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Combination Methods for Screening Marine Actinomycetes Producing Potential Compounds as Anticancer Farida, Yuyun; Widada, Jaka; Meiyanto, Edy
Indonesian Journal of Biotechnology Vol 12, No 2 (2007)
Publisher : Universitas Gadjah Mada

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Abstract

Marine actinomycetes is a robust source of secondary metabolites including anticancer compounds . The objective of this research was to select marine actinomycetes producing potential compounds as anticancer used combination methods that consist of amplification PKS I (polyketide synthases type I) and NRPS (non ribosomal peptide synthetases) genes, analysis the diversity of secondary metabolites and genetic. Selected isolates were used for cytotoxicity assay. PKS I and NRPS genes were amplified using sets of degenerate primers. K1F and M6R were used for amplify ketosynthase and methyl-malonyl-CoA transferase modules of PKS I gene which targeted sequences 1200-1400 bp. A3F and A7R were used for amplify adenilation domains of NRPS gene which targeted sequences 700-800 bp. The diversity of secondary metabolites was analized by TLC and densitometry of ethyl acetate extracts. Genetic diversity was analized by repetitive DNA fingerprinting using BOXA1R primers. The cytotoxicity of secondary metabolites on T47D and MCF7 breast cell lines cancer was measured by MTT assay method. Fifty two marine actinomycetes isolates were screened using combination methods. Ten isolates were detected encoding both PKS I and NRPS genes, whereas 11 isolates were detected encoding the NRPS gene. The screening by analysis of secondary metabolites and genetic diversity methods were obtained 6 selected isolates for cytotoxicity assay, which consist of 3 isolates encoding both PKS I and NRPS genes and 3 isolates encoding NRPS gene.Isolate 1 had high cytotoxicity with the IC50 on T47D cell was 19 μg/ml and the IC50 on MCF7 cell was 7 g/ml. This findings suggests that combination methods were effective and efficient way to select marine actinomycetes producing potential compounds as anticancer.
Cloning of Thermostable DNA Polymerase Gene from a Thermophilic Brevibacillus sp. Isolated from Sikidang Crater, Dieng Plateu, Central Java Witasari, Lucia Dhiantika; Prijambada, Irfan Dwidya; Widada, Jaka; Arif Wibawa, Dionysius Andang
Indonesian Journal of Biotechnology Vol 15, No 2 (2010)
Publisher : Universitas Gadjah Mada

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Abstract

Thermostable DNA polymerase has an important role for amplifying small amount of DNA through polymerase chain reaction (PCR). Thermophillic bacteria Brevibacillus sp. was isolated from Sikidang Crater, Dieng Plateu, Central Java. Previous study showed that crude protein of the isolate could be used in PCR. Unfortunately, like most native thermostable enzymes, the thermostable DNA polymerase of the isolate is synthesized in a very low level and therefore is cumbersome to purify. The purpose of this research is to clone thermostable DNA polymerase gene of the isolate. The DNA polymerase gene was amplified by means of PCR using spesific primers. The amplified fragment was then isolated, purified, and ligated into the pGEM-T cloning vector. The recombinant plasmid was then transformed to competent E. coli JM109 cells using heat shock method. The cloned thermostable DNA polymerase gene from the thermophilic isolate was then characterized for its nucleotide base sequence. The result showed that the DNA Pol I gene was successfully be amplified from the isolate DNA genom, resulting in ± 2,7 kb DNA fragment in length. Sequence analysis of segment of targeted gene showed high similarity to that of thermostable DNA polymerase genes from other Bacillus.Key words : Thermostable DNA Pol I, Brevibacillus sp., PCR, cloning
Isolation and Screening of Antimicrobial Producing-Actinomycetes Symbionts in Nudibranch ., Riyanti; Widada, Jaka; Rajasa, Ocky Karna
Indonesian Journal of Biotechnology Vol 14, No 1 (2009)
Publisher : Universitas Gadjah Mada

