SEBAGAI BIOMATERIAL
SEKOLAH PASCASARJANA INSTITUT PERTANIAN BOGOR
BOGOR 2012
45
DAFTAR PUSTAKA
Aini NH. 2009. Pengaruh beberapa konsentrasi media dan lamanya perebusan
kokon Attacus atlas L. terhadap kualitas filamen yang dihasilkan [skripsi]. Bogor: Departemen Biologi Fakultas Matematika dan Ilmu Pengetahuan
Alam Institut Pertanian Bogor.
Apriyantono A, Fardiaz D, Puspitasari NL, Yasni S, Budiyanto S. 1989. Analisis Pangan. Bogor: IPB Press.
Aramwit P, Sangcakul A. 2007. The effect of sericin cream on wound healing in rats. Biosci. Biotechnol. Biochem. 71(10):2473-2477.
Aramwit P, Kanokpanont S, Punyarit P, Srichana T. 2010. The effect of sericin from various extraction methods on cell viability and collagen production. Int. J. Mol. Sci. 11:2200-2211.
Atmosoedarjo S, Kartasubrata J, Kaomini M, Saleh W, Moerdoko W. 2000. Sutera Alam Indonesia. Jakarta: Yayasan Sarana Wana Jaya.
Brasla A, Matei A. 1997. Pelatihan Pembibitan Ulat Sutera II Oleh Ahli Dari Rumania. Jawa Tengah: PPUS Candiroto.
Cazes J. 2005. Encyclopedia of Chromatography Second Edition (Volume one). Florida: Taylor and Francis Group Florida USA.
Chang-Kee H, Yuk-Hyun J, Sung-Hee L, Geum-Ju P, Deock-Hyoung C. 2002. Anticancer agents containing antigenotoxic and immunostimulative peptides produced from the hydrolysate of silkworm cocoon. Patent No. WO02076487.
Cui X, Urita S, Imanishi S, Nagasawa S, Suzuki K. 2009. Isolation and charactherization of 41 kDa sericin from the wild silkmoth Antheraea yamamai.J. Insect Biotechnology and Sericology 78:11-16.
Dash R, Ghosh SK, Kaplan DL, Kundu SC. 2007. Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta. Comparative Biochem and Physiology 147:129-134.
Dash R, Mandal M, Ghosh SK, Kundu SC. 2008. Silk sericin protein of tropical Tasar silkworm inhibits UVB- induce apoptosis in human skin keratinocytes. Mol Cell Biochem 311(1-2):111-119.
46 Fabiani C, Pizzichini M, Spadoni M, Zeddita G. 1996. Treatment of waste water from silk degumming processes for protein recovery and water reuse. Desalination 105:1-9.
Faatih M. 2005. Aktivitas antimikrobia kokon Attacus atlas. J Penelitian Sains dan Teknologi 6(1):35-48.
Gaspersz V. 1992. Tehnik analisis dalam penelitian percobaan. Bandung: Tarsito. Gulrajani ML, Purwar R, Joshi M. 2008. A value added finish from silk degumming waste liquor. Department of Textile Technology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi.
Hagerman AE. 2002. The Tannin Handbook. http://chemistry.muohio.edu [30 Maret 2012].
Invitrogen. Tissue extraction reagent 1. www.invitrogen.com [13 Maret 2012]. Kato et al. 1998. Silk protein, sericin, inhibits lipid peroxidation and tyrosinase
activity. Bioscience Biotech Biochem 62(1):145-147.
Kato H. 2000. Structure and thermal properties of Anaphe, Cricula and Attacus cocoon filaments. Int. J Wild Silkmoth and Silk 5:11-20.
Kwang et al. 2003. Preparation of self-assembled silk sericin nanoparticles. Int J Bio Macromol 32:36-42.
Laemmli UK. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Lehninger AL. 1982. Dasar-dasar biokimia jilid 2. Jakarta: Erlangga.
Lenth RV. 2010. Response surface methods in R, using rsm. The University of Iowa, United States of America.
Mattjik AN, Sumertajaya IM. 2002. Perancangan percobaan dengan aplikasi SAS dan minitab jilid 1. Bogor: IPB Press.
