Indonesian Journal of Biotechnology
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Agrobacterium-Mediated Stable Transformation of Medicinal Plant Andrographis paniculata Callus Expressing β-glucuronidase (GUS) Gene
No full textThis study was carried out to establish a stable genetic transformation in callus culture of Andrographispaniculata mediated by Agrobacterium tumefaciens. The leaf disks of A. paniculata were infected with A. tumefaciensLBA4404 carrying a binary vector pCAMBIA1304 that contain β-glucuronidase (GUS) and hygromycinphosphotransferase (hpt) genes. The infection was conducted by dipping method for one hour, followed byco-cultivation in the dark for three days. To examine transient GUS expression, the co-cultivated leaf disks wereassayed for β-glucuronidase activity and to obtain stable transformed callus, the co-cultivated leaf disks wereselected on the callus induction medium which contain 20 mg/l hygromycin for selection. The transformedcallus was periodically subcultured every three weeks into the fresh selection medium over the 15 weeksperiod. To test a stable transformation, the callus was subjected to PCR analysis for GUS gene detection. Theresults indicated that the co-cultivated leaf disks expressed GUS activity and proliferated to produce callus onthe selective medium. Analysis of PCR on the transformed callus indicated the presence 976 bp fragment thatconfi rmed the presence of β-glucuronidase gene. These fi ndings imply that the β-glucuronidase was stably integrated into A. paniculata callus culture.Keywords: Andrographis paniculata, Agrobacterium tumefaciens, andrographollide, transformed callus,β-glucuronidase gene
Legume Nodulating Bacterium, Achromobacter xylosoxidans Found in Tropical Shrub Agroecosystem, Sumatera, Indonesia
No full textLegume nodulating bacteria (LNB), known also as rhizobia, are soil bacteria, which are able to form rootnodules and fi x nitrogen in the leguminous plants. The LNB availability in the soil depends on the type ofagroecosystem, where plant grows. In this study, we isolated LNB from the shrub agroecosystem in Sumatera,Indonesia, and obtained four selected bacterial strains. Among them, the isolate UGM48a formed root nodulein Macroptilium atropurpureum and showed highest number of nitrogenase activity. UGM48a also contains nifHand nodA genes. An analysis of the PCR-amplifi ed 16S rDNA and BLASTn analysis showed that UGM48adisplayed 96% similarity with Achromobacter xylosoxidans. In addition, UGM48a were successfully nodulatedGlycine max (L.) merr var. wilis. This is the fi rst report detecting A. xylosoxidans as nodule-forming species forGlycine max possesing the positive copy of nodA gene. Keywords : Legume Nodulating Bacteria, shrub agroecosystem, Achromobacter xylosoxidans, nodA, Glycine ma
Partial Purifi cation, Stability Analysis, and Preservation of Xylanase from Xylanolytic Alkalophylic Bacteria
No full textA xylanase, which produces xylose from oat spelt xylans, was isolated from the culture medium of xylanolytic alkalophylic bacteria mutant. The enzyme was purifi ed by ammonium sulphate with level 30, 40, 50, 60, 70, 80, and 90%. The purify of the fi nal preparation was demonstrated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The molecular masses of the purifi ed xylanase were 137.61 and 165.34 kDa. Result of ammonium sulphate saturation with the highest activity was used as standart for saturation for enzyme production and preservation, using corn, tapioca, soy bean meal and gaplek fl our as carriers. Addition of 60% ammonium sulphate showed the highest xylanase activity (62.03 U/g), and produced 89.40% enzyme recovery. Tapioca, as a carrier, produced the highest xylanase activity. Key words: preservation, purifi cation, stability analysis, xylanase
Chemosystematic of Enterobacteriaceae Familia Obtained from Blood Cultures Based on Total Protein Profiles