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Abstract

         The aims of  this study were to isolate and to screen actinomycetes associated with sea slug which have the ability to produce antimicrobial compound, especially against MDR strains. Actinomycetes were isolated from nudibranchs collected from Bandengan coastal waters and the Panjang island, Jepara, Central Java. Actinomycete isolates were assayed for their antimicrobial activity against MDR strains (MDR 6 E. coli, MDR 7 Enterobacter sp., MDR 13 Proteus sp., MDR 14 Staphylococcus sp.). The genetic diversity of the active isolates was analyzed by using repetitive DNA fingerprinting.  Antimicrobial activity was also performed on the  ethyl acetate bacterial extract.  The amplification of Polyketide Synthase-I (PKS-I) and Non-Ribosomal Peptide Synthetase (NRPS) genes was carried out to estimate the genetic potency of actinomycetes. The most active actinomycete isolate was sequenced based on 16S rDNA approach. General profile of antimicrobial substances was analyzed by using Thin Layer Chromatography (TLC). A total 27 isolates were obtained from nudibranchs Jorunna sp. and 12 isolates from Chromodoris sp.  Ten isolates exhibited antimicrobial activity. Five representative isolates were selected based on rep-PCR analysis.  Three ethyl acetate extracts exhibited antimicrobial activity against MDR 7, MDR 13, and MDR 14, except MDR 6. NPC 8 isolates significantly inhibited the growth of the tested strain   and amplified NRPS gene fragment. Molecular identification revealed that isolate NPC 8 closely related to Streptomyces sp with a high homology of 96%.
16s rRNA Sequence Analysis and Ammonium Excretion Ability of Nitrogen Fixing Bacteria Isolated from Mineral Acid Soil H, Hartono; Widada, Jaka; Kabirun, Siti
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

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Abstract

Nitrogen fixing bacteria defined as bacteria which is capable to transform free nitrogen molecules into ammonium v (PCR). Nitrogenase activity of these selected isolates was measured using Acetylene Reduction Assay (ARA). The ability of these selected isolates in ammonium excretion was qualitatively and quantitavely measured using Nessler reagent and spectrophotometry method respectively. Taxonomic position of the selected bacteria were determined based on their 16S rRNA sequence analysis. Genetic diversity analysis of these 15 isolates of nitrogen fixing bacteria yield eight selected bacteria for subsequent analysis. Sequence of nifH gene from all of these selected bacteria were successfully amplified. Nitrogenase assay of these selected bacteria revealed 6 isolates with high nitrogen fixation capasity namely GMA3, GMA5, GMA6, GMA9, GMA12 AND GMA 13.</div><div>Ammonium excretion analysis revealed 4 isolates which have remarkable ability of producing high level of ammonium namely GMA1, GMA3, GMA6, and GMA9. The 16S rRNA sequence analysis shown that isolates GMA3, GMA5, GMA11 and GMA12 had a close relationship with Brevibacillus formosus strain DSM 9885T, Flexibacter canadensis strain ISSDS-428, Rhizobium tropici strain rif 200849, and Azotobacter tropicalis strain RBS. Respectively, isolate GMA1 and GMA13 had a close relationship with Sthenotropphomonas sp. Strain MFC-C, while isolate GMA6 and GMA9 had a close relationship to Azotobacter vinelandii strain ISSDS-428.</div>, string),(105, en_US, subject, nitrogen fixing bacteria, ammonium excretion, identification, string),(105, en_US, sponsor, , string),(107, en_US, title, Effect of Probiotic Lactobacillus sp. Dad13 on Humoral Immune Response of Balb/C Mice Infected with Salmonella typhimurium, string),(107, en_US, abstract, An indigenous strain of lactic acid bacterium (LAB) identified as Lactobacillus spp. Dad13 (Dad13), isolated from traditional fermented buffalo milk, was found to be potential as probiotic. The aim of this research was to study the effect of probiotic Dad13 on humoral immune response of Balb/C mice infected with Salmonella typhimurium. Thespecific objective was to find out the effect of different Dad13 consumption time (before and along with infection of S. typhimurium) on the humoral immune response of Balb/C mice. The experiment was conducted by in vivo trial on 20<br />males of Balb/C mice, age of 6-8 weeks, fed with AIN-93 standard diet. The mice were assigned into 4 groups. Each group received the following treatments, ie. Dad13 only, Dad13 before infection, Dad13 along with infection and Salmonella infection only. A volume of 100 μl Dad13 cell suspensions (1010 CFU/ml) were given by oral forced feeding daily for a week, at week 3 for group before infection and at week 4 for group of Dad13 only and Dad13 along with infection. Salmonella infection (109 CFU/ml) was given once orally at week 4 to all groups except group treated with Dad13 only. The humoral immune response of Balb/C mice was detected 2 weeks after infection by measuring the titers of IgG and IgA specific from serum and mucosal intestinal liquid samples using Enzyme-linked Immunosorbent Assay (ELISA) method. The result indicated that humoral immune response of Balb/C mice consuming Dad13 before and along with Salmonella infection were significantly different (p<0.05). Dad13 consumption along with Salmonella infection increased circulated IgG and IgA as well as secretory IgA. It can be concluded that Dad13 probiotic feeding along with infection increased humoral immune response more significantly compared to that before infection.
An Actinomycetes Producing Anticandida Isolated from Cajuput Rhizosphere: Partial Identification of Isolates and Amplification of pks-I genes ., Alimuddin; Asmara, Widya; Widada, Jaka; ., Mustofa; Nurjasmi, Reni
Indonesian Journal of Biotechnology Vol 15, No 1 (2010)
Publisher : Universitas Gadjah Mada

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Abstract

Actinomycetes have been the most prolific producer of various kinds of antifungal metabolites, and many of them are described as being produced by polyketide synthetases (pks). We present strain of Actinomycetes producing anticandida isolated from rhizosphere plant for amplification of Pks-I genes. The isolate was obtained from Wanagama I Forest UGM Yogyakarta. Gene of seven isolates, from total of 173 isolates, were amplified using degenerate primer to detect the presence of pks genes. One strain that is named Streptomyces sp. GMR-22 was partialy identified as anticandida producing actinomycete. The strain shown the strongest activity against Candida albicans. Based on bioautography assay, one spot active with Rf 0.57 was appeared as bright yellow by cerrium sulphate but it was and not visible on UV254 and 366 lights. Key words : pks genes, anticandida, Streptomyces sp GMR-22, rep-PCR, cajuput rhizosphere
Diversity of Dibenzofuran-Utilizing Bacteria Isolated by Direct-Plating and Enrichment Methods Prijambada, Irfan Dwidya; Widada, Jaka; Kusumaningtyas, Pintaka; Suryawan, Dhani
Indonesian Journal of Biotechnology Vol 17, No 1 (2012)
Publisher : Universitas Gadjah Mada

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Abstract

The effect of enrichment bias on the diversity of Dibenzofuran (DBF)-degrading bacteria recovered from soil was evaluated by direct plating, plating after in-soil adaptation, and plating after batch culture enrichment. Among colonies appeared on Bushnell Haas agar with DBF as the sole carbon source, 119 colonies (49, 38, and 32 from direct plating, plating after in-soil adaptation, and plating after batch culture enrichment, respectively) were arbitrarily selected based on the appearance of the colonies. Total DNA were then extracted from the rest of the colonies and analyzed for their diversity using Ribosomal Intergenic Spacer Analysis (RISA). Number of DNA bands obtained from direct plating was higher than the ones obtained after in-soil enrichment and batch culture enrichment. The RISA bands obtained from direct plating were also found to be distributed more evenly than the ones obtained after in-soil enrichment and batch culture enrichment. Dominant bands were observed on RISA from samples obtained after in-soil enrichment and batch culture enrichment. Out of 119, only 9 isolates were consistently able to grow on Bushnell-Haas broth with DBF as the sole carbon source as indicated by broth turbidity. All of the isolates were obtained from soil samples which were enriched in a batch culture. Some of the isolates were able to degrade more then 80 % DBF in the minimal medium.
FITOREMEDIASI KANDUNGAN KROMIUM PADA LIMBAH CAIR MENGGUNAKAN TANAMAN AIR Safarrida, Anna; ., Ngadiman; Widada, Jaka
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol 2, No 2 (2015): December 2015
Publisher : Badan Pengkajian dan Penerapan Teknologi (BPPT)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (358.874 KB) | DOI: 10.29122/jbbi.v2i2.509

