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Articles by issue : Vol 6, No 1 (2012): March 2012
7
Articles
Cloning of α-L-arabinofuranosidase Genes and Its Expression in Escherichia coli: A Comparative Study of Recombinant Arabinofuranosidase Originatingin Bacillus subtilis DB104 and Newly Isolated Bacillus licheniformis CW1

NURCHOLIS, MOCHAMAD ( Universitas Brawijaya ) , NURHAYATI, NIKNIK, HELIANTI, IS, ULFAH, MARIA, WAHYUNTARI, BUDIASIH, WARDANI, AGUSTIN KRISNA

Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

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Abstract

Arabinofuranosidase (Abfa) is one of the most important enzymes involved in degradation of lignocelullose biomass.  Two genes encoding α-L-Arabinofuranosidase (abfA), each from Bacillus subtilis DB104 (abfAa1) and an indigenous Indonesian B. licheniformis CW1 (abfAb3), were cloned by the PCR approach  and expressed in Escherichia coli. Sequences analysis of abfAa1 and abfAb3 revealed that each consists of 1721 and 1739 base pairs long DNA, respectively. Each clone contains a hypothetical open reading frame of 1503 and 1509 bp that encode an Abfa protein of 500 and 502 amino acids for abfAa1 and abfAb3, respectively. The deduced amino acid sequence of AbfaB3 shares 75% identity to that of AbfaA1. The recombinant enzymes were expressed constitutively in E. coli. Partial characterization of those enzymes revealed that the AbfaA1 and AbfaB3 were optimally active at 50 ºC and 60 ºC at pH 6, respectively. Thermostability studies of the recombinant enzymes with p-nitrophenyl α-L-arabinofuranoside at their optimal conditions showed that up to 50% AbfaA1 activity was lost after 5 h incubation at 50  ºC, whereas the AbfaB3 retained its activity over 75% after 12 h pre-incubation oat 60 ºC. This thermostability study of recombinant AbfaB3 showed for the first time that the arabinofuranosidase from B. licheniformis is a thermostable enzyme. The recombinant enzyme showed a higher optimal reaction temperature (60 ºC) in comparison to the previously reported thermostable arabinofuranosidase. The thermostable AbfaB3 has a potential to be applied to the degradation of lignocellulose biomass synergistically with thermostable xylanases, for instance in the production of xylo-oligosaccharides.

Molecular Analysis of Immune-Escape Mutants of Hepatitis B Virus from Local Clinical Samples

JINATA, CHANDRA, GIRI-RACHMAN, ERNAWATI ARIFIN, RETNONINGRUM, DEBBIE SOEFIE

Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

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Abstract

Small hepatitis B surface antigen (sHBsAg) is used as a component of hepatitis B vaccine. Even though this vaccine is known to be effective in preventing hepatitis B disease, natural mutation may induce Hepatitis B Virus (HBV) to form immune-escape mutant. This mutant is not only capable of infecting hepatitis B-vaccinated people, but also causing commercial diagnostic assay failure. Immune-escape mutant is generally detected from amino acid change at Major Hydrophilic Region (MHR) of sHBsAg while the change occurred outside the region may also lead to immune-escape mutant formation. This research was aimed to investigate the presence of HBV immune-escape mutants in local clinical samples in Indonesia. sHBsAg gene of seventeen HBV samples from local patients were amplified by polymerase chain reactions then subjected to two-directional sequencing. The DNA sequences later were analyzed by bioinformatics programs. Fifteen out of seventeen samples were genotype B and subtype adw2, while the other two were genotype C and subtype adrq+. Among fifteen genotype B samples, twelve of them were not immune-escape mutants, two were immune-escape mutants that have been previously reported (Gln129Arg and Met133Leu), and one was a mutant outside MHR that has not been previously reported as an immune-escape mutant (Tyr161Ser). Both samples of genotype C group were not immune-escape mutants. As conclusion, by investigating seventeen local clinical HBV samples, it was known that two of seventeen samples were confirmed as immune-escape mutants and one of seventeen samples was a mutant outside MHR.

Cloning and Expression of Nonstructural Protein NS1 of Dengue Virus Serotype 2

DEWI, BETI ERNAWATI, FITHRIYAH, FITHRIYAH, RUKMANA, ANDRIANSJAH, PAISAL, PAISAL, LARASATI, DEKA, SUDIRO, TJAHJANI MIRAWATI

Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

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Abstract

Early diagnosis of dengue virus (DENV) infection is affirmative for patient management and control of the disease. Detection of nonstructural-1 (NS1) antigen has been proven to provide early detection of DENV infection. Commercial NS1 antigen assays are available in Indonesia with variable sensitivity. In an attempt to develop an NS1-based diagnostic test, we successfully cloned NS1 gene of DENV2 to a glutathione Stransferase- based vector pGEX6P-1 in Escherichia coli system. The recombinant protein (pG2NS12) was expressed in E. coli BL21. After induction with isopropyl-β-D-thiogalactoside 0.1 mM for 4 h at 25 °C a recombinant protein GST-NS1 with molecular size of approximately 75 kDa was  obtained. The fusion protein was insoluble and found in the pellet fraction of the cell lysate. Addition of lysozyme (10 mg mL-1) and DNase-I (7.2 mg mL-1) in the lysis buffer was necessary to collect proteins from the pellet fraction. The proteins in the cell pellet were fractionated through Sephadex-G100 column, and GST-NS1 was further purified with Glutathione-Sepharose 4B beads. To obtain pure recombinant NS1 protein to be used in the immunization of mice, the fusion protein was cut with PreScission Protease® by addition of 0.075% Triton-X 100 was necessary to cut the fusion protein. We found that antibodies that recognized the recombinant NS1 protein and DENV2 virus were produced in mice immunized with purified NS1 protein. Therefore, our recombinant NS1 could be used to produce antibody that is potentially useful for developing diagnostic assay to determine the presence of dengue virus NS1 antigen in patient sera.