Mahendran B, Ghosh SK, Kundu SC. 2006. Molecular phylogeny of silk producing insect bases on internal transcribed spancer DNA1. J Biochem and Mol Bio 39(5):522-529.
Masahiro S, Hideyuki Y, Norihisa K. 2000. Consumption of silk protein, sericin elevates intestinal absorption of zinc, iron, magnesium and calcium in rats. Nutrition Research 20:1505-1511.
47 Masakazu T, Kazuhisa T, Hideyuki Y, Hiroshi T, Shigeru N. 2003. The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture. Biotech Letters 25:1805-1809.
Myers RH. 1971. Response surface methodology. Boston: Allyn and Bacon Inc. Okamoto H, Ishikawa E, Suzuki Y. 1982. Structural analysis of sericin genes. J
Bio Chem 257(24):15192-15199.
Padamwar MN, Pawar AP. 2004. Silk sericin and its applications. J Scientific and Industrial Research 63:323-329.
Padamwar MN, Pawar AP, Daithankar AV, Mahadik KR. 2005. Silk sericin as a moisturizer: an in vivo study. J Cosmetic Dermatology 4:250-257.
Peigler R. 1989. A Revision Of The Indo-Australian Genus Attacus. California: The Lepidoptera Research Foundation, Inc. Beverly Hill.
Risnasari I. 2002. Tanin. Universitas Sumatera Utara digital library.
Salanger JL. 2002. Surfactants types and uses. Venezuela: Universidad De Los Andes.
Shakhashiri. 2009. Ethanol. www.scifun.org [27 September 2011].
Sihombing DTH. 1999. Satwa Harapan I. Bogor: Pustaka Wirausaha Muda. Solihin DD, Fuah AM. 2010. Budidaya Ulat Sutera Alam Attacus atlas. Jakarta:
Penebar Swadaya.
Suriana. 2011. Karakterisasi serat sutera dan gen penyandi fibroin pada ulat sutera liar Cricula trifenestrata Helfer (Lepidoptera:Saturniidae) [disertasi]. Bogor: Program Pascasarjana, Institut Pertanian Bogor.
Suryani A, Dadang, Setyadjit, Tjokrowardojo AS, Kurniadji MNN. 2008. Sintesis alkil poliglikosida (APG) berbasis alkohol lemak dan pati sagu untuk formulasi herbisida. J. Pascapanen 5(1):10-20.
Sutera Indonesia. 2004. Mencermati Sutera Liar. Tabloid Sutera Indonesia:5. Takasu Y, Hata T, Uchino K, Zhang Q. 2010. Identification of Ser2 proteins as
major sericin components in the non-cocoon silk of Bombyx mori. Insect Biochem and Mol Biol 40:339-344.
Tokutake S. 1980. Isolation of the smallest component of silk protein. J Biochem 187:413-417.
48 Tripoulas NA, Samols D. 1986. Developmental and hormonal regulation of sericin RNA in the silkworm, Bombyx mori. Developmental Biology 116:328-336.
Vaithanomsat P, Malapant T, Apiwattanapiwat W. 2008. Silk degumming solution as substrate for microbial protease production. J Nat Sci 42:543- 551.
Wei T, Li MZ, Xie RJ. 2005. Preparation and structure of porous silk sericin materials. Macromolecular Materials and Engineering 290:188-194.
Wikipedia. Surface tention. www.wikipedia.org [8 maret 2012].
Wu JH, Wang Z, Xu SY. 2007. Preparation and characterization of sericin powder extracted from silk industry wastewater. Food Chem 103: 1255-1262.
Yamada H, Tsubouchi K. 2001. Characterization of silk proteins in the cocoon fibers of Cricula trifenestrata. Int J Wild Silkmoth & Silk 6:47-54.
Zhang YQ. 2002. Applications of natural silk protein sericin in biomaterials. Biotech Adv 20(2):91-1.
Zhang et al. 2004. Immobilization of L-asparaginase on the microparticles of the natural silk sericin protein and its characters. Biomaterials25:3751-3759. Zhaorigetu S, Yanaka N, Sasaki M, Watanabe H, Kato N. 2003. Silk protein,
sericin, suppresses DMBA-TPA-induced mouse skin tumorigenesis by reducing oxidative stress, inflammatory responses and endogenous tumor promoter TNF-α. Oncology 10:537-543.