Abstract

Existence of heavy metals in industrial waste is gaining global attention since their negative impact to environment. One of the efforts to solve the problem was to use plant to absorb metal in liquid medium, known as rhizofiltration. This research was aimed to select aquatic plants which showed relative resistantce and susceptibility to chromium. Four species of aquatic plants (Pistia stratiotes, Eichhornia crassipes, Lemna minor and Salvinia sp.) were grown in artificial medium (Hoagland) supplemented with 0, 1, 2, 4 and 8 ppm chromium. The plants resistance and absorption toward chromium was observed based on the morphology and chromium content in their biomass. Based on their resistance to and absorption of chromium, the selected plants were tested further in liquid waste of tanning industry. In Hoagland medium, Salvinia sp. demonstrated 67.2% higher resistance and absorption toward chromium while that of P. stratiotes 20.3% lower compared to other plants which were tested. This result could be applicable in reducing such environmental pollutant as the heavy metal chromium from industrial waste. Keywords: Phytoremediation, chromium, Hoagland medium, aquatic plants, liquid waste ABSTRAKLogam berat dalam limbah industri merupakan bahan pencemar lingkungan yang mendapatkan perhatian global. Salah satu upaya untuk mengatasi masalah tersebut adalah memanfaatkan tanaman untuk menyerap logam dalam medium cair atau dikenal sebagai fitoremediasi. Penelitian ini bertujuan untuk mengetahui tanaman air lokal yang tahan dan peka secara relatif terhadap kromium. Empat spesies tanaman air (Pistia stratiotes, Eichhornia crassipes, Lemna minor, dan Salvinia sp.) ditumbuhkan pada medium buatan (Hoagland) yang dipasok kromium 0, 1, 2, 4, dan 8 ppm. Pengujian toleransi tanaman dan serapan terhadap kromium dilakukan berdasarkan pengamatan morfologis serta analisis kadar kromium dalam biomasa. Berdasarkan daya tahan dan serapan kromium, tanaman terseleksi diujikan lebih lanjut dalam limbah cair industri penyamakan kulit. Dalam medium Hoagland, Salvinia sp. mempunyai ketahanan dan serapan kromium lebih tinggi sebesar 67,2% sedangkan P. stratiotes mempunyai ketahanan dan serapan kromium lebih rendah sebesar 20,3% dibandingkan tanaman lain yang diujikan. Hasil penelitian ini dapat diterapkan untuk mengurangi bahan pencemar lingkungan berupa logam berat kromium dari limbah industri.Kata Kunci: Fitoremediasi, kromium, medium Hoagland, tanaman air, limbah cair
PURIFICATION AND CHARACTERIZATION OF THE NEWLY THERMOSTABLE PROTEASE PRODUCED BY Brevibacillus thermoruber LII ISOLATED FROM PADANG CERMIN HOTSPRING, INDONESIA Zilda, Dewi Zeswita; Harmayani, Eni; Widada, Jaka; Asmara, Widya; Irianto, Hari Eko; Patantis, Gintung; Fawzya, Yusro Nuri
Squalen, Buletin Pascapanen dan Bioteknologi Kelautan dan Perikanan Vol 9, No 1 (2014): May 2014
Publisher : Research and Development Center for Marine and Fisheries Product Processing and Biotechnol

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15578/squalen.v9i1.91

Abstract

Thermo stability is among of the vital enzyme characteristics for industrial application. Brevibacillus thermoruber LII was obtained as a potential isolate from the previous researchwhich screened the potential thermostable protease producing bacteria from Indonesian hotspring.The newly thermostable protease produced by thermophilic Brevibacillus thermoruber LII hadbeen purified and characterized. It was predicted that the pure enzyme obtained from Brevibacillusthermoruber LII was homo hexameric, having molecular weight of 36 kDa unit protein and itsnative was 215 kDa. In addition, it was also a neutral metalo serine protease according tobiochemical tests that it was totaly inhibited by PMSF (Phenylmethanesulfonyl fluoride) and EDTA(Ethylenediaminetetraacetic acid). It showed optimum activity at pH of 8 and active in acidic buffer(up to pH of 4). All of metal ion in the form of chloride salt (2.5 mM) which were tested on theenzyme enhanced the enzyme activity but Li2+. Ca2+ion increased the activity and the stability ofenzyme against thermal. The enzyme also showed the stability against solvent. The protease LIIhad optimum temperature at 60oC without CaCl 2and 80 – 85oC with addition of 2.5 mM CaCl 2. TheK Mand V maxvalues for the purified protease LII were 27.2 mg/ml or 0.362 – 0.272 M for substrateHammersteinCasein (MM 75–100 kDa) and 261.1 µg/minute/ml, respectively.
Diversity of Culturable Actinomycetes from Deepsea Floor of Makassar Strait, Indonesia Hatmanti, Ariani; Lisdiyanti, Puspita; Widada, Jaka; Wahyuono, Subagus
Oseanologi dan Limnologi di Indonesia Vol 3, No 2 (2018)
Publisher : Oseanologi dan Limnologi di Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/oldi.v3i2.205

