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Diversity of Actinomycetes at Several Forest Types in Wanagama I Yogyakarta and Their Potency as a Producer of Antifungal Compound Nurjasmi, Reni; Widada, Jaka; N, Ngadiman
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (332.685 KB)

Actinomycetes are bacterial groups that produce many secondary metabolites, which different biological activities, such as antifungi, antibacteria, antivirus, antitumor, etc. Actinomycetes are widely distributed in soil and their diversity is influenced by type of forest. The aim of this study is to investigate diversity of actinomycetes in several forest types of Wanagama I forest in Yogyakarta and their potency as a producer of antifungal compound. Soil samples under the forest of Tectona grandis, Swietenia macrophylla King, Bamboosa vulgaris, Melaleuca leucadendron, and Gliricidia maculata were used as sources of soil bacteria. Bacteria and actinomycetes communities were analyzed through culture-independent approach by RISA and nested-PCR RISA using actinomycetes spesific primer (F243), respectively. Through culture-dependent approach, isolated actinomycetes diversity were analyzed by identification of morphology (colony and cell), genetic (BOX element by rep-PCR), and secondary metabolites (thin layer chromatography). In addition, isolates were assayed for their antifungal activity against Saccharomyces cerevisae, Candida albicans, Fusarium oxysporum and Aspergillus flavus. The presence of Polyketide Synthase-I (PKS-I) and NonRibosomal Peptide Synthetase (NRPS) genes were amplified by PCR to study their correlation with antifungal activity of the actinomycete isolates. The results showed that types of forest influence diversity of rhizobacteria especially actinomycetes. According to culture-independent approach, relatively, com-</div><div>munity of rhizobacteria from the highest were soil under the forest of B. vulgaris, G. maculata, T. grandis, S.macrophylla King, and M. leucadendron, respectively. Meanwhile, community of actinomycetes from the highest were soil under the forest of G. maculata, B. vulgaris, M. leucadendron, S. macrophylla King, and T. grandis, respec- tively. Fourty-three morphologically different isolates were found by using culture-dependent approach consisting of 17 isolates were found in soil under the forest of M. leucadedron, each of 9 isolates in G. maculata and T. grandis, 6 isolates in S. macrophylla King. and 2 isolates in B. vulgaris. More diversity of secondary metabolites were observed in soil actinomycetes under the forest of M. leucadendron. Of the 43 isolates, 100% were active against S.cerevisae, 37.20% against C. albicans, 95.30% against F. oxysporum, and 83.70% against A. flavus. Antifungal activity of actinomycete isolates did not always have correlation with the presence of PKS-I and NRPS.

Isolation and Analysis of DNA Fragment of Genes Related to Kopyor Trait in Coconut Plant S, Sukendah; Volkaert, Hugo; S, Sudarsono
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (316.744 KB)

Kopyor coconut is a natural mutant that has abnormal endosperm development. For the first time several genes that were suspected to be related to kopyor trait were identified based on the chemical compounds of the endosperm that different from that of normal coconut. Sucrose synthase (SUS), Stearoyl acyl carrier protein desaturase (SACPD), and Absicid acid insensitive (ABI) genes were isolated and analyzed. Four DNA fragments with length of 746, 738, 780, and 687 bp (CnSus1A, CnSus1B, CnSus2A, and CnSus2B) were obtained from SUS gene. Sequence analysis at DNA and amino acid level showed that CnSus1A, CnSus1B, CnSus2A, and CnSus2B were classified into monocot SUS group with nongrass SUS type. Isolation of SACPD gene resulted in one DNA fragment with DNA length of 716 bp. CnSacpd shared a high homology with SACPD gene of oil palm and soybean. Isolation of ABI gene resulted in two DNA fragments, CnAbi3A and CnAbi3B, with DNA length of 760 and 728 bp, respectively. CnAbi3A and CnAbi3B showed a high homology with ABI3 gene of several plants. All DNA fragment obtained from SUS, SACPD, ABI genes were used as templates to design spesific markers for each corresponding gene. There were 7 specific primer sets designed, i.e., CnSUS1A, CnSUS1B, CnSUS2A, CnSUS2B, CnSACPD, CnABI3A, and CnABI3B.<

Combination PT-PCR with in vitro Transcription-Translation System: Rapid and Simple Approach for Monoclonal Antibody Production Ali, Muhamad
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (428.952 KB)

We have developed a simple and efficient system for screening and generation of monoclonal antibodies, which can bypass conventional monoclonal antibody production system that includes time-consuming, labor-intensive establishment and cultivation. Our method consist of: (1) cDNA synthesis from single B cells of immunized mouse, followed by (ii) PCR amplification of the Lc and Hc (Fd portion) cDNAs separately using low concentration (0,05 &igrave;M) of the respective cDNA-specific primers with 5&rsquo; homotags in the presence of the homotag-specific primer (0,5 &igrave;M), (iii) overlapping PCR of the amplified Lc and Hc cDNAs with the cassettes for the T7 promoter (T7P) and T7 terminator (T7T) to construct the following expression units: T7P-Lc-T7T and T7P-Hc-T7T, and (iv) in vitro expression of these units using an Escherichia coli S30 extract followed by ELISA screening without</div><div>purification. Light-and heavy-chain cDNA were amplified and expressed using in vitro system, thus avoiding time consuming steps of cloning and bacterial expression. Having successfully amplified and expressed Lc and Hc genes from single B cells in rapid, simple, versatile, labor-saving, and cost effective, this method seem to be useful</div><div>and applicable for the high-throughput monoclonal antibody generation. 

Construction and Immunogenicity Testing of Salmonella, STM1 Vaccine Vector Expressing HIV-1 Antigen Bachtiar, Endang Winiati; Smooker, Peter; Coloe, Peter J
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

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Objective of this study: to determine the ability of Salmonella enterica serovar Typhimurium STM1 as a delivery vehicle for the HIV p24 gene and HIV env gene. The STM1 delivery HIV-p24 vaccination was carried out in the form of a recombinant or a DNA vaccine whereas only a DNA vaccine was used for HIV env. Naked DNA vaccination was also tested and immune responses were evaluated following immunisation in mouse model. Results: vaccination cellular immune responses induced by recombinant p24 STM1 (STM1/pHly-p24) were greater than those elicited by the p24 DNA vaccine in STM1 (STM1/VR-p24), (but statistically not significance) than those induced by oral vaccination. However, IgA responses induced by oral vaccination with either a recombinant or DNA vaccine of p24 in STM1 are higher than those induced by IP vaccination. Conclusions: This result confirms</div><div>other studies that Salmonella was able to deliver HIV antigens to the immune system and induced specific immune responses to the HIV antigen.

Decolorization of Remazol Briliant Blue R by Laccase from White Rot Fungus Polyporus sp. S133 Hadibarata, Tony; Tachibana, Sanro
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

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The decolourization of the recalcitrant dye RBBR by the culture filtrate of Polyporus sp. S133 and its isolatedlaccase was investigated. The laccase alone decolorized RBBR. A small molecular weight redox mediator (HBT) wasnecessary to increase the decolorization. The purified laccase totally decolorized the dye of 200 mg l-1 initialconcentration of RBBR when only 1.5 U ml-1 of laccase was used in the reaction mixture. The effects of differentphysicochemical parameters were tested and optimal decolorization rates occurred at pH 5 and at a temperature of 50°C. The effect of surfactants on the decolourization of RBBR was tested with Tween 80, Tween 20, and Brij 35. It wasdemonstrated that Tween 80 was inhibiting substrate for the decolorization while Tween 80 and Brij 35 was noinhibiting effect for the decolorization. Provided that all of the condition is included, it is suggested that laccase maybe suitable for the wastewater treatment of similar anthraquinone dyes.Keywords: Decolorization; Laccase; Remazol Brilliant Blue R (RBBR); Polyporus sp. S133

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

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (283.87 KB)

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 &mu;l 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&lt;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.

16s rRNA Sequence Analysis and Ammonium Excretion Ability of Nitrogen Fixing Bacteria Isolated from Mineral Acid Soil H. Hartono; Jaka Widada; Siti Kabirun
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

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 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.

Diversity of Actinomycetes at Several Forest Types in Wanagama I Yogyakarta and Their Potency as a Producer of Antifungal Compound Reni Nurjasmi; Jaka Widada; N. Ngadiman
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