49
LAMPIRAN
50 Lampiran 1 Tahapan ekstraksi protein serisin dari kelenjar sutera tengah
Attacus atlas (Invitrogen 2012)
Tahapan ekstraksi:
- Penimbangan KSA
- Penambahan 1 ml tissue extraction reagent Iper 0,1 g KSA - Homogenisasi dengan mortar
- Sentrifugasi pada 10.000 rpm selama 5 menit
- Supernatan dikoleksi dan simpan pada suhu (-20oC) sebagai protein serisin.
Hasil homogenisasi Supernatan
anterior
posterior KSA
51 Lampiran 2 Tahapan ekstraksi protein serisin dari kulit kokon
Tahapan preparasi sampel (Solihin & Fuah 2010):
- Kokon dibersihkan dari floss (serabut kokon) dan sisa pupa, yang selanjutnya disebut kulit kokon
- Kulit kokon dibersihkan dengan air tanpa sabun, kemudian dikeringanginkan - Kulit kokon dipotong-potong sekitar 1 cm2 supaya tidak voluminous
- Kulit kokon yang telah dipotong-potong siap untuk proses degumming.
Tahapan ekstraksi protein serisin: 1. Teknik degumming
- Fisik (Padamwar & Pawar 2004)
- Potongan kulit kokon (1 cm2) sebanyak 0,04 g/ml dimasukkan dalam tabung yang telah diisi DW 150 ml
- Tutup rapat tabung dan masukkan dalam autoklaf
- Autoklaf di setting suhu dan waktunya sesuai yang diinginkan
- Tabung diangkat dari autoklaf setelah proses selesai dan didinginkan dalam suhu ruang
- Isi tabung kemudian disaring untuk memisahkan fibroin dan larutan - Larutan inilah yang disebut larutan hasil degumming yang akan diisolasi
protein serisinnya. - Kimia (Suriana 2011)
Proses sama seperti teknik degumming secara fisik tetapi ditambah basa kuat NaOH pada larutan sebelum masuk autoklaf.
52 2. Isolasi protein serisin
Tahapan isolasi protein (Wu et al. 2007):
- Penambahan etanol dingin (-18oC) sebesar 75% v/v pada larutan hasil degumming yang kemudian disebut larutan mixed
- Larutan mixed kemudian disimpan semalam pada suhu (-25oC)
- Larutan yang selanjutnya di settling pada suhu 4oC selama 72 jam untuk diambil endapannya
- Endapan kemudian dievaporasi pada suhu 50oC dan tekanan 7 mbar untuk menghilangkan etanol
53 Lampiran 3 Tahapan uji tegangan permukaan
Tahapan uji tegangan permukaan:
- Penambahan crude protein serisin dalam DW (ml) yang kemudian dimasukkan ke dalam gelas beaker 100 ml
- Gelas beaker ditempatkan pada tensiometer
- Pengukuran tegangan permukaan sampel dilakukan sampai penarik tegangan permukaan (ring) keluar dari permukaan air, dan nilainya ditunjukkan oleh penunjukkan jarum tensiometer pada angka tertentu dalam dynes/cm.