Abstract

With regard to collaboration research called Widya Nusantara Exploration (EWIN) in May-June 2013 and November 2014, a study on isolation of actinomycetes from sediments of Makassar Strait have been conducted. Actinomycetes is one of microbe which has an excellent track record in producing antimikrob and other active substances. But due to terrestrial actinomycetes has been widely explored, then recently researchers began focusing on wide variety of extreme environments, such as marine environment, to screening aktinomisetes in producing new secondary metabolites. A total of 36 strains of actinomycetes were isolated from 10 samples obtained from deepsea floor in Makassar Strait, Indonesia, Direct Dillution Method were best used to isolate the actinomycetes compare to Sodium Dodecyl Sulfida – Yeast Extract Method (SDS-YE Method) and Rehidration Centrifugation Method (RC Method). NBRC-802 media and Actinomycetes Isolation Agar(AIA)(Himedia)media were used as the isolation media. All the isolates were identified by morphological characteristic and by analysis of 16S rRNA gene sequence. Actinomycetes isolated from deepsea floor of Makassar Strait have been dominated by Micromonospora (58%), Verrucosispora (14%)Streptomyces (8%) and Luteipulveratus (5%), however genus Nocardiopsis, Micrococcus, Gordonia, Kytococcus, and Arthrobacter were not dominant (3%). Station 25 in 1.547 m depth was the most abundant of actinomycetes, 18 strains and dominated by the genus Micromonospora which is isolated using Direct Dillution Method and both NBRC 802 or AIA media.
Pengaruh Fungi Mikoriza Arbuskular Dalam Medium Zeolit Terhadap Pertumbuhan dan Intensitas Penyakit Bercak Daun Pada Bibit Kakao Sariasih, Yenny; Hadisutrisno, Bambang; Widada, Jaka
Jurnal Agro Teknologi Tropika Vol 1, No 1 (2012)
Publisher : Jurnal Agro Teknologi Tropika