Actinomycetes are bacterial groups that produce many secondary metabolites, which different biological activities, such as antifungi, antibacteria, antivirus, antitumor, etc. Actinomycetes are widely distributed in soil and their diversity is influenced by type of forest. The aim of this study is to investigate diversity of actinomycetes in several forest types of Wanagama I forest in Yogyakarta and their potency as a producer of antifungal compound. Soil samples under the forest of Tectona grandis, Swietenia macrophylla King, Bamboosa vulgaris, Melaleuca leucadendron, and Gliricidia maculata were used as sources of soil bacteria. Bacteria and actinomycetes communities were analyzed through culture-independent approach by RISA and nested-PCR RISA using actinomycetes spesific primer (F243), respectively. Through culture-dependent approach, isolated actinomycetes diversity were analyzed by identification of morphology (colony and cell), genetic (BOX element by rep-PCR), and secondary metabolites (thin layer chromatography). In addition, isolates were assayed for their antifungal activity against Saccharomyces cerevisae, Candida albicans, Fusarium oxysporum and Aspergillus flavus. The presence of Polyketide Synthase-I (PKS-I) and NonRibosomal Peptide Synthetase (NRPS) genes were amplified by PCR to study their correlation with antifungal activity of the actinomycete isolates. The results showed that types of forest influence diversity of rhizobacteria especially actinomycetes. According to culture-independent approach, relatively, com-</div><div>munity of rhizobacteria from the highest were soil under the forest of B. vulgaris, G. maculata, T. grandis, S.macrophylla King, and M. leucadendron, respectively. Meanwhile, community of actinomycetes from the highest were soil under the forest of G. maculata, B. vulgaris, M. leucadendron, S. macrophylla King, and T. grandis, respec- tively. Fourty-three morphologically different isolates were found by using culture-dependent approach consisting of 17 isolates were found in soil under the forest of M. leucadedron, each of 9 isolates in G. maculata and T. grandis, 6 isolates in S. macrophylla King. and 2 isolates in B. vulgaris. More diversity of secondary metabolites were observed in soil actinomycetes under the forest of M. leucadendron. Of the 43 isolates, 100% were active against S.cerevisae, 37.20% against C. albicans, 95.30% against F. oxysporum, and 83.70% against A. flavus. Antifungal activity of actinomycete isolates did not always have correlation with the presence of PKS-I and NRPS.

Isolation and Analysis of DNA Fragment of Genes Related to Kopyor Trait in Coconut Plant S. Sukendah; Hugo Volkaert; S. Sudarsono
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

Kopyor coconut is a natural mutant that has abnormal endosperm development. For the first time several genes that were suspected to be related to kopyor trait were identified based on the chemical compounds of the endosperm that different from that of normal coconut. Sucrose synthase (SUS), Stearoyl acyl carrier protein desaturase (SACPD), and Absicid acid insensitive (ABI) genes were isolated and analyzed. Four DNA fragments with length of 746, 738, 780, and 687 bp (CnSus1A, CnSus1B, CnSus2A, and CnSus2B) were obtained from SUS gene. Sequence analysis at DNA and amino acid level showed that CnSus1A, CnSus1B, CnSus2A, and CnSus2B were classified into monocot SUS group with nongrass SUS type. Isolation of SACPD gene resulted in one DNA fragment with DNA length of 716 bp. CnSacpd shared a high homology with SACPD gene of oil palm and soybean. Isolation of ABI gene resulted in two DNA fragments, CnAbi3A and CnAbi3B, with DNA length of 760 and 728 bp, respectively. CnAbi3A and CnAbi3B showed a high homology with ABI3 gene of several plants. All DNA fragment obtained from SUS, SACPD, ABI genes were used as templates to design spesific markers for each corresponding gene. There were 7 specific primer sets designed, i.e., CnSUS1A, CnSUS1B, CnSUS2A, CnSUS2B, CnSACPD, CnABI3A, and CnABI3B.<

Combination PT-PCR with in vitro Transcription-Translation System: Rapid and Simple Approach for Monoclonal Antibody Production Muhamad Ali
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

We have developed a simple and efficient system for screening and generation of monoclonal antibodies, which can bypass conventional monoclonal antibody production system that includes time-consuming, labor-intensive establishment and cultivation. Our method consist of: (1) cDNA synthesis from single B cells of immunized mouse, followed by (ii) PCR amplification of the Lc and Hc (Fd portion) cDNAs separately using low concentration (0,05 ìM) of the respective cDNA-specific primers with 5’ homotags in the presence of the homotag-specific primer (0,5 ìM), (iii) overlapping PCR of the amplified Lc and Hc cDNAs with the cassettes for the T7 promoter (T7P) and T7 terminator (T7T) to construct the following expression units: T7P-Lc-T7T and T7P-Hc-T7T, and (iv) in vitro expression of these units using an Escherichia coli S30 extract followed by ELISA screening without</div><div>purification. Light-and heavy-chain cDNA were amplified and expressed using in vitro system, thus avoiding time consuming steps of cloning and bacterial expression. Having successfully amplified and expressed Lc and Hc genes from single B cells in rapid, simple, versatile, labor-saving, and cost effective, this method seem to be useful</div><div>and applicable for the high-throughput monoclonal antibody generation.

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