Jarum
54 Lampiran 4 Analisis ragam pengaruh kemurnian etanol terhadap rendemen
protein serisin Source DF SS MS F P Sampel 1 0,0006 0,0006 0,04 0,846 Error 4 0,0557 0,0139 Total 5 0,0563 S = 0,1180 R-Sq = 1,07% R-Sq(adj) = 0,00%
Individual 95% CIs For Mean Based on Pooled StDev
Level N Mean StDev ---+---+---+---+
E1 3 1,0233 0,0379 (---*---)
E2 3 1,0033 0,1626 (---*---)
---+---+---+---+ 0,90 1,00 1,10 1,20 Pooled StDev = 0,1180
55 Lampiran 5 Analisis ragam pengaruh rasio volume NaOH 0,1 N dan jenis
spesies ulat sutera terhadap rendemen protein serisin
Source DF SS MS F P rasio NaOH 1 9,22 9,22 1,76 0,242 spesies 1 1350,18 1350,18 258,07 0,000 Error 5 26,16 5,23 Total 7 1385,56 S = 2,287 R-Sq = 98,11% R-Sq(adj) = 97,36% Individual 95% CIs For Mean Based on rasio Pooled StDev
NaOH Mean ---+---+---+---+-
R1 16,0200 (---*---)
R2 18,1675 (---*---)
---+---+---+---+-
15,0 17,5 20,0 22,5 Individual 95% CIs For Mean Based on Pooled StDev spesies Mean ---+---+---+---+ S1 4,1025 (---*---) S2 30,0850 (---*--) ---+---+---+---+ 8,0 16,0 24,0 32,0
56 Lampiran 6 Analisis ragam pengaruh konsentrasi NaOH terhadap rendemen
protein serisin Source DF SS MS F P kode spl 5 64,452 12,890 13,00 0,004 Error 6 5,950 0,992 Total 11 70,402 S = 0,9958 R-Sq = 91,55% R-Sq(adj) = 84,50%
Individual 95% CIs For Mean Based on Pooled StDev
Level N Mean StDev ---+---+---+---+---
N1 2 5,865 0,262 (---*---) N2 2 6,780 0,424 (---*---) N3 2 8,740 2,065 (---*---) N4 2 7,395 0,445 (---*---) N5 2 11,690 0,339 (---*---) N6 2 11,840 1,061 (---*---) ---+---+---+---+--- 5,0 7,5 10,0 12,5 Tukey 95% Simultaneous Confidence Intervals Individual confidence level = 99,27% kode spl = N1 subtracted from: kode spl Lower Center Upper ---+---+---+---+-
N2 -3,049 0,915 4,879 (---*---) N3 -1,089 2,875 6,839 (---*---) N4 -2,434 1,530 5,494 (---*---) N5 1,861 5,825 9,789 (---*---) N6 2,011 5,975 9,939 (---*---) ---+---+---+---+- -5,0 0,0 5,0 10,0 kode spl = N2 subtracted from: kode spl Lower Center Upper ---+---+---+---+-
N3 -2,004 1,960 5,924 (---*---) N4 -3,349 0,615 4,579 (---*---) N5 0,946 4,910 8,874 (---*---) N6 1,096 5,060 9,024 (---*---) ---+---+---+---+- -5,0 0,0 5,0 10,0 kode spl = N3 subtracted from: kode spl Lower Center Upper ---+---+---+---+-
N4 -5,309 -1,345 2,619 (---*---)
N5 -1,014 2,950 6,914 (---*---)
N6 -0,864 3,100 7,064 (---*---)
---+---+---+---+-
-5,0 0,0 5,0 10,0 kode spl = N4 subtracted from: kode spl Lower Center Upper ---+---+---+---+-
N5 0,331 4,295 8,259 (---*---)
N6 0,481 4,445 8,409 (---*---)
---+---+---+---+-
-5,0 0,0 5,0 10,0 kode spl = N5 subtracted from: kode spl Lower Center Upper ---+---+---+---+-
N6 -3,814 0,150 4,114 (---*---)
---+---+---+---+- -5,0 0,0 5,0 10,0
57 Lampiran 7 Rendemen protein serisin pada optimasi 1
Suhu (oC)
Waktu (menit)
Rendemen protein dalam kulit kokon
tanpa AFB (%)
Rendemen protein dalam kulit kokon dengan AFB (%) 110,00 30,00 5,708 6,196 110,00 50,00 7,691 8,356 120,00 30,00 7,511 8,154 120,00 50,00 9,565 10,326 115,00 40,00 7,776 8,436 115,00 40,00 6,667 7,274 115,00 40,00 6,487 7,077 115,00 40,00 5,824 6,372 115,00 40,00 7,185 7,861 122,07 40,00 8,605 9,264 107,93 40,00 5,352 5,877 115,00 54,14 8,596 9,380 115,00 25,86 7,883 8,789
58 Lampiran 8 Analisis response surface methodology (RSM) pada optimasi 1
tanpa penambahan AFB
Estimate Std. Error t value Pr(>|t|) (Intercept) 6.86113 0.37166 18.461 1.63e-06 *** blockB2 -0.18302 0.41549 -0.440 0.67501 x1 0.63077 0.26281 2.400 0.05329 . x2 1.03483 0.26281 3.938 0.00765 ** x1:x2 0.01788 0.37164 0.048 0.96318 x1^2 0.73738 0.28263 2.609 0.04017 * x2^2 0.10691 0.28263 0.378 0.71826 Signif. codes: *** . ** . * . 5 . .
