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Articles by issue : Vol 8, No 4 (2014): December 2014
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Articles
Cloning, Overexpression, and Purification of PhoR CytoplasmicDomain Protein from Mycobacterium tuberculosis strain H37Rv

ROKA AJI, OKTIRA, PASCAPURNAMA, DYSHELLY NURKARTIKA, PRATAMA, FENRYCO, IHSANAWATI, IHSANAWATI, MOEIS, MAELITA RAMDHANI, GIRI-RACHMAN, ERNAWATI ARIFIN

Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

Tuberculosis still becomesa major health problem in the world. This infectious disease is caused by Mycobacterium tuberculosis (Mtb). Novel anti-tubercular drug is urgently needed to counter multidrug resistant cases and Mtbs spread. The cytoplasmic domain of PhoR histidine kinase, a part of the two-component system PhoR-PhoP in Mtb, is one of the potential candidates for anti-tubercular drug target.Three dimensional protein of drug target is needed to screen potential drug candidate using rational drug design approaches. Previous studies have successfully characterized and isolated putative cytoplasmic domain of PhoR (CytoPhoR) from Mtb strain H37Rv. This study aimed to clone, overexpress and purify of CytoPhoR protein. CytoPhoR was fused with thioredoxin protein in expression vector pET32b and overexpressed in Escherichia coli (E.coli)BL21 (DE3) as soluble fraction by induction  1 mM IPTG. Purification of his-tagged CytoPhoR was carried out using IMAC Ni-NTA Agarose his-tag affinity column. SDS-PAGE analysis showed that another protein was co-purified (~35 kDa) along with the CytoPhoR protein. Subsequent protein purification using DEAE-ion exchange column generate a strong single band of 37 kDa on SDS–PAGE which is indicated as CytoPhoR protein. The purified CytoPhoR protein was successfully obtained and can be used for further analysis on determining three dimensional structure of CytoPhoR protein.

Isolation and Molecular Identification of Endophytic Bacteria from Durian Arillus (Durio zibethinus Murr.) var. Matahari

SUHANDONO, SONY, UTARI, INDAH BUDI

Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

Endophytes are plant-associated microorganisms that able to form colonies in internal tissue and considered as an important component of biodiversity. However, information about the existence of naturally fruits-associated endophytic bacteria at different life-history stages of hosts is very limited. Durian (Durio zibethinus Murr.) is an exotic tropical fruits with a high economical value, but the occurrence and functional role of associated endophytes remains unexplored. A total of sixteen endophytic bacterial isolates were identified by 16S rRNA sequence analysis from ripe and unripe stages of Durian fruits var. Matahari. These isolates belonged to the genus Staphylococcus, Bacillus, Enterobacter, Moraxella, Gordonia, Salmonella, Rhizobium, Brachybacterium, Kocuria, and Klebsiella. This is the first report of an endophytic bacterial spesies residing in Durian arillus. This research also indicated potency of culturable endophytes from Durian fruits in plant growth promotion.

Characterization of chaperone-like activity of small heat shock protein (sHSP) isolated from Indonesian Traditional Food (Tempoyak ) Lactobacillus plantarum U10

HASLIA, MARGARETA, MUSTOPA, APON ZAENAL, BUDIARTO, BUGI RATNO, WIDYASTUTI, UTUT

Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

The characterization of small heat shock protein (sHSP) from tempoyak-originated Lactobacillus plantarum was investigated. The heat adaptive response proteins were ranging from 18 kDa to 51 kDa. Interestingly, the Intercellular Protein (IP) fraction of heat shocked-L.plantarum U10 exhibited chaperone like activity by the ability to prevent loss of proteinase K activity from denaturation. Furthermore, The sHSP gene that related to the predicted sHSP ±18 kDa protein were successfully identified by PCR method and this gene has 423 bp size. The sHSP gene has 140 amino acids (with unique motive at C-terminus T-L-P-K amino acid sequence) and has closely 100% identity with those L.plantarum isolated from food or non-food environment. Moreover, the gene encoding sHSP ±18 kDa protein was indeed up-regulated after L.plantarum U10 treated by heat shocking as proven by Reverse Transcriptase-PCR. This result suggested that sHSP ±18 kDa in our study may confers a survival advantage on Lactobacillus plantarum and capable of protecting the cell against under temperature stress.

Codon Optimization and Chaperone Assisted Solubilization of Recombinant Human Prethrombin-2 Expressed in Escherichia coli

SILABAN, SARONOM, MAKSUM, IMAN PERMANA, GHAFFAR, SHABARNI, HASAN, KHOMAINI, ENUS, SUTARYA, SUBROTO, TOTO, SOEMITRO, SOETIJOSO

Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

Prethrombin-2 (PT2) is a thrombin precursor, which plays a role in the conversion of fibrinogen into fibrin during blood clotting process. Previous study reported that the expression of human prothrombin-2 (rhPT2) in Escherichia coli formed inclusion bodies. The aim of this study was to establish a strategy to express a soluble rhPT2 in E. coli. This study was animed to design and codon optimize human prethrombin-2 gene as well as to optimize the expression condition using four strains of E. coli. The codon adaptation index (CAI) of the unoptimized hpt2 gene was 0.336, with 56.8% GC content. After optimization, the CAI of optimized hpt2 became 1.000 with 53.1% GC content. The optimized gene was successfully cloned into pTWIN1 expression vector. Expression analysis indicated that only E. coli ArcticExpress strain could successfully express a soluble recombinant rhPT2 protein, with only part of rhPT2 being expressed in insoluble form. However, the rest of the E. coli strains used in the experiments failed to express the rhPT2 in soluble form. We are deducing that the success in achieving soluble expression was not only due to the availability of chaperonins Cpn60/Cpn10, which played a crucial role in the protein folding in E. coli ArcticExpress strain, but also due to the codon optimization of hpt2 gene.

Cloning and Expression of Endoglucanase Gene from Thermophilic Bacteria Bacillus sp. RP1

MOEIS, MAELITA RAMDANI, NATALIA, DESSY, NINGRUM, RAHMA WIDYA, DWIJAYANTI, ARI

Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

An endoglucanase gene from glycoside hydrolase family 5, had been isolated from Bacillus sp. RP1 and cloned into Escherichia coli. The cloned gene comprised the promoter, coding sequence and terminator of the gene.  This gene encoded a protein with 499 amino acid residues (Mr=55.2 kDa) with a typical Bacillus signal peptide. The recombinant endoglucanase (EG) had optimum activity at pH 5.0 and 50 °C. The recombinant EG was expressed in the extracellular, intracellular, and periplasmic fractions with the highest total activity (60.15%) in the intracellular fraction, measured at three hours after isopropyl-β-Dthiogalactopyranoside (IPTG) induction. Three hours after the addition of 1% carboxymethyl cellulose (CMC), there was a two-fold increase in intracellular EG specific activity compared to the uninduced cells. Three hours after the addition of 1 mM IPTG, 1% glucose, 1% galactose or 1% cellobiose the intracellular EG specific activity decreased compared to the uninduced cells.

Cloning, Sequencing, and Expression of the Gene Encoding a Family 9 Cellulase from Bacillus licheniformis F11 in Escherichia coli and Bacillus megaterium, and Characterization of the Recombinant Enzymes

HELIANTI, IS, ULFAH, MARIA, NURHAYATI, NIKNIK, MULYAWATI, LLINA

Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

 A gene encoding cellulase belonging to the glycosyl hydrolase family 9 along with its native promoter was isolated from Bacillus licheniformis F11, cloned in Escherichia coli DH5 α and subcloned by transconjugation to Bacillus megaterium MS941. Functionality of the encoded protein was proven both in heterologous hosts, E. coli and B. megaterium. In the latter, the gene product was found in the extracellular fraction expressing a high specific activity; whereas in E. coli the protein was not secreted into the medium, and rather, showed a lower specific activity. The optimum temperature of the recombinant enzyme expressed in the hosts range from 65-75 ºC; whereas the optimum pH is 6. The recombinant enzyme was stable between 50-60 ºC and in a broad pH range (pH 5 - 9). Addition of Ca2+ and Fe3+ enhanced the enzyme activity, whereas EDTA and Cu2+  had the opposite effect. Lichenin, rather than carboxyl methyl cellulose, is the preferred substrate.

Effect of Lactic Acid Filtrate and Bacteriocins of Lactobacillus acidophillus on Phagocytosis Activity of Macrophages Cell againts Enteropathogen Escherichia coli (EPEC)

HERAWATI, IIS, HILMI, DIKI, FAUZIAH, PRIMA NANDA

Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

Immunity development known as one of effective ways in avoiding infection. Antibacterial agent product isolated from Lactobacillus acidophillus has been  reported can activate T lymphocyte as part of adaptive immunity. This experimental study aimed at investigation of lactic acid and bacteriocins filtrate from L. acidophillus in modulating phagocytosis activity of human macrophages infected by enteropathogenic Escherichia coli (EPEC). Each of human macrophages culture was supplemented with lactic acid and bacteriocins filtrate at concentration of 3.125, 6.25, and 12.5 µg mL-1 as well as control without filtrate addition and incubated for 24 h. Macrophages culture was then infected with EPEC for 30 minutes and was microscopically observed after being stained by Giemsa. Percentage of phagocytosis activity was gained from active macrophages in 100 observed cells. Macrophages cultures supplemented with bacteriocins filtrate showed augmented phagocytosis activity while cultures supplemented with lactic acid filtrate showed decreased phagocytosis activity. ANOVA analysis showed significant difference in phagocytosis activity of macrophage cultures supplemented with lactic acid (p=0,038) and bacteriocins (p=0,016 and 0,023). Tukey HSD analysis for phagocytosis activity of macrophage cultures supplemented by bacteriocins, each group of treatment showed significant difference againts control. In conclusion, lactic acid from  L.acidophillus has no effect in modulation of macrophages phagocytosis activity while bacteriocins can improve phagocytic activity. Bacteriocins from L. acidophillus then can be suggested to have a role as immunomodulator.