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Abstract

In Indonesia, cocoa (Theobromae cacao L.) is one of the nations third-largest contributor of foreign exchange, but the problem that arises on cocoa plantations in Indonesia is the difficulty of obtaining a healthy cacao seedlings in large numbers for the rejuvenation of cocoa cropping. One of methods to obtained healthy cacao seedlings with good performance in large numbers is application of arbuscular mycorrhizal fungi (AMF). This researches aims to observe the role of AMF mass production in the zeolite medium on the growth and leaf spot intensity of cocoa seedlings. The researches were conducted at the field condition in Sleman, Yogyakarta and Mycological Laboratory of the Faculty of Agriculture and the Laboratory of Phytochemistry, Faculty of Pharmacy, University of Gadjah Mada, Yogyakarta. The main ingredient of the study is AMF mass production in the zeolite medium. The observed variables were the growth of cocoa seedlings, the number of spores from each location, the cocoa seedlings height, leaf numbers, wet weight, dry weight and root length of seedlings of cocoa, symptom and intensity of leaf spot disease, detection of salicylic acid (SA) manually and TLC methods, and environmental factors which include: temperature, humidity and light intensity. The results indicated that the only real difference in the height and number of leaves, whereas other variables were not significantly different. This is because AMF spores from all locations are derived from the similarity of the two genera, namely Glomus sp., and Gigaspora sp., and was almost the same amount. Leaf spot disease symptoms appeared only a few of the cocoa seedlings, but more due to unfavorable environmental factors and conditions are weak pathogens. Plant defense responses had not been established because the salicylic acid content in leaves of cocoa seedlings at 12 weeks has not been detected.
Co-Authors , Tamad . Tohari A. Alimuddin Achmad Dinoto Adi Laksono Ahmad Romdhon Akira Hosoyama Akira Hosoyama Ali Ikhwan Alim Isnansetyo Amekan, Yumechris Angga Prasetya Anna Safarrida Anna Safarrida, Anna Annisa Yusuf, Wahida Ariani Hatmanti Arif Muliawan Arifah Khusnuryani Arifah Khusnuryani Ariyanti, Nur Fitriana Asrul Asrul Asrul Asrul Atsushi Yamazoe Atsushi Yamazoe Aziz Purwantoro Azwar Maas Bambang Hadisutrisno Bambang Hadisutrisno Bambang Hadisutrisno Bambang HADISUTRISNO Bambang Hariwiyanto Bambang Hariwiyanto Bambang Hendro Sunarminto Bambang Hendro sunarminto Bostang Radjagukguk Camelia Herdini Christanti Sumardiyono Denny Irawati Dewi Seswita Zilda DEWI SESWITA ZILDA Dhani Suryawan Dhani Suryawan, Dhani Diani Mentari Diannastiti, Fani Aulia Didik Indradewa Didik Indradewa Didik Indradewa Dinar Mindrati Fardhani Dionysius Andang Arif Wibawa Dionysius Andang Arif Wibawa, Dionysius Andang Dody Kastono Dolly Iriani Damarjaya Dolly Iriani Damarjaya, Dolly Iriani Donny Widianto Donny Widianto Donny Widianto Donny Widianto Dyah Weny Respatie Edy Meiyanto Eka Tarwaca Susila Putra Eko Hanudin Eko Hanudin EKO IRIANTO Ema Damayanti Ema Damayanti Endang Semiarti Endang Sutriswati Rahayu Eni Harmayani ENI HARMAYANI Erni Martani Erni Martani Eti Nurwening Sholikhah Fatturahman Ridwan, Nur Febriansah , Rifki Galang Indra Jaya Ganis Lukmandaru Gintung Patantis Gintung Patantis GINTUNG PATANTIS Hadi, Ismanurrahman Hari Eko Irianto Hartono Hartono H, Hartono Hera Nirwati Hideaki Nojiri Hideaki Nojiri Indun Dewi Puspita IRFAN D. PRIJAMBADA Irfan Dwidya Prijambada Irwan Suluk Padang Jauhari Syamsiyah Joko Sulistyo Kana Ninomiya Keishi Senoo Keishi Senoo, Keishi Khoirun Nisa Lucia Dhiantika Witasari Lucia Dhiantika Witasari, Lucia Dhiantika M. Mustofa Maria Gratias Mariyatun Mariyatun, Mariyatun Masagus Muhammad Prima Putra Masaya Nishiyama Masaya Nishiyama, Masaya Ma’as, Azwar Melki Melki Mirtani Naima Mohamad Aji Ikhrami Muhammad Dylan Lawrie Muhammad Nur Cahyanto Muhammad Saifur Rohman Mujiyo Mujiyo Mukhlissul Faatih Mukhlissul Faatih1, Mukhlissul Mulyadi Mulyadi Mulyadi Mulyadi Mulyono Mulyono Murwantoko . Mustofa M, Mustofa Mustofa Mustofa Mustofa Mustofa N. Ngadiman Naima, Mirtani Nastiti Wijayanti Nastiti Wijayanti Nastiti Wijayanti Ngadiman Ngadiman . Ngadiman ., Ngadiman Ngadiman N, Ngadiman Noviyanto, Amir Nunuk Supriyatno Nur Edy Nur Prihatiningsih Nurfiani, Sri Ocky Karna Radjasa Oedjijono Oedjijono, Oedjijono Pintaka Kusumaningtyas Prijambada, Irfan Dwidja PUSPITA LISDIYANTI Putra, Sukmana Siswandana Putu Sudira R. Riyanti Rahayu, Endang Sutriswati Reni Nurjasmi Reni Nurjasmi, Reni Riska Wulansari Ristiarini, Susana Riyanti Riyanti Rusdi Evizal Saipul Abbas Sarto SATRIYAS ILYAS Shigeto Otsuka Shigeto Otsuka, Shigeto Shinta Hartanto Shogo Matsumoto Sigit Sunarta, Sigit SITI KABIRUN Siti Kabirun Siti Kabirun Siti Subandiyah Sofia Mubarika Haryana Sri Hastuti Sri Nopitasari Sri Nuryani Hidayah Utami Sri Nuryani Hidayah Utami Sri Nuryani Hidayah Utami Sri Suryanti Sri Suryanti Sri Wedhastri Stalis Norma Ethica Subagus Wahyuono Sudadi Sudadi Suhartiningsih Dwi Nurcahyanti Sumarno Sumarno Sumarno Sumarno Suryanti Suryanti Suryanti Suryanti Susila Herlambang TOHARI TOHARI Tohari Tohari Tomy Listyanto Tomy Listyanto Tri Harjaka Tri Joko Raharjo Tri Rini Nuringtyas Tri Wibawa Tri Wibawa Triwibowo Yuwono Triwidodo Arwiyanto Wangi, Dyah Sekar A P Widya Asmara WIDYA ASMARA Wulansari, Riska Yani Lestari Nuraini Yani Lestari Nuraini, Yani Lestari Yasushi Yoshioka Yenny Sariasih Yose Rizal Yuli Setiawati Yuliana Prahastiwi Yuliana Yuliana Prahastiwi, Yuliana Yusro Nuri Fawzya YUSRO NURI FAWZYA Yusro Nuri Fawzya Yuuki Asano Yuyun Farida Yuyun Farida, Yuyun Zilda, Dewi Zeswita