DAFTAR ISI
Bab
I
II
III
Halaman DAFTAR TABEL ...........................................................................
ix
DAFTAR GAMBAR .......................................................................
x
DAFTAR LAMPIRAN ...................................................................
xii
PENDAHULUAN 1.1 Latar Belakang ........................................................................ 1.2 Identifikasi Masalah ................................................................ 1.3 Tujuan Penelitian..................................................................... 1.4 Kegunaan Penelitian................................................................ 1.5 Pendekatan Masalah ................................................................
1 2 2 3 3
KAJIAN PUSTAKA 2.1 Selulosa ................................................................................... 2.2 Enzim Selulase ........................................................................ 2.3 Bakteri Laut ............................................................................. 2.4 Bakteri Selulolitik ................................................................... 2.5 Penapisan Gen dengan Metode PCR ....................................... 2.6 Primer ...................................................................................... 2.7 Karakterisasi In Silico .............................................................
6 11 16 18 20 23 26
BAHAN DAN METODE 3.1 Tempat dan Waktu Penelitian ................................................. 3.1.1 Tempat Penelitian.................................................................... 3.1.2 Waktu Penelitian ..................................................................... 3.2 Alat dan Bahan Penelitian ....................................................... 3.2.1 Alat Penelitian ......................................................................... 3.2.2 Bahan Penelitian...................................................................... 3.3 Metode Penelitian.................................................................... 3.4 Prosedur Penelitian.................................................................. 3.4.1 Sterilisasi Alat dan Medium .................................................... 3.4.2 Peremajaan Biakan dan Kultur Padat ...................................... 3.4.3 Uji Aktivitas Selulolitik .......................................................... 3.4.4 Teknik Molekuler dengan Polymerase Chain Reaction (PCR) dan Bioinformatik ........................................................ 3.4.5 Service Sekuensing ................................................................. 3.4.6 Karakterisasi Secara In Silico (Analisis Bioinformatik Sekuen Gen Endoglukonase) .................................................. 3.5. Analisis Data ...........................................................................
vii
28 28 28 28 28 30 32 32 32 33 33 35 40 40 40
IV
V
HASIL DAN PEMBAHASAN 4.1 Peremajaan Biakan dan Kultur Padat ...................................... 4.2 Hasil Uji Aktivitas Selulolitik ................................................. 4.3 Teknik Molekuler dengan Polymerase Chain Reaction (PCR) dan Bioinformatik ........................................................ 4.3.1 Isolasi DNA Genom Bakteri ................................................... 4.3.2 Desain Primer Gen Pengkode Endoglukanase ........................ 4.3.3 Penapisan Gen Pengkode Endoglukanase Menggunakan Metode PCR ............................................................................ 4.3.4 Analisis Hasil Sekuensing Gen Pengkode Endoglukanase ..... 4.3.5 Analisis Hasil Karakterisasi In Silico Sekuen Gen Pengkode Endoglukanase ........................................................................
41 44 47 47 50 54 58 61
KESIMPULAN DAN SARAN 5.1 Kesimpulan.............................................................................. 5.2 Saran ........................................................................................
70 70
DAFTAR PUSTAKA ..............................................................................
71
LAMPIRAN ............................................................................................
80
RIWAYAT HIDUP .................................................................................
131
viii
DAFTAR TABEL
No
Judul
Halaman
1.
Hidrolisis Berbagai Substrat oleh Enzim Selulase ........................
14
2.
Substrat Selulosa Berdasarkan Kelarutan Air dan Jenis Enzim Selulase .........................................................................................
15
3.
Komponen Reaksi PCR .................................................................
38
4.
Pengaturan Program PCR ..............................................................
38
5.
Uji Aktivitas Selulolitik Pada Isolat Bakteri B.subtilis dan Bakteri B.thuringiensis ..............................................................................
45
6.
Primer Gen Endoglukanase Bakteri B.subtilis Desain Primer3 ....
50
7.
Primer Gen Endoglukanase Bakteri B.thuringiensis Desain Primer Degenerate ....................................................................................
53
Hasil Analisis Pensejajaran Berganda (BLASTX) Urutan Sekuen Hasil Sekuensing Template Bakteri B.subtilis dengan Data GeneBank ......................................................................................
60
Hasil Analisis Pensejajaran Berganda (BLASTX) Urutan Sekuen Hasil Sekuensing Template Bakteri B.thuringiensis dengan Data GeneBank..............................................................................
60
Matriks Perbandingan Sekuen Protein Gen Pengkode Endoglukanase Dari Bakteri B.subtilis dan B.thuringiensis .........
69
8.
9.
10.
ix
DAFTAR GAMBAR
No
Judul
Halaman
1.
Struktur Kimia Selulosa...................................................................
7
2.
Rumus Struktur α – selulosa ............................................................
8
3.
Rumus Struktur β – selulosa ............................................................
8
4.
Ikatan Hidrogen Intra dan Antar Rantai Selulosa ............................
10
5.
Model Fibril Struktur Supramolekul Selulosa ................................
10
6.
Skema Tahap-Tahap Pemecahan Selulosa ......................................
12
7.
Klasifikasi Enzim Selulase ..............................................................
14
8.
Prinsip dan Cara Kerja PCR ............................................................
22
9.
Elektroforegram Amplifikasi Fragmen Penyandi Kitinase Bakteri Kitinolitik.........................................................................................