Residual standard error: 0.7433 on 6 degrees of freedom Multiple R-squared: 0.8242, Adjusted R-squared: 0.6485 F-statistic: 4.69 on 6 and 6 DF, p-value: 0.04099
Analysis of Variance Table Response: yield
Df Sum Sq Mean Sq F value Pr(>F) block 1 0.0341 0.0341 0.0618 0.81199 FO(x1, x2) 2 11.7481 5.8741 10.6327 0.01066 TWI(x1, x2) 1 0.0013 0.0013 0.0023 0.96318 PQ(x1, x2) 2 3.7609 1.8804 3.4038 0.10281 Residuals 6 3.3147 0.5525 Lack of fit 3 1.4129 0.4710 0.7429 0.59355 Pure error 3 1.9018 0.6339
Stationary point of response surface: x1 x2
-0.3693974 -4.8088952 Stationary point in original units: time temp 36.30603 90.95552 Eigenanalysis: $values [1] 0.7375092 0.1067817 $vectors [,1] [,2] [1,] -0.99989949 0.01417747 [2,] -0.01417747 -0.99989949
59 Lampiran 9 Analisis response surface methodology (RSM) pada optimasi 1
dengan penambahan AFB
Estimate Std. Error t value Pr(>|t|) (Intercept) 7.45845 0.40999 18.192 1.78e-06 *** blockB2 -0.13608 0.45835 -0.297 0.7766 x1 0.64606 0.28991 2.228 0.0674 . x2 1.08982 0.28991 3.759 0.0094 ** x1:x2 0.00300 0.40997 0.007 0.9944 x1^2 0.82984 0.31178 2.662 0.0374 * x2^2 0.07261 0.31178 0.233 0.8236 --- Signif. codes: *** . ** . * . 5 . . Residual standard error: 0.8199 on 6 degrees of freedom Multiple R-squared: 0.8137, Adjusted R-squared: 0.6274 F-statistic: 4.368 on 6 and 6 DF, p-value: 0.04792
Analysis of Variance Table Response: yield
Df Sum Sq Mean Sq F value Pr(>F) block 1 0.0082 0.0082 0.0122 0.91578 FO(x1, x2) 2 12.8388 6.4194 9.5484 0.01366 TWI(x1, x2) 1 0.0000 0.0000 0.0001 0.99440 PQ(x1, x2) 2 4.7736 2.3868 3.5502 0.09607 Residuals 6 4.0338 0.6723 Lack of fit 3 1.8466 0.6155 0.8443 0.55369 Pure error 3 2.1872 0.7291
Stationary point of response surface: x1 x2
-0.3757147 -7.4965849 Stationary point in original units: time temp 36.24285 77.51708 Eigenanalysis: $values [1] 0.8298440 0.0726094 $vectors [,1] [,2] [1,] -0.999998038 0.001980896 [2,] -0.001980896 -0.999998038
60 Lampiran 10 Analisis ragam titik pusat optimasi 1 terhadap rendemen
protein serisin Source DF SS MS F P sampel 1 0,949 0,949 1,64 0,236 Error 8 4,631 0,579 Total 9 5,580 S = 0,7608 R-Sq = 17,01% R-Sq(adj) = 6,64%
Individual 95% CIs For Mean Based on Pooled StDev
Level N Mean StDev +---+---+---+---
O11 5 6,7878 0,7363 (---*---)
O12 5 7,4040 0,7847 (---*---)
+---+---+---+--- 6,00 6,60 7,20 7,80
61 Lampiran 11 Rendemen protein serisin pada optimasi 2
Suhu (oC) Waktu (menit) Rendemen protein dalam kulit kokon tanpa AFB
(%)
Rendemen protein dalam kulit kokon dengan AFB
(%) 110,00 30,00 5,388 5,848 110,00 50,00 4,923 5,349 120,00 30,00 7,253 7,874 120,00 50,00 12,034 12,991 115,00 40,00 10,160 11,022 115,00 40,00 9,075 9,901 115,00 40,00 9,426 10,283 115,00 40,00 9,252 10,123 115,00 40,00 8,697 9,515 122,07 40,00 10,540 11,348 107,93 40,00 4,399 4,831 115,00 54,14 9,075 9,903 115,00 25,86 6,649 7,413
62 Lampiran 12 Analisis response surface methodology (RSM) pada optimasi 2
tanpa penambahan AFB
Estimate Std. Error t value Pr(>|t|) (Intercept) 9.34250 0.24250 38.526 2.04e-08 *** blockB2 -0.05095 0.27110 -0.188 0.85711 x1 0.96854 0.17148 5.648 0.00132 ** x2 2.20792 0.17148 12.876 1.35e-05 *** x1:x2 1.31148 0.24248 5.408 0.00165 ** x1^2 -0.79415 0.18441 -4.306 0.00506 ** x2^2 -0.99062 0.18441 -5.372 0.00171 ** Signif. codes: *** . ** . * . 5 . .
