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Identification of Drought Tolerant Related Insertional Mutant Lines Using PEG 6000
No full textDrought is one of the most important abiotic stresses in rice (Oryza sativa) productivity. The development of drought tolerant cultivars are therefore highly desireable. We have developed insertional mutant based on the Japonica rice cv Nipponbare rice by transposons Ac/Ds insertions containing activation-tag and gene trap. Screening of the mutant population for drought tolerant related phenotypes is of our priority. The screening protocol based on PEG 6000 has been developed and was being used to screen 70 mutant lines tocharacterize their responds to the treatment based on different parameters (number of leaf, total weight, plant height, root length and number of germinating seeds). These characters were used to score the Degradation Index and Vigour Index. Results showed varyingresponds of the lines to the osmotic pressure. Some lines showing a good performance indicated by lower Degradation Index and higher Vigour Index have been identified. Some inconsistencies in the performances scored by both indices were thought to be due to seed quality
Site-Directed Mutagenesis of Glu-269 L-Arabinose Isomerase from Geobacillus stearothermophilus Isolated from Tanjung Api Poso, Indonesia
No full textIndustrializing of tagatose requires enzymes that meet to industrial need such as thermophile, slightly acidic and metal independent. Previously, we cloned, sequenced and expressed L-arabinose isomerase from Geobacillus stearothermophilus isolated from Tanjung Api, Poso, Indonesia. Based on DNA alignment analysis, the gene had high homology with those of G. stearothermophilus T6 (Gene Bank Acc No: AAD45718) which has optimum activity at high temperature and alkaline condition. In this paper, we described site-directedmutagenesis approach to mutate Glu-269 (Q269) to Lys-269 (K269) to decrease the optimum pH of the strain. Sequencing result showed that mutagenesis had been successful to mutate amino acid at position 269 from glutamine (Q) into lysine (K). Expression of mutant Q269 showed protein with molecular mass ~56 kDa. 
The Effect of Increase in NaCl Concentration on Growth and Proline Content of Purple Yam (Dioscorea alata L.) Grown In Vitro
No full textTuber of purple yam (Dioscorea alata L.) has been used as an alternative food in some areas in Indonesia. The tuber contains high carbohydrate, low glycemic index and gluten free, therefore, study on genetic improvement of this species is needed to increase the productivity and to find out new cultivars which can be cultivated in marginal lands. This research was aimed to investigate the effect of NaCl concentration on growth and proline content of purple yam grown in vitro. Shoot tips were cultured on MS (Murashige and Skoog) medium supplemented with NaCl at concentrations of 25; 50; 100; 200 and 250 mM. After six weeks in culture, height of shoots, number of nodes, number of leaves, as well as proline content were recorded. The results showed that shoots grown on MS medium supplemented with NaCl at 25 and 50 mM had better growth compared to control. The best medium for its growth was MS containing 50 mM of NaCl. Increase in NaCl level’s resulted in decrease of growth. The LD50 value was obtained at 183 mM of NaCl. The Highest proline concentration was achieved by shoots grown on the medium supplemented with 100 mM of NaCl. This result indicated that purple yam was tolerant to the increase of NaCl concentration up to 100 mM, on MS medium without addition of plant growth regulators
Identification of Cadalene-β-Carboxylic Acid from Barks of Bawang Hutan (Scorodocarpus borneensis Becc.)
No full textOne sesquiterpene compound has isolated and identified from ethylacetate extract of bawang hutan (Scorodocarpus borneensis Becc.) barks. The barks were macerated with methanol, and then partitioned with mixture of ethylacetate-water (1:1). Fractionation of the ethylacetate phase by column chromatography gave pure compound. Based on data interpretation from of ultra-violet (UV) spectra, Fourier Transform Infra Red (FT-IR), NMR 1D (1H and 13C-NMR); NMR 2D (HMQC, COSY, HMBC) and comparison with literature, the pure isolated compound was determined as a sesquiterpene compound, cadalene-β-carboxylic acid which exhibit LC50 of 42.32 ppm
Expression of An Immunogenic Intimin Fragment of EHEC O157:H7 in Escherichia coli Periplasm under The Control of A Rhamnose-Based Regulated Promoter
No full textIntimin is the main adhesin of Enterohemorrhagic E. coli (EHEC) O157:H7 bacteria which are the most common leading infectious cause of bloody diarrhea and acute kidney failure in children who develop hemolytic uremic syndrome (HUS). Intimin is required for persistent bacterial colonization to eukaryotic host cell and its receptor-binding activity is localized at the C-terminus 282 amino acids (Intimin282). Thus, Intimin282 is an attractive antigen candidate that could be useful in vaccine and diagnostic systems against EHEC infections. Previous studies had reported expression of Intimin in E. coli cytoplasm using commonly used prokaryotic expression systems. However, it usually encountered several problems, i.e. low expression level, leaky expression, inclusion body formation, and truncated protein. The pRHA vector, which is tightly regulated by Lrhamnose and D-glucose, represents a viable alternative E. coli expression system to overcome such problems. Moreover, E. coli periplasm has an advantage of maintaining protein functionality by providing an oxidative environment that is more efficient than cytoplasm. However, to date there is no study about Intimin expressionusing pRHA expression system and/or in E. coli periplasm. Accordingly, we constructed a recombinant pRHA vector harbouring the respective gene to investigate the expression of an immunogenic Intimin fragment of EHEC O157:H7 in E. coli periplasm. The gene encoding His6-tagged Intimin282 (Int282) together with pelB signal sequence was cloned into the pRHA vector, subsequently expressed in E. coli JM109 and purified. Expression and purification of Int282 were verified by SDS-PAGE and Western blot. The result showed that Int282 was successfully expressed in E. coli periplasm with a protein size of approximately 32 kDa, which corresponded with the predicted size of the protein based on its amino acid sequence
Glucoamylase Production by Aspergillus awamori KT-11 In Solid State Fermentation Using Cassava Peel as Substrate
No full textCassava has long been known as one of the main staple food in Indonesia. Whereas the cassava peel contains starch of approximately 72%, it is still underrated as a carbohydrate source for fermentation.The utilization of cassava peel as a substrate in solid state fermentation potentially replaces rice as a carbon source leading to more cost-effective production. This study aims at producing glucoamylase by means of solid state fermentation using Aspergillus awamori KT-11 and cassava peel as substrate. The study demonstrated that medium composition and drying technique affected the production of glucoamylase. The highest glucoamylase activities were identified when cassava peel and mineral media was used in fermentation, compared to only cassava peel; the combination of cassava peel, mineral, and rice bran; rice media or a mixture of rice, mineral and rice bran. Freeze-dried glucoamylase, furthermore, exhibited higher specific activity in contrast to the oven-dried one, with 452 U/mL and 365 U/mL, respectively. In conclusion, cassava peel plus mineral is a better substrate for glucosamine production by A. awamori KT-11 in solid state fermentation. Besides, powdered glucoamylase had been demonstrated to be capable of hydrolyzing starch-based biomass
