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Characterization of Extracellular Penicilin G Acylase Produced by A New Local Strain of Bacillus subtilis BAC4

HAYATI Journal of Biosciences Vol 15, No 2 (2008): June 2008
Publisher : Bogor Agricultural University, Indonesia

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Abstract

Penicillin G acylase (PGA) which catalyses penicillin G hydrolysis reaction is a key enzyme for the industrial production of penicilin G derivatives used in therapeutics. A new local strain of Bacillus subtilis BAC4 was found capable of producing extracellular PGA. However, characteristics of this extracellular PGA are not known. The goal of this research was to characterize the extracellular PGA produced by B. subtilis BAC4. Enzyme production was carried out by batch fermentation, followed by enzyme purification and characterization of the PGA. The PGA activity was determined by the Kornfeld method, with optimal activity for hydrolysing penicillin G observed at 43 oC and pH 8.5. The activation energy of penicillin G hydrolysis by the PGA of B. subtilis BAC4 was determined as 4.9 kcal.mol-1 and Vmax and Km values were found to be 0.7 µmole.min-1.mg-1 and 3.5 mM respectively. PGA catalytic activity was competitively inhibited by phenylacetic acid with an inhibition constant, Ki(PAA) , of 347.2 mM. It was concluded that the extracellular PGA of B. subtilis BAC4 can hydrolyse penicillin G efficiently. Key words: PGA, extracellular, Bacillus, local

PRODUKSI ANTIBIOTIKA OLEH Bacillus subtilis M10 DALAM MEDIA UREA-SORBITOL

REAKTOR Volume 13, Nomor 3, Juni 2011
Publisher : Dept. of Chemical Engineering, Diponegoro University

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Abstract

PRODUCTION OF ANTIBIOTICS BY Bacillus subtilis M10 IN UREA-SORBITOL MEDIUM. Infection diseases still become the main health problems that suffered by people in Indonesia. Besides, there were many pathogen bacteria found to be resistant to the some antibiotics. Therefore, the efforts to get a new antibiotic require to be done continuously. A new local strain of Bacillus subtilis BAC4 has been known producing an antibiotic that inhibit Serratia marcescens ATCC 27117 growth. To make efficient the local strain, mutation on Bacillus subtilis BAC4 was done by using acridine orange and a mutant cell of Bacillus subtilis M10 that overproduction for producing antibiotic was obtained. Nevertheless, the production kinetics of antibiotic by this mutant has not been reported. The objective of this research was to study the production kinetics of antibiotic by Bacillus subtilis M10 mutant. The production of antibiotic was conducted using batch fermentation and antibiotic assay was performed with agar absorption method using Serratia marcescens ATCC 27117 as bacteria assay. Research result provided that Bacillus subtilis M10 mutant with overproduction of antibiotic produced an antibiotic since 8th hour’s fermentation and optimum of it production was at 14th hours after inoculation.  Penyakit infeksi masih menjadi masalah yang utama diderita oleh masyarakat Indonesia. Di samping itu, banyak bakteri patogen yang ditemukan resisten terhadap beberapa antibiotika. Oleh karena itu, upaya-upaya untuk mendapatkan antibiotika baru perlu dilakukan secara terus-menerus. Suatu galur lokal baru Bacillus subtilis BAC4 teridentifikasi memproduksi senyawa antibiotika yang menghambat pertumbuhan Serratia marcescens ATCC27117. Untuk memberdayakan galur tersebut, terhadap Bacillus subtilis BAC4 dilakukan mutasi dengan larutan akridin oranye dan diperoleh mutan Bacillus subtilis M10 yang memproduksi antibiotika berlebihan. Namun, kinetika produksi antibiotika oleh Bacillus subtilis M10 belum pernah dilaporkan. Penelitian ini bertujuan untuk mempelajari kinetika produksi antibiotika oleh mutan Bacillus subtilis M10. Bacillus subtilis M10 difermentasikan ke dalam media urea-sorbitol dan diamati kemampuan produksi antibiotikanya menggunakan Serratia marcescens ATCC 2711 sebagai bakteri uji. Hasil penelitian menunjukkan bahwa mutan Bacillus subtilis M10 memproduksi antibiotika sejak jam ke 8, dan produksi optimumnya terjadi pada jam ke 14 setelah inokulasi.

Improving the Effectiveness of Crude-Oil Hydrocarbon Biodegradation Employing Azotobacter chroococcum as Co-Inoculant

Microbiology Indonesia Vol 1, No 1 (2007): April 2007
Publisher : Indonesian Society for microbiology

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Abstract

Azotobacter chroococcum has a great potential as biosurfactant producing bacteria and was used as co-inoculant to promote the rate of hydrocarbon biodegradation. The rate of hydrocarbon biodegradation were 0.01212, 0.01582, and 0.01766 per day for Acinetobacter sp., Bacillus cereus and the consorsium culture respectively. On the other hand, the rates of biodegradation using Azotobacter as co-inoculant were 0.1472, 0.01612, and 0.02709 g per day. Azotobacter chroococcum co-inoculant has the capability of increasing biodegradation efficiency of crude oilhydrocarbon. The biodegradation efficiency of petroleum hidrocarbon was increated by 13.4, 14.6, and 14.4% within the Petrobacter cultures.

Analisis DNA Bacillus sp. BAC4 Hasil Polymerase Chain Reaction (PCR) dengan Primer-Primer yang Dirancang dari Gen pga

Jurnal Kedokteran Maranatha Vol 8, No 1 (2008)
Publisher : Universitas Kristen Maranatha

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Abstract

Bacillus sp. BAC4 DNA was amplified using PCR method employing primers designed from pga genes of 5 bacteria (Arthrobacter viscosus, Bacillus megaterium, Escherichia coli, Kluyvera citrophila, and Providentia rettgeri). Analysis  of DNA base direct sequencing of PCR products showed the highest homology of Bacillus sp. BAC4 DNA with Bacillus subtilis DNA at the region from citG to yirG.  This fact states that primers designed from pga genes of 5 bacteria has not been successful in amplifying Bacillus sp. BAC4 pga gene, and that Bacillus sp.  BAC4 pga gene is predicted to be quite different from the existing data of pga genes available at Genbank.  Analysis of DNA base direct sequencing amplified using the pga primers shows the highest homology to the DNA of Bacillus subtilis.

Fractionation and activity assay of the root extract of Piper sarmentosum Roxb. ex Hunter against Candida albicans

INDONESIAN JOURNAL OF PHARMACY Vol 15 No 2, 2004
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

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Abstract

Piper sarmentosum Roxb. ex Hunter (“sirih duduk”) has long been used as a traditional medicine against fungoid diseases, especially at vagina and foot-sole. The investigation of the bioactive compounds of P. sarmentosum roots has not infections been carried yet. This research was then aimed to fractinate and to examine the antifungus activity of the extract of P. sarmentosum roots.The extraction was performed by using organic solvents such as methanol, ethyl acetate, chloroform, benzene, and n-hexane. The bioactivity tests were performed against C. albicