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Kuriki, T., Imanaka, T., (1999), The Concept of the α-Amylase Family: Structural Similarity and Common Catalytic Mechanism, Journal of Bioscience and Bioengineering, 87 (5), 557565 Legin, E., Ladrat, C., Godfroy, A., Barbier, G., Duchiron, F., (1997), Thermostable amylolitic enzymes of thermophilic microorganisms from deep-sea hydrothermal vents, Animal biology, 320, 893-898 Lo, H.F., Lin, L. L., Chiang, W. Y., Chie, M. C., Hsu, W. H.and Chang, C. T., (2002), Deletion analysis of the C-terminal region of the á-amylase of Bacillus sp. strain TS-23. Archives Microbiology, 178, 115–123 Madigan, Michael; Martinko, John (editors), (2005). Brock Biology of Microorganisms, 11th ed., Prentice Hall Mamo, G., Gessesse A., (1999), Purification and characterization of two raw-starch-digesting thermostable α-amylases from a thermophilic Bacillus, Enzyme and Microbial Technology, 25, 433–438 Mitsuiki, S., Mukae, K ., Sakai, M., Goto, M., Hayashi, S., Furukawa, K. , (2005), Comparative characterization of raw startch hidrolizing α-amylase from various Bacillus strain. Enzyme and Microbial Technology, 37, 410-416 Najafi, M.F., Deobagkar, D., Deobagkar, D, (2005a), Purification and characterization of an extracellular α-amylase from Bacillus subtilis AX20. Protein Expression & purification, 41, 349-354 Najafi, Mohsen F., Kembhavi, A., (2005), One step purification and characterization of an extracellular α-amylase from marine Vibrio sp., Enzyme and Microbial Technology, 36: 535–539 Nazmi, A. R., Reinisch, T., Hinz, H. J., (2006), Ca-binding to Bacillus licheniformis α-amylase (BLA), Archives of Biochemistry and Biophysics, 453, 16–23 Nielsen, J. E., Borchert, T. V., Vriend, G., (2001), The determinants of α-amylase pH-activity profiles, Protein Engineering, 147 (7), 505-512 Oates, C. G., (1997), Towards an understanding of starch granule structure and hydrolysis, Trends in Food Science and Technology, 8, 375-382 Ohdan, K., Kuriki, T., Takata, H., Kaneko, H. and Okada, S., (2000), Introduction of raw starchbinding domains into Bacillus subtilis α-amylase by fusion with the starch-binding domain of Bacillus cyclomaltodextrin glucanotransferase. Applied Environment Microbiology, 66, 3058-3064 Parker, R., Ring, S. G., (2001), Aspects of the Physical Chemistry of Starch, Journal of Cereal Science, 34, 1–17 Robyt, J., French, D., (1963), Action pattern and specificity of an amylase from Bacillus subtilis. Archives Biochemistry Biophysics, 100, 451-467 38
Robyt, John F., (1998), Essentials of Carbohydrate Chemistry, New York: Springer Sivaramakrishnan, S., Gangadharan, D., Madhavan, K. N., Soccol C. R., and Pandey A., (2006), α-Amylases from Microbial Sources – An Overview on Recent Developments, Food Technology Biotechnology, 44 (2), 173–184 Tester, R. F., Qi, X., Karkalas, J., (2006), Hydrolysis of native starches with amylases, Animal Feed Science and Technology van der Maarel, M.J.E.C., van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H., Dijkhuizen, L., (2002), Properties and applications of starch-converting enzymes of the α-amylase family. Journal of Biotechnology, 94, 137-155 Vidilaseris, K., (2007), Karakterisasi α-Amilase Pendegradasi Pati Mentah Bakteri Laut Bacillus subtilis ALSHL3, Skripsi, Institut Teknologi Bandung, Bandung Wai, S. N., Mizunoe, Y., Yoshida S., (1999), How Vibrio cholerae survive during starvation, FEMS Microbiology Letters, 180, 123-131 Wiseman, A., (1986), Handbook of enzyme biotechnology, 2nd Ed., John Willey & Sons, New York, 26–34 Yune Li, J., I Yeh, A., (2003), Effects of starch properties on rheological characteristics of starch/meat complexes, Journal of Food Engineering, 57, 287–294 Zorov, I. N., (2006), Hydrolysis of Wheat Flour with Amylase Preparations and Individiual Enzymes, Applied Biochemistry and Microbiology, 42, 616-619
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Lampiran A. Kurva pertumbuhan Vibrio sp. B10.2.8 Tabel A.1 Densitas Optik (OD600) tiap waktu pertumbuhan Vibrio sp. B10.2.8 Waktu (jam) 0 3 6 9 12 15 16 18 19 22 25
Densitas Optik (OD600) OD1 OD2 0.049 0.031 0.459 0.481 0.244 0.197 0.356 0.339 0.500 0.509 0.485 0.498 0.541 0.542 0.530 0.535 0.563 0.542 0.571 0.646 0.484 0.433
OD rata-rata 0.040 0.470 0.221 0.348 0.505 0.492 0.542 0.533 0.553 0.609 0.459
Standar Deviasi 0.013 0.016 0.033 0.012 0.006 0.009 0.001 0.004 0.015 0.053 0.036
fp (kali) 1 1 4 4 5 5 5 5 5 5 6
OD600 x fp 0.040 0.470 0.882 1.390 2.523 2.458 2.708 2.663 2.763 3.043 2.751
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Lampiran B. Profil sekresi α-amilase Vibrio sp. B10.2.8 Tabel B.1 Aktivitas α-amilase Vibrio sp. B10.2.8 tiap waktu pertumbuhan Waktu (jam) 0 3 6 9 12 15 16 18 19 22 25
Asampel (λ600) 1 2 3 0.839 0.853 0.852 0.687 0.646 0.686 0.600 0.578 0.552 0.355 0.377 0.392 0.273 0.297 0.302 0.238 0.247 0.251 0.271 0.302 0.290 0.266 0.251 0.274 0.205 0.214 0.235 0.307 0.287 0.300 0.219 0.222 0.221
Akontrol (λ600) 1 2 3 0.847 0.840 0.866 0.697 0.687 0.709 0.695 0.669 0.654 0.630 0.621 0.631 0.606 0.630 0.616 0.588 0.618 0.616 0.628 0.629 0.625 0.588 0.598 0.602 0.603 0.614 0.591 0.577 0.631 0.618 0.597 0.621 0.599
Aktivitas (U/mL) 1 2 3 1.89 -3.10 3.23 2.87 11.94 6.49 27.34 27.20 31.19 87.30 78.58 75.75 109.90 105.71 101.95 119.05 120.06 118.51 113.69 103.97 107.20 109.52 116.05 108.97 132.01 130.29 120.47 93.59 109.03 102.91 126.63 128.50 126.21
Aktivitas rata-rata 0.68 7.10 28.58 80.55 105.85 119.21 108.29 111.52 127.59 101.84 127.12
Std. Deviasi 3.33 4.56 2.26 6.02 3.98 0.79 4.95 3.94 6.22 7.78 1.22
Pengujian aktivitas enzim dilakukan dengan menggunakan metode Fuwa. 1 unit aktivitas didefinisikan sebagai penurunan absorban sebesar 10% per ml enzim yang digunakan pada kondisi percobaan.. Perhitungan aktivitas enzim dengan metode Fuwa:
⎛ (A kontrol − A sampel ) ⎞ ⎜ ⎟ A kontrol ⎠ Unit aktivitas (U/ml) = ⎝ × faktor pengenceran enzim 10% × Venzim
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Lampiran C. Penentuan konsentrasi protein Penentuan konsentrasi protein dilakukan dengan menggunkan metode Bradford. Standar yang digunakan adalah BSA dari konsentrasi 2-16 μg/mL. Tabel C.1 Absorbansi larutan standar BSA pada λ595 [BSA] (μg/mL) 2 4 6 8 10 12 14 16
A595 0.125 0.224 0.426 0.442 0.615 0.703 0.850 0.831
0.137 0.295 0.255 0.484 0.461 0.650 0.785 0.941
0.084 0.237 0.290 0.520 0.643 0.761 0.726 0.881
Std. Deviasi 0.028 0.038 0.090 0.039 0.098 0.056 0.062 0.055
Arata-rata 0.115 0.252 0.324 0.482 0.573 0.705 0.787 0.884
1 0.9 0.8 0.7
A595
0.6 0.5 0.4
y = 0.0553x + 0.0175
0.3
R2 = 0.9953
0.2 0.1 0 0
5
10
15
20
[BSA] (μg/mL)
Gambar C.1 Kurva standar BSA
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Lampiran D. Pengaruh pH terhadap aktivitas α-amilase Tabel D.1 Data pengaruh pH terhadap aktivitas α-amilase Vibrio sp. B10.2.8 pH 5 5.5 6 6.5 7 7.5 8
1 0.463 0.293 0.300 0.287 0.359 0.348 0.565
Asampel(λ600) 2 3 0.458 0.494 0.349 0.359 0.331 0.327 0.291 0.306 0.388 0.403 0.351 0.367 0.596 0.582
1 0.696 0.641 0.667 0.609 0.672 0.628 0.891
Akontrol (λ 600) 2 3 0.680 0.703 0.657 0.683 0.711 0.687 0.618 0.633 0.638 0.689 0.637 0.648 0.880 0.878
Aktivitas (U/mL) 1 2 3 66.95 65.29 59.46 108.58 93.76 94.88 110.04 106.89 104.80 105.75 105.83 103.32 93.15 78.37 83.02 89.17 89.80 86.73 73.18 64.55 67.43
Aktivitas rata-rata 63.90 99.07 107.25 104.96 84.85 88.57 68.38
Std.Deviasi 3.94 8.25 2.64 1.43 7.56 1.62 4.39
Pengujian aktivitas enzim dilakukan dengan menggunakan metode Fuwa.
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Lampiran E. Pengaruh suhu terhadap aktivitas α-amilase Tabel E.1 Pengaruh suhu terhadap aktivitas α-amilase Vibrio sp. B10.2.8 Suhu (°C) 30 40 50 60 70 80
Asampel (λ 600) 1 2 3 0.271 0.268 0.266 0.208 0.210 0.215 0.175 0.186 0.200 0.277 0.291 0.302 0.454 0.442 0.410 0.524 0.599 0.517
Akontrol (λ 600) 1 2 3 0.662 0.666 0.671 0.642 0.638 0.648 0.639 0.625 0.624 0.601 0.630 0.622 0.590 0.657 0.596 0.568 0.598 0.522
Aktivitas (U/mL) 1 2 3 118.13 119.52 120.72 135.20 134.17 133.64 145.23 140.48 135.90 107.82 107.62 102.89 46.10 65.45 62.42 15.49 -0.33 1.92
Aktivitas rata-rata 119.45 134.34 140.53 106.11 57.99 5.69
Std.Deviasi 1.30 0.79 4.66 2.79 10.41 8.56
Pengujian aktivitas enzim dilakukan dengan menggunakan metode Fuwa.
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Lampiran F. Kemampuan hidrolisis pati mentah F.1 Kurva Standar Glukosa Tabel F.1 Data absorbansi larutan standar glukosa pada λ620 [glukosa] (mg/L) 25 50 75 100 150
0.149 0.277 0.474 0.497 0.853
A620 0.149 0.333 0.448 0.543 0.838
0.144 0.321 0.472 0.574 0.803
Std.Deviasi 0.003 0.029 0.014 0.039 0.026
Arata-rata 0.147 0.310 0.465 0.538 0.831
0.9 0.8 0.7
A620
0.6 0.5 0.4
y = 0.0053x + 0.0327
0.3
R = 0.9914
2
0.2 0.1 0.0 0
50
100
150
200
[Glukosa] (mg/L)
Gambar F.1 Kurva standar glukosa F.2 Kemampuan α-amilase Vibrio sp. B10.2.8 dalam menghidrolisis pati mentah Tabel F.2 Data hasil hidrolisis pati mentah oleh α-amilase Vibrio sp. B10.2.8 Pati mentah Maizena Beras Tapioka Kentang
fp (kali) 10 10 10 10
Asampel (λ620) 1 2 3 0.564 0.464 0.195 0.221 0.401 0.387 0.220 0.864 0.222 0.506 1.200 1.023
Arata-rata 0.408 0.336 0.435 0.910
Akontrol (λ620) 1 2 3 0.590 0.779 0.757 0.838 0.938 0.957 0.559 0.249 0.756 1.124 0.933 0.141
Arata-rata 0.709 0.911 0.521 0.733
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Sagu
10
0.054
0.058
0.800
0.304
1.119
1.109
1.089
1.106
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Lampiran G. Fraksinasi amonium sulfat Konsentrasi awal amonium sulfat (% jenuh) pada 0°C 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
Konsentrasi akhir amonium sulfat (% jenuh) pada 0°C 20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
559 526 493 460 427 395 362 329 296 263 230 197 164 132 99 66 33 0
603 570 536 503 469 436 402 369 335 302 268 235 201 168 134 101 67 34 0
650 615 581 547 512 478 445 410 376 342 308 273 239 205 171 137 103 68 34 0
697 662 627 592 557 522 488 453 418 383 348 313 279 244 209 174 139 105 70 35
Berat amonium sulfat yang ditambahkan (gram) dalam 1 liter larutan 106 79 53 26 0
134 108 81 54 27 0
164 137 109 82 55 27 0
194 166 139 111 83 56 28 0
206 197 169 141 113 84 56 28 0
258 229 200 172 143 115 86 57 29 0
291 262 233 204 175 146 117 87 58 29 0
326 296 266 237 207 179 148 118 89 59 30 0
361 331 301 271 241 211 181 151 120 90 60 30 0
398 368 337 306 276 245 214 184 153 123 92 61 31 0
436 405 374 343 312 280 249 218 187 156 125 93 62 31 0
476 444 412 381 349 317 285 254 222 190 159 127 95 63 32 0
516 484 452 420 387 355 323 291 258 226 194 161 129 97 62 32 0
100
0
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48