Residual standard error: 0.485 on 6 degrees of freedom Multiple R-squared: 0.9782, Adjusted R-squared: 0.9564 F-statistic: 44.92 on 6 and 6 DF, p-value: 9.991e-05 Analysis of Variance Table
Response: yield
Df Sum Sq Mean Sq F value Pr(>F) block 1 0.157 0.1572 0.6683 0.444885 FO(x1, x2) 2 46.497 23.2484 98.8475 2.556e-05 TWI(x1, x2) 1 6.880 6.8799 29.2518 0.001651 PQ(x1, x2) 2 9.860 4.9298 20.9604 0.001963 Residuals 6 1.411 0.2352 Lack of fit 3 0.644 0.2146 0.8393 0.555541 Pure error 3 0.767 0.2557
Stationary point of response surface: x1 x2
3.374335 3.348060 Stationary point in original units: time temp 73.74335 131.74030 Eigenanalysis: $values [1] -0.2293262 -1.5554372 $vectors [,1] [,2] [1,] -0.7576786 -0.6526279 [2,] -0.6526279 0.7576786
63 Lampiran 13 Analisis response surface methodology (RSM) pada optimasi 2
dengan penambahan AFB
Estimate Std. Error t value Pr(>|t|) (Intercept) 10.166877 0.264077 38.500 2.05e-08 *** blockB2 0.004915 0.295225 0.017 0.98726 x1 1.017511 0.186734 5.449 0.00159 ** x2 2.360736 0.186734 12.642 1.50e-05 *** x1:x2 1.404000 0.264062 5.317 0.00180 ** x1^2 -0.845349 0.200820 -4.209 0.00563 ** x2^2 -1.129685 0.200820 -5.625 0.00135 ** --- Signif. codes: *** . ** . * . 5 . .
