkan dalam tabung reaksi AT0 • Untuk mendeaktivasi enzim

• Untuk menghomogenkan
• ditambah 3 mL TCA 20% • Untuk menghasilkan kondisi optimum
• divortek
aktivitas enzim
• diinkubasi selama 30 menit dalam penangas
air 35°C
• ditambahkan 4 mL kasein 2% • Kasein sebagai protein target untuk
• divortek aktivitas enzim
• direndam dalam air es selama 15 menit • Untuk menghomogenkan
• disentrifuge 5 menit • Untuk mengendapkan protein
• Untuk memisahkan peptida pendek
Supernatan AE0 larut air dan protein
Residu
 Metode (Penentuan Aktivitas Enzim)
4 mL kasein 2%

• dimasukkan dalam tabung reaksi AT20
• diinkubasi selama 5 menit dalam penangas
air 35°C
• ditambahkan 2 mL buffer fosfat 0,1 M pH 8
• divortek
• ditambahkan 1 mL tripsin 0,4%
• divortek
• diinkubasi selama 20 menit dalam penanga
s air 35°C
• ditambah 3 mL TCA 20%
• divortek
• direndam dalam air es selama 15 menit
• disentrifuge 5 menit
• Kasein sebagai protein target untuk aktivitas enzim
• Untuk menghasilkan kondisi optimum aktivitas enzim
• Untuk menghasilkan kondisi optimum aktivitas enzim
• Untuk menghomogenkan
• Tripsin sebagai kontrol positif aktivitas protease
• Untuk menghomogenkan
• Untuk memberi enzim waktu beraktivitas selama 20
menit pada suhu optimumnya
• Untuk menghentikan kerja enzim
• Untuk menghomogenkan
• Untuk mengendapkan protein
• Untuk memisahkan peptida pendek larut air dan
protein

Supernatan AT Resid
20 u
 Metode (Penentuan Aktivitas Enzim)
4 mL kasein 2%

• Kasein sebagai protein target untuk aktivitas enzim

• dimasukkan dalam tabung reaksi AE20
• Untuk menghasilkan kondisi optimum aktivitas enzim
• diinkubasi selama 5 menit dalam penangas air
35°C
• Untuk menghasilkan kondisi optimum aktivitas enzim
• ditambahkan 2 mL buffer fosfat 0,1 M pH 8
• divortek
• Untuk menghomogenkan
• ditambahkan 1 mL ekstrak kasar protease
• Tripsin sebagai kontrol positif aktivitas protease
• divortek
• Untuk menghomogenkan
• diinkubasi selama 20 menit dalam penangas a
• Untuk memberi enzim waktu beraktivitas selama 20 menit
ir 35°C
pada suhu optimumnya
• ditambah 3 mL TCA 20%
• Untuk menghentikan kerja enzim
• divortek
• Untuk menghomogenkan
• direndam dalam air es selama 15 menit
• Untuk mengendapkan protein
• disentrifuge 5 menit
• Untuk memisahkan peptida pendek larut air dan protein

Supernatan AE20 Residu

 Metode (Penentuan Konsentrasi Peptida Pendek
Metode ANSON)

2 mL supernatan AT0; AE0; AT20 dan AE20

• Untuk menetralkan suasana asam dari TCA

• ditambahkan 4 mL NaOH 0,5 M
• Untuk membentuk kompleks biru
• ditambah 1 mL reagen Folin-Ciocalteu
• Untuk menghomogenkan
• divortek
• Untuk memberi waktu agar reaksi berjalan
• didiamkan selama 5 menit
sempurna
• diukur absorbansinya pada panjang
• Untuk mengukur intensitas warna / absorbansi
gelombang 650 nm
dari kompleks hasil reaksi

Hasil
Metode (Penentuan Konsentrasi Protein Metode Lowry)
BSA 200 μg/mL
• diencerkan dengan akuades untuk membuat
masing-masing 1 mL larutan standar 0; 20; 40
; 60; 80; 100 dan 200 μg/mL
@1 mL BSA 0; 20; 40; 60; 80; 100 dan 200 μg/mL
• ditambahkan 5 mL reagen Biuret
• diinkubasi pada suhu kamar selama 10 menit
• ditambahkan 0,5 mL reagen Folin-Ciocalteu
• diaduk
• diinkubasi pada suhu kamar selama 30 menit
• diukur absorbansinya pada panjang gelombang
750 nm
Hasil
• Untuk membuat kurva kalibrasi standar serapan kompl
eks protein nantinya pada berbagai konsentrasi
• Agar bereaksi membuat kompleks dengan protein
• Agar reaksi berjalan sempurna
• Untuk meningkatkan sensitivitas warna Biuret agar hasi
l pengukurannya bagus
• Untuk menghomogenkan
• Agar reaksi berjalan sempurna
• Untuk mengukur intensitas warna / absorbansi dari ko
mpleks hasil reaksi
 Metode (Penentuan Konsentrasi
Protein Metode Lowry)

@1 mL tripsin dan ekstrak kasar enzim

• ditambahkan 5 mL reagen Biuret • Agar bereaksi membuat kompleks dengan protein

• diinkubasi pada suhu kamar selama 10 menit
• ditambahkan 0,5 mL reagen Folin-Ciocalteu
• diaduk
• diinkubasi pada suhu kamar selama 30 menit
• diukur absorbansinya pada panjang
gelombang 750 nm
• Agar reaksi berjalan sempurna
• Untuk meningkatkan sensitivitas warna Biuret agar
hasil pengukurannya bagus
• Untuk menghomogenkan
• Agar reaksi berjalan sempurna
• Untuk mengukur intensitas warna / absorbansi dari
kompleks hasil reaksi

Hasil
 Hasil
Absorbansi
Kode tabung
650 nm Nomor Tabun Konsentrasi ( Absorbansi 7

AT20 0,328 g μg/mL) 50 nm

AT0 0,015 1 0 0,000

AE20 0,368 2 20 0,075

AE0 0,083 3 40 0,189

4 60 0,196
5 80 0,329
6 100 0,466
7 120 0,496
Tripsin - 0,065
Ekstrak kasar - 0,505
 Analisis Data dan Pembahasan

Absorbansi sampel−Absorbansi kontrol

Aktivitas enzim =
waktu

AT20−AT0 0,328 − 0,015

 Aktivitas tripsin = = = 0,01565 Unit
waktu 20

AE20−AE0 0,368 − 0,083

 Aktivitas ekstrak kasar enzim = = = 0,01425 Unit
waktu 20
 Analisis Data dan Pembahasan

Kurva Standar Protein

0.5

0.4
Absorbansi 750 nm

0.3
y = 0.0044x - 0.0122
R² = 0.9617
0.2

0.1

0
0 20 40 60 80 100 120

-0.1
Konsentrasi BSA (μg/mL)
 Analisis Data dan Pembahasan
 Kadar protein dari ekstrak kasar enzim
 Kadar tripsin y = 0,0044X – 0,012
y = 0,0044X – 0,012 0,505 = 0,0044X – 0,0122
0,065 = 0,0044X – 0,0122 X=
0,505+0,0122
= 117,545 μg/mL
0,0044
0,065+0,0122
X= = 17,545 μg/mL
0,0044

kadar spesifik ekstrak kasar enzim

kadar spesifik tripsin aktivitas ekstrak kasar enzim (U) 0,01425 μg/mL 103 μg
= = x =
kadar ekstrak kasar enzim 117,545 μg/mL 1 mg
aktivitas tripsin (U) 0,01565 μg/mL 103 μg
= = x = 0,121 U/mg
kadar tripsin 17,545 μg/mL 1 mg

0,892 U/mg
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