No full textThe purpose of this study was to determine the chemosystematic of 14 strains of bacteria in blood cultures from Semarang using 1 reference strain S. typhi NCTC 786, based on the total protein profi les with the similarity relationship analysis based on Simple Matching Coeffi cient (SSM) analysis and algorithm methodof unweighted pair group with averages (UPGMA) presented in a dendrogram. The results showed that thechemosystematic based on the total protein profi les using SDS-PAGE method can classify the member ofbacterial strains of each species. The Clusters respectively consist of 4 strains of S. typhi (similarity: 89.7%),2 strains of Ser. marcescens (similarity: 89.7%), two strains of E. coli, and one strain of Salmonella ssp, S. typhi NCTC 786 (similarity: 100%). Those three incorporated clusters had the similarity value of 75.3%. Those four strains of Ent. cloacae composed in one cluster (similarity: 100%) are incorporated in a cluster consisting of one strain of Kleb. pneumoniae (similarity: 92.9%). Both clusters were incorporated in a cluster consisting of S. typhi NCTC 786 (similarity: 67.9%). Key words: Enterobacteriaceae, chemosystematic, blood cultures, protein profil
The Diversity of Legume-Nodulating Bacteria from Several Agroecosystems in Sumberjaya, Lampung
No full textBacteria that capable of forming root nodules on legumes are known as Rhizobia. They have also known as Legume- Nodulating Bacteria (LNB). They can fi x nitrogen from the atmosphere. Diversity of Legume-Nodulating Bacteria is affected by biotic factors (such as their genetic factors, plants, and competition with the other soil microbes) and abiotic factors (such as land use, soil’s temperature, pH, chemistry and soil’s properties). The aim of this experiment is to know the diversity of eleven Legume- Nodulating Bacteria based on their phenotypic and genotypic characters. The eleven LNB used in this experiments were isolated from several agroecosystems in Sumberjaya, Lampung. The analysis of these LNB diversity were carried out by characterizing both phenotypic and genotypic properties. The diversity analysis showed that the eleven LNB isolates had high diversity, based on nodule formation, and classifi ed into two groups of cross inoculation group. Key words: Rhizobia, phenotypic diversity, genotypic diversit
Cytotoxic Activity of Tegari (Dianella nemorosa Lam.) Methanol Extract Against HeLa Cells
No full textDianella nemorosa Lam. also known as tegari belonging to the Liliaceae family. This plant has been utilized for Papua traditional medicine as well as anticancer agent. This research examined potential cytotoxic activity of tegari (D. nemorosa) leaves extract against cervical cancer cell line (HeLa). Methanol extract was obtained by extracting the leaves powder using methanol. Extract was then applied into HeLa cell line to find out the cytotoxic activity. MTT [3-(4,5-dimetilthiazol-2-il)2,5-difeniltetrazolium bromida) assay was used to measure the cytotoxic activity. The result indicated that D. nemorosa leaves extract possessed cytotoxic activity in HeLa cell line with IC50 values were 685,69 µg/ml, 506,43 µg/ml and 708 µg/ml at the incubation period of 24, 48 and 72 h respectively. The strongest cytotoxic was showed by methanol extract incubated in 48 h
Genetic Relatedness among Duku, Kokosan, and Pisitan in Indonesia Based on Random Amplified Polymorphic DNA Markers
No full textGenetic relatedness among duku, kokosan, and pisitan from Indonesia were investigated using random amplified polymorphic DNA (RAPD) markers. Eleven primers (OPA-01, OPA-02, OPA-10, OPB-07, OPB-11, OPB-12, OPB-15, OPT-16, OPU-14, OPU-19, and OPU-20) were used for amplification and yielded a total of 174 DNA bands, of which 167 were polymorphic. Primer OPA-10, OPB-11, OPB-12, OPB-15, and OPU-19 produced all of the polymorphic DNA bands. The size of the amplified DNA fragments ranged from 41-1546 bp. The dendrogram separated into two clusters at a genetic similarity coefficient of 0.76. The cluster 1 consisted of subclusters duku and several pisitan (pisitan OKI, pisitan Sleman, pisitan Hatu, pisitan Punggur, and pisitan Tanjung), and cluster 2 consisted of subclusters kokosan and pisitan. In the kokosan subclusters, including duku Drendan. Dendrogram supported the determination of taxonomic status of duku, kokosan, and pisitan as one species, namely Lansium domesticum Corr. and its divided into two groups, namely L. domesticum ’duku group’ and L. domesticum ’pisitan-kokosan group’. Thus, RAPD analysis was useful tool for determining the genetic variation and the genetics relatedness among duku, kokosan, and pisitan in Indonesia.Key words: duku, kokosan, pisitan/langsat, genetic relatedness, RAP
Paternity Analysis of Tea (Camellia sinensis L. Kuntz) Hybrids Using Isozyme Marker