Residual standard error: 0.5281 on 6 degrees of freedom Multiple R-squared: 0.9776, Adjusted R-squared: 0.9552 F-statistic: 43.66 on 6 and 6 DF, p-value: 0.0001085 Analysis of Variance Table
Response: yield
Df Sum Sq Mean Sq F value Pr(>F) block 1 0.108 0.1079 0.3870 0.556763 FO(x1, x2) 2 52.859 26.4296 94.7584 2.89e-05 TWI(x1, x2) 1 7.885 7.8849 28.2697 0.001800 PQ(x1, x2) 2 12.208 6.1040 21.8849 0.001752 Residuals 6 1.673 0.2789 Lack of fit 3 0.839 0.2797 1.0056 0.498209 Pure error 3 0.834 0.2781
Stationary point of response surface: x1 x2
3.036414 2.931730 Stationary point in original units: time temp 70.36414 129.65865 Eigenanalysis: $values [1] -0.2712658 -1.7037682 $vectors [,1] [,2] [1,] -0.7741088 -0.6330525 [2,] -0.6330525 0.7741088
64 Lampiran 14 Analisis ragam titik pusat optimasi 2 terhadap rendemen
protein serisin Source DF SS MS F P sampel 1 1,793 1,793 5,95 0,041 Error 8 2,412 0,301 Total 9 4,205 S = 0,5491 R-Sq = 42,64% R-Sq(adj) = 35,47%
Individual 95% CIs For Mean Based on Pooled StDev
Level N Mean StDev ---+---+---+---+----
O21 5 9,322 0,541 (---*---)
O22 5 10,169 0,557 (---*---)
---+---+---+---+----
9,00 9,50 10,00 10,50 Pooled StDev = 0,549 Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of sampel Individual confidence level = 95,00% sampel = O21 subtracted from: sampel Lower Center Upper -+---+---+---+---
O22 0,0460 0,8468 1,6476 (---*---)
-+---+---+---+--- -0,70 0,00 0,70 1,40
65 Lampiran 15 Rendemen protein serisin pada optimasi 3
Suhu (oC)
Waktu (menit)
Rendemen protein dalam kulit kokon tanpa AFB (%)
Rendemen protein dalam kulit kokon dengan AFB (%) 110,00 30,00 0,446 0,487 110,00 50,00 0,483 0,528 120,00 30,00 0,719 0,781 120,00 50,00 0,738 0,801 115,00 40,00 0,273 0,299 115,00 40,00 0,230 0,252 115,00 40,00 0,244 0,266 115,00 40,00 0,242 0,264 115,00 40,00 0,251 0,275 122,07 40,00 0,695 0,759 107,93 40,00 0,352 0,388 115,00 54,14 0,451 0,508 115,00 25,86 0,515 0,573
66 Lampiran 16 Analisis response surface methodology (RSM) pada optimasi 3
tanpa penambahan AFB
Estimate Std. Error t value Pr(>|t|) (Intercept) 0.271592 0.020589 13.191 1.17e-05 *** blockB2 -0.059012 0.023017 -2.564 0.042687 * x1 -0.004256 0.014559 -0.292 0.779857 x2 0.126611 0.014559 8.697 0.000128 *** x1:x2 -0.004612 0.020587 -0.224 0.830168 x1^2 0.143656 0.015657 9.175 9.44e-05 *** x2^2 0.163920 0.015657 10.470 4.46e-05 *** ---
Signif. codes: *** . ** . * . 5 . .
Residual standard error: 0.04117 on 6 degrees of freedom Multiple R-squared: 0.9764, Adjusted R-squared: 0.9528 F-statistic: 41.41 on 6 and 6 DF, p-value: 0.0001265 Analysis of Variance Table
Response: yield
Df Sum Sq Mean Sq F value Pr(>F) block 1 0.002865 0.002865 1.6901 0.2412894 FO(x1, x2) 2 0.128368 0.064184 37.8587 0.0003958 TWI(x1, x2) 1 0.000085 0.000085 0.0502 0.8301681 PQ(x1, x2) 2 0.289878 0.144939 85.4916 3.896e-05 Residuals 6 0.010172 0.001695 Lack of fit 3 0.009170 0.003057 9.1479 0.0509345 Pure error 3 0.001002 0.000334
Stationary point of response surface: x1 x2
0.008616798 -0.386077421 Stationary point in original units: time temp 40.08617 113.06961 Eigenanalysis: $values [1] 0.1641787 0.1433969 $vectors [,1] [,2] [1,] -0.1116648 -0.9937459 [2,] 0.9937459 -0.1116648
67 Lampiran 17 Analisis response surface methodology (RSM) pada optimasi 3
dengan penambahan AFB
Estimate Std. Error t value Pr(>|t|) (Intercept) 0.294658 0.020772 14.185 7.67e-06 *** blockB2 -0.058656 0.023222 -2.526 0.0449 * x1 -0.003864 0.014688 -0.263 0.8013 x2 0.136470 0.014688 9.291 8.80e-05 *** x1:x2 -0.005250 0.020771 -0.253 0.8089 x1^2 0.160672 0.015796 10.171 5.26e-05 *** x2^2 0.177177 0.015796 11.216 3.00e-05 *** --- Signif. codes: *** . ** . * . 5 . .