23
10. Diagram Alir Penelitian ...................................................................
32
11. Hasil Peremajaan Biakan dan Kultur Padat Isolat Bakteri B.subtilis
43
12. Hasil Peremajaan Biakan dan Kultur Padat Isolat Bakteri B.thuringiensis .................................................................................
44
13. Uji Aktivitas Selulolitik Hasil Pewarnaan Red congo (Isolat B.1.2)
45
14. Uji Aktivitas Selulolitik Hasil Pewarnaan Red congo (Isolat C2)...
46
15. Elektroforegram Hasil Isolasi DNA Genom Bakteri B.subtilis .......
49
16. Elektroforegram Hasil Isolasi DNA Genom Bakteri B.thuringiensis
49
17. Ilustrasi Penempelan Primer Spesifik Gen Endoglukanase Pada Sekuen Konsensus Bakteri B.subtilis ..............................................
51
18. Ilustrasi Penempelan Pasangan Primer pada Struktur Utama Domain Fungsional Gen Endoglukanase Pada Sekuen Konsensus Bakteri B.subtilis .......................................................................................... 52 19. Ilustrasi Penempelan Primer Degenerate Gen Endoglukanase Pada Sekuen Hasil Contig Ketiga Database Bakteri B.thuringiensis .......
53
20. Ilustrasi Penempelan Pasangan Primer pada Struktur Utama Domain Fungsional Gen Endoglukanase Pada Sekuen Hasil Contig Bakteri B.thuringiensis ................................................................................. 54 21. Elektroforegram Amplikon Gen Endoglukanase Bakteri B.subtilis Suhu Annealing 600C (B.1.2) ..........................................................
x
56
22. Hasil Purifikasi Amplikon Gen Endoglukanase Bakteri B.subtilis (B.1.2) Oleh 1st BASE .....................................................................
57
23. Elektroforegram Amplikon Gen Endoglukanase Bakteri B.thuringiensis Suhu Annealing 550C (C2) .....................................
57
24. Hasil Purifikasi Amplikon Gen Endoglukanase Bakteri B.thuringiensis (C2) oleh 1st BASE .................................................
58
25. Struktur Domain Fungsional Sekuen Gen Pengkode Endoglukanase Bakteri B.subtilis .............................................................................
61
26. Sekuen Asam Amino Gen Pengkode Endoglukanase Bakteri B.subtilis ..........................................................................................
62
27. Struktur Domain Fungsional Sekuen Gen Pengkode Endoglukanase Bakteri B.thuringiensis ....................................................................
63
28. Kelompok Domain Glyco Hydro 8 (GH 8) .....................................
63
29. Sekuen Asam Amino Gen Pengkode Endoglukanase Bakteri B.thuringiensis .................................................................................
64
30. Daerah Sisi Aktif Gen Pengkode Endoglukanase Bakteri B.subtilis
65
31. Daerah Sisi Aktif Gen Pengkode Endoglukanase Bakteri B.thuringiensis .................................................................................
66
32. Motif Sekuen Protein Gen Pengkode Endoglukanase Bakteri B.subtilis ..........................................................................................
67
33. Motif Sekuen Protein Gen Pengkode Endoglukanase Bakteri B.thuringiensis .................................................................................
68
xi
DAFTAR LAMPIRAN
No
Judul
Halaman
1.
Tahap Melakukan Desain Primer3 Bakteri B.subtilis......................
80
2.
Tahap Melakukan Desain Primer Degenerate Bakteri B.thuringiensis 89
3.
Datasheet Oligonukleotida Primer Gen Endoglukanase Bakteri B.subtilis dan B.thuringiensis ............................................................... 102
4.
Alat-alat Peremajaan Biakan dan Kultur Padat .................................... 103
5.
Alat-alat Uji Aktivitas Selulolitik ......................................................... 104
6.
Alat-alat Kultur Cair dan Isolasi DNA Genom .................................. 105
7.
Alat-alat Amplifikasi ............................................................................ 106
8.
Alat-alat Elektroforesis ......................................................................... 107
9.
Bahan-bahan Peremajaan Biakan dan Kultur Padat ............................. 108
10. Bahan-bahan Uji Aktivitas Selulolitik .................................................. 109 11. Bahan-bahan Isolasi DNA Genom dan Kultur Cair ............................. 110 12. Bahan-bahan Amplifikasi ..................................................................... 111 13. Bahan-bahan Elektroforesis .................................................................. 112 14. Proses Peremajaan Biakan dan Kultur Padat ........................................ 113 15. Hasil Peremajaan Biakan dan Kultur Padat .......................................... 114 16. Hasil Pewarnaan Gram Bakteri ............................................................ 115 17. Proses Uji Aktivitas Selulolitik ............................................................ 116 18. Bagan Alir Kultur Cair dan Isolasi DNA Genom ................................. 117 19. Penapisan Gen Endoglukanase ............................................................. 119 20. Bagan Alir Elektroforesis ..................................................................... 120 21. Analisis Hasil Sekuensing Gen Endoglukanase ................................... 122 22. Hasil Sekuensing Sampel B.1.2 ............................................................ 127 23. Hasil BLAST Sampel B.1.2 ................................................................. 128 24. Hasil Sekuensing Sampel C2 ................................................................ 129 25. Hasil BLAST Sampel C2 ..................................................................... 130
xii