Isolation and Identification of Endophytic Fungi from Srikaya Plants (Annona squamosa) Having Potential Secondary Metabolites as Anti-Breast Cancer Activity

YUNIANTO, PRASETYAWAN, ROSMALAWATI, SYOFI, RACHMAWATI, INDRA, SUWARSO, WAHYUDI PRIYONO, SUMARYONO, WAHONO

Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

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Abstract

Annonaceous acetogenin was extracted from Annona squamosa (Srikaya) seeds. It has cytotoxic activity against cancer cells and lower toxicity compared to other cancer drugs. Endophyte from Annonaceae is expected to have similar extracted metabolites to the host, thus increasing the economic value. This research is a preliminary study to obtain active compounds with potential as anti-cancer agents from endophytic fungi of Srikaya plants. Four endophytic fungal strains were isolated from Srikaya plants (Annona squamosa) and identified based on 28S rDNA sequence. The isolates are SKY II.3.1, SKY I.1.2, SKY II.3.2, and SKY III.3.1, and have similarity with Fusarium sp Vega760, Fusarium sp NRRL 22354 NRRL223, Nectria rigidiuscula, and Fusarium sp BOL35, respectively. The identified isolates were fermented in liquid media for three weeks. The liquid and mycelium were extracted using ethyl acetate. Whole extract of each fermented isolate was partitioned and evaporated to obtain ethyl acetate extract. Cytotoxicity assay of ethyl acetate extract was carried out at level 100 ppm by Methyl Thyazole Tetrazolium (MTT) viability test towards MCF-7 (breast cancer cell). The result indicated that each ethyl acetate extract could inhibit the viability of cell MCF-7 with 11.34, 99.78, 91.48, and 96.84%, for SKY II.3.1, SKY I.1.2, SKY II.3.2, and SKY III.3.1, respectively. Based on the results of cytotoxicity assay on MCF-7 breast cancer cells, endophytic fungi isolates SKY I.1.2, SKY II.3.2, and SKY III.3.1 are potential as sources of anti-breast cancer compounds.

Molecular Identification of Yeasts Isolated from Dadih by RFLP-PCR and Assessment on Their Ability in Utilizing Lactate

JATMIKO, YOGA DWI, LOPES, MIGUEL DE BARROS, BARTON, MARY D

Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

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Abstract

A wide variety of yeasts have been involved in traditional fermented foods, and potentially contributed to the development of product properties. The existence of indigenous yeasts in dadih, a traditionally fermented form of buffalo milk of West Sumatera, has been reported but an accurate identification is still required to be conducted. This study was aimed to identify yeasts isolated from dadih using a molecular approach, and to evaluate their lactate utilization. A total of 51 isolates were characterized and identified as Pichia jadinii and Candida stellimalicola using PCR amplification of the 5.8S - internal transcribed spacer region combined with restriction fragment length polymorphism (RFLP) analyses and gene sequencing. The former species were the dominant one in this tested product. Their ability to utilize lactate was demonstrated, indicating that they could modify the sensory characteristics of dadih, and hence interact with the indigenous lactic acid bacteria in dadih. The restriction profiles of the dadih yeasts can be used as a data base for rapid identification of yeasts in the future. Further work is still needed to elucidate the dadih yeast ecology.

Sub-Acute Toxicity of Pigment Derived from Penicillium resticulosum in Mice

SOPANDI, TATANG, WARDAH, WARDAH

Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

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Abstract

Pigments derived from Penicillium have different toxicities depending on the pigment components. This study was intended to evaluate the sub-acute toxicity of oral exposure of Balb/c mice to Penicillium resticulosum pigment. A total of 50 healthy adult male and female mice were divided into 5 treatment groups and different doses of pigment (0, 125, 250, 500, and 1000 mg kg-1 weight) were orally administered. Oral feeding of pigment with doses 125 to 1000 mg kg-1 body weight daily to adult mice did not cause mortality nor any clinical abnormalities. There were no significant differences in body, liver and kidney weights, nor liver and kidney functions of mice when pigment was given orally with intake doses of 125 to 1000 mg/kg body weight daily for 28 d in comparison to mice without pigment intake (control groups). There is a slight difference in liver histopathology of mice exposed to 500and 1000 mg/kg body weight of pigment for 28 d in comparison to mice control groups, although there were no differences in kidney histopathology. Thus, we can conclude that the pigment of P. resticulosum can be cathegorized as low toxic pigment and well tolerated at dose below 500 mg kg-1 body weight daily for 28 d.

Application of Phenol Pretreatment for the Isolation of Rare Actinomycetes from Indonesian Soil

ISTIANTO, YUDHIE, KOESOEMOWIDODO, RADEN SETYO ADJI, WATANABE, YOSHIO, PRANAMUDA, HARDANING, MARWOTO, BAMBANG

Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

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Abstract

Phenol treatment was applied for isolation of rare Actinomycetes using 25 soil samples collected from Pulau Seribu, Tanjung Redep, Manokwari, and Halmahera. The samples were air-dried and suspended in 1.5 % (w/v) phenol solution at 30 oC for 30 minutes, and subsequently cultured on plates of humic acid-vitamin agar (HVA) medium supplemented with cycloheximide (50 μg/mL) and nystatin (50 μg/mL). A total of 61 isolates were obtained and the most dominant isolates were not Streptomyces (only 24.6%), whereas other genera such as Micromonospora, Actinomadura, Microbispora and Polymorphospora were isolated with ratios of 49.2%, 13.1%, 9.8%, and 3.3%, respectively.