Residual standard error: 0.04154 on 6 degrees of freedom Multiple R-squared: 0.9797, Adjusted R-squared: 0.9594 F-statistic: 48.32 on 6 and 6 DF, p-value: 8.084e-05 Analysis of Variance Table
Response: yield
Df Sum Sq Mean Sq F value Pr(>F) block 1 0.00228 0.002277 1.3194 0.2944183 FO(x1, x2) 2 0.14909 0.074545 43.1960 0.0002739 TWI(x1, x2) 1 0.00011 0.000110 0.0639 0.8088926 PQ(x1, x2) 2 0.34886 0.174428 101.0751 2.395e-05 Residuals 6 0.01035 0.001726 Lack of fit 3 0.00913 0.003043 7.4514 0.0665894 Pure error 3 0.00123 0.000408
Stationary point of response surface: x1 x2
0.005734926 -0.385038919 Stationary point in original units: time temp 40.05735 113.07481 Eigenanalysis: $values [1] 0.1775840 0.1602642 $vectors [,1] [,2] [1,] -0.1533750 -0.9881681 [2,] 0.9881681 -0.1533750
68 Lampiran 18 Analisis ragam titik pusat optimasi 3 terhadap rendemen
protein serisin Source DF SS MS F P Sampel 1 0,001346 0,001346 4,80 0,060 Error 8 0,002245 0,000281 Total 9 0,003590 S = 0,01675 R-Sq = 37,48% R-Sq(adj) = 29,66%
Individual 95% CIs For Mean Based on Pooled StDev
Level N Mean StDev ---+---+---+---+---
O31 5 0,24800 0,01589 (---*---)
O32 5 0,27120 0,01757 (---*---)
---+---+---+---+---
0,240 0,255 0,270 0,285 Pooled StDev = 0,01675 Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Sampel Individual confidence level = 95,00% Sampel = O31 subtracted from: Sampel Lower Center Upper --+---+---+---+---
O32 -0,00123 0,02320 0,04763 (---*---)
--+---+---+---+--- -0,020 0,000 0,020 0,040
69 Lampiran 19 Kurva standar marker bobot molekul pada separating gel
12,5% dan stacking gel 4%
Rf marker = [panjang marker pada gel : CBB]
Dimana: Panjang marker diukur dari garis atas separating gel
CBB adalah batas akhir running elektroforesis (ada di bawah separating gel)
Log BM adalah log bobot molekul marker.
y = -1,177x + 1,998 R² = 0,978 0,00 0,50 1,00 1,50 2,00 2,50 0,00 0,50 1,00 L o g B M Rf marker Kurva Standar log BM Linear (log BM)
70 Lampiran 20 Perhitungan bobot molekul sampel
Bobot molekul sampel dihitung dari persamaan linier kurva standar (Lampiran 19). Persamaan linier kurva standar:
Y = -1,77x + 1,998 Dimana: Y = log bobot molekul sampel
71 Lampiran 21 Kromatogram analisis asam amino dengan hidrolisis asam
72 Lampiran 22 Kromatogram analisis asam amino dengan hidrolisis basa
73 Lampiran 23 Analisis ragam uji tegangan permukaan larutan dengan kadar
protein serisin berbeda
Source DF SS MS F P Kode sampel 2 111,740 55,870 167,61 0,000 Error 6 2,000 0,333
Total 8 113,740
S = 0,5774 R-Sq = 98,24% R-Sq(adj) = 97,66%
Individual 95% CIs For Mean Based on Pooled StDev
Level N Mean StDev ---+---+---+---+-
TP1 3 51,267 0,929 (--*--) TP2 3 57,467 0,306 (--*--) TP3 3 59,567 0,208 (--*---) ---+---+---+---+- 52,5 55,0 57,5 60,0 Pooled StDev = 0,577 Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Kode sampel Individual confidence level = 97,80% Kode sampel = TP1 subtracted from: Kode sampel Lower Center Upper +---+---+---+---
TP2 4,753 6,200 7,647 (---*---)
TP3 6,853 8,300 9,747 (---*---)
+---+---+---+---
-3,5 0,0 3,5 7,0 Kode sampel = TP2 subtracted from: Kode sampel Lower Center Upper +---+---+---+---
TP3 0,653 2,100 3,547 (---*---)
+---+---+---+--- -3,5 0,0 3,5 7,0