No full textTea plant has been categorized as self-incompatible crop. This is the reason behind the high genetic diversity. Natural pollination is possible to occur and the male parent is usually unknown, therefore, there is a need of method to identify male parent of hybrids through paternity analysis. Isozyme markers have been successfully used for paternity analysis due to their co-dominant polymorphism. This research aimed to predict male parents of hybrids by figuring out the mating system through isozyme banding patterns. In this experiment, seven enzyme systems were evaluated, of which only two of the enzyme systems i.e. esterase and shikimate dehydrogenase showing clear band pattern of Est-1, Est-2, and Shd-1 loci. The mating system of tea could be categorized as a mixed mating model, with high estimated out-crossing rate of 98.6 %. The pollen contributors were not always originated from the vicinity of the female parents.Key words: isozyme markers, paternity analysis, te
Identification of Pediococcus Strains Isolated from Feces of Indonesian Infants With in vitro Capability to Consume Prebiotic Inulin and to Adhere on Mucus
No full textThe aim of this experiment was to identify isolates obtained from feces of Indonesian infants and to evaluate their capability as probiotics. Identification of isolates was carried out based on morphology, physiology and biochemical identifications, and molecular identification based on 16S rRNA sequence. Morphological and physiological identification was carried out based on Gram staining, shape, motility, spore formation and catalase production. Biochemical identifications based on production of CO2 and NH3 from glucose, the ability to grow on different temperature (10 and 45°C) and pH (4.4 and 9.6), and different salt concentration (6.5 and 18%). Probiotics capability of isolates was assayed on the ability to grow on low pH (pH 2.0), on different bile salts concentration (0.3; 0.5; 1.0 and 1.5%), the capacity to grow on media with inulin as the only carbon source, and in vitro adhesion ability on porcine mucin. Morphological, physiological and biochemical identification suggest that all of isolates belong to lactic acid bacteria. Further molecular identification of five isolates showedthat isolates AA, BE and BK were strains of Pediococcus acidilactici (similarity 99%), while isolate AP and AG were strains of Lactobacillus casei (similarity 99-100%). Probiotic assays showed that more than 80% of cells of Pediococcus acidilactici isolates AA, BE and BK were viable after grown on pH 2.0 for 90 min, and around 80% of cells from the same isolates were survived on media supplemented with bile salt 1.5% for 2 h. All of isolates had high adhesion capacity as seen by more than 75% of cells attached on pig gastric mucin. Investigationof isolates to grow on inulin showed Pediococcus acidilactici isolate BE was able to consume inulin as the only carbon source. It is concluded that Pediococcus acidilactici isolate BE was a candidate probiotics and subject to further in vivo evaluation using animal models to examine their beneficial health effects.Key word : Pediococcus acidilactici, Lactobacillus casei, human origin and probiotics
Production and Optimization of Oleic Acid Ethyl Ester Synthesis Using Lipase From Rice Bran (Oryza sativa L.) and Germinated Jatropha Seeds (Jatropha curcas L.) by Response Surface Methodology
No full textRecently, the fatty acid ethyl ester has been synthesized in place of fatty acid methyl ester since ethanol has been more renewable. In this research, oleic acid ethyl ester (OAEE) was synthesized using germinated jatropha seeds (Jatropha curcas.L) and rice bran (Oryza sativa) as source of lipase. The objective of the research was to optimize the synthesis conditions using Response Surface Methodology. Factors, such as crude enzyme concentration, molar ratio of oleic acid to ethanol, and the reaction time, were evaluated. The results show that lipase from germinated jatropha seeds had the hydrolitic and esterifi cation activity about 6.73 U/g and 298.07 U/g, respectively. Lipase from rice bran had the hydrolitic and esterifi cation activity about 10.57 U/g and 324.03 U/g, respectively. The optimum conditions of esterifi cation reaction using germinated jatropha seed lipase as biocatalyst were crude enzyme concentration of 0.31 g/ml, molar ratio of oleic acid to ethanol of 1 : 1.81, and reaction time of 50.9 min. The optimum conditions of esterifi cation reaction using rice bran lipase were crude enzyme concentration of 0.29 g/ml, molar ratio of oleic acid to ethanol of 1 : 2.05, and reaction time of 58.61 min. The obtained amounts of OAEE were 810.77 μmole and 626.92 μmole for lipases from rice bran and germinated jatropha seed, respectively