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Lampiran 1. Uji Biokimia Isolat MBXi-K4 No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Jenis Uji Oksidase Motilitas Nitrat Lisin Ornitin H2S Glukosa Manitol Xilosa ONPG Indol Urease Voges-Proskauer (VP) Sitrat TDA Gelatin Malonat Inositol Sorbitol Rhamnosa Sukrosa Laktosa Arabinosa Adonitol Rafinosa Salisin Arginin Bentuk sel Pewarnaan Gram Pigmen
Hasil + + + + + + + Bulat/coccus + Kuning
Dari hasil uji biokimia tersebut, Isolat MBXi-K4 adalah : Staphylococcus aureus
63
Lampiran 2. Isolat MBXi-K4 M
G Gambar 1. Isoolat MBXi-K K4 umur 24 jjam.
Gambar 2. Pewarnaan Gram isolatt MBXi-K4
64
Lampiran 3. Komponen Reagen Dinitrosalisilic Acid (DNS) (Miller, 1959) Bahan NaOH padat NaK Tartarat Na2SO3 Dinitrosalisilic Acid (DNS) aquades
Jumlah 10 gram 182 gram 0,5gram 10 gram Ditera sampai 1000 ml
Sebanyak 10 gram NaOH, 182 gram NaK Tartarat dan 5 gram Na2SO3 dilarutkan dalam 500 ml aquades.
Ditambahkan 10 gram asam dinitrosalisilat
sedikit demi sedikit sambil diaduk dengan pengaduk bermagnet (magnetic stirrer). Setelah larut ditambahkan aquades dan ditera sampai volume 1000 ml. Larutan disimpan dalam botol gelap pada suhu dingin. Lampiran 4. Penentuan Aktifitas Xilanase Bahan
Jumlah
kontrol
sampel
Blanko
Larutan substrat dalam buffer Enzim
100 µl
Ya
Ya
-
100 µl
Ya(setela h DNS) Ya
Ya
-
Ya(setelah Inkubasi) -
Ya
1000 µl DNS Aquades
ya
-
Aktivitas xilanase (U/ml) dihitung berdasarkan rumus: Aktifitas Xilanase (unit/mL) =
(Csp − Ckt)×1000× fp T × BMXilosa
Keterangan : Csp
= Kadar xilosa sampel
Ckt
= Kadar xilosa kontrol
T
= waktu inkubasi (30 menit)
Fp
= faktor pengenceran
BM xilosa= Berat molekul xilosa : 150,3 g/mol 65
Lampiran 5. Kurva standar xilossa
Lampiran 6. Kompossisi Reagen Bradford (B Bradford, 1976) Bahan B Coomassie C B Brilliant Bluee G250 Ethanol E 95% Asam A fosfat 85% 8 Aquades A
jumlah 100 mg 50 ml 100 ml Ditera sampai s 1000 ml
Sebaanyak 100 mg m CBB G-250 ditambahhkan kedalam m 50 ml ethaanol 95% daan 100 ml asaam fosfat 85%, diadukk sampai CB BB benar-beenar larut dan d homogenn. Kemudian ditambahkaan aquades ditera d sampaai 1000 ml diaduk sam mpai homogeen dan disaring menggunaakan kertas saring s Whatm man no 1 unntuk memisaahkan partikeel yang tidak larut. Larutaan disimpann dalam botol gelap padaa suhu dinginn.
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Lampiran 7. Kurva Standar S Prootein
Lampiran 8. Kompossisi gel dan pereaksi p un ntuk elektrooforesis Komposisi pereaksi unttuk elektrofooresis 1. Larutan A (30 % (b//v) akrilamidd; 0,8% (b/v) bis-akrilam mid) Sebanyak 14,6 gram m akrilamid dan 0,4 graam bis-akrilaamid dilarutkkan dalam 50 5 ml kem mudian diadukk dengan peengaduk maggnetik hingga larut dan homogen. h 2. Larutan B (buffer geel pemisah , TrisCl 2 M, pH 8,8) Sebanyaak 75 ml laarutan TrisC Cl pH 8,8 dan 4 ml larutan SDS 10% (b/vv) ditambah hkan aquadees hingga volume total 100 ml. 3. Larutan C (buffer geel penahan , TrisCl 1 M, pH 6,8) Sebanyaak 50 ml laarutan Tris Cl pH 6,8 dan 4 ml larutan SD DS 10% (b/vv) ditambah hkan aquadees hingga volume total 100 ml. 4. Ammon nium per sulffat 10% (b/vv) Sebanyaak 0,1 gram ammonium a p persulfat dilaarutkan dalaam 1 ml aquaades. 5. Buffer elektroforesis e s Sebanyaak 1,803 gram Tris-basee; 8,648 gram m lisin dan 0,6 gram SD DS dilarutkaan dalam 60 00 ml aquaddes lalu diteraa hingga pH H 8,3 dengan HCl 1 M. 67
6. Buffer sampel Untuk SDS PAGE: 0,3 ml TrisCl 1 mM pH 6,8; 2,5 ml gliserol 50% (v/v); 1,0 ml SDS 10% (b/v) ; 0,25 ml 2-merkaptoethanol ; 0,5 ml biru bromfenol 1% (b/v) dan 0,45 ml aquades.
Komposisi gel penahan dan pemisah SDS PAGE Komponen
Gel
pemisah
Gel penahan (4%)
(10%) Larutan A Larutan B Larutan C Aquades Ammonium 10% TEMED
persulfat
3,333 ml 2,500 ml 4,057 ml 0,100 ml 0,010 ml
0,67 ml 1,25 ml 3,00 ml 50,0 µl 5,00 µl
Lampiran 9. Pereaksi dan Prosedur Pewarnaan Perak 1. Larutan fiksasi Sebanyak 25% methanol dan 12% asam asetat dilarutkan dalam aquabedestilata 2. Larutan methanol 50% dan 30% Sebanyak 50 ml methanol dilarutkan dalam 50 ml aquabidestilata (50%) dan sebanyak 30 ml methanol dilarutkan dalam 70 ml aquadestilata (70%). 3. Larutan pengembang Sebanyak 0,1 g Na2S2O3.5H2O dilarutkan dalam 500 ml aquabidestilata 4. Larutan perak nitrat Sebanyak 0,4 g AgNO3 dan 70 µl formaldehide dilarutkan dalam 250 ml aquabidestilata. Prosedur pewarnaan perak 1. Fiksasi gel dalam larutan fiksasi, selama 1 jam 2. Rehidrasi dalam larutan ethanol 50% selama 20 menit 3. Gel dicuci dengan ethanol 30% selama 20 menit (dilakukan 2 kali) 4. Gel direndam dalam larutan enhancer (larutan Na2S2O3) selama 1 – 2 menit 68
5. Gel dicuci dalam aqua bebas ion selama 20 detik (dilakukan 3 kali) 6. Gel diwarnai dengan larutan silver (larutan 0,1 g AgNO3 dalam 50 ml aqua bebas ion, ditambah 17,5 µl formaldehide 7. Gel dicuci dengan aqua bebas ion selama 20 detik (dilakukan 2 kali) 8. Gel direndam dalam larutan developer (larutan 1,5 g NaCO3 dalam 25 ml aqua bebas ion, ditambah 12 µl formaldehide) hingga muncul pita-pita proteinnya. 9. Reaksi dihentikan dengan larutan fiksasi
Lampiran 10. Kurva standar marker elektroforesis (Pharmacia) dan perhitungan bobot molekul sampel (SDSPAGE) Protein
BM
Log BM
phosporilase b Albumin Ovalbumin Carbonic anhidrase Trypsin inhibitor Lysozyme Sampel gel filtrasi
97000 66000 45000 30000 20100 14400 47905,57
4.987 4.820 4.653 4.477 4.303 4.158 4,680
Band (cm)
Rf
0.5 1.2 2 2.6
0.11627907 0.27906976 0.46511627 0.60465116
1,8
0,418605
Kurva standar marker elektroforesis (SDS PAGE) 5,1 5
Log BM
4,9 y = ‐1,023x + 5,108 R² = 0,995
4,8 4,7
Rf
4,6 4,5 4,4 0
0,2
0,4
0,6
0,8
jarak Rf
69
Lampiran 11. Kurva standar marker zimogram ( LMW Fermentas) dan kurva standar zimogram
Protein
BM
ß-galactosidase Bovine Serum Albumin Ovalbumin Lactate dehydrogenase RE-ase Bsp981 ß-Lactoglobulin Lysozyme Sampel gel filtrasi
Log BM
116000 66200 45000 35000 25000 18400 14400 21631,9
5.06446 4.82086 4.65321 4.54407 4.39794 4.26482 4.15836 4,33509
Band (cm)
Rf
0.2 0.5 1.2 1.6 2.1 2.9
0.03125 0.07813 0.18750 0.25000 0.32813 0.45313
3,8
0.59375
Kurva standar bobot molekul zimogram 4,9 4,8
Log BM
4,7
y = ‐1,023x + 4,952 R² = 0,993
4,6 4,5
log BM
4,4 4,3 4,2 4,1 0
0,2
0,4
0,6
0,8
1
jarak Rf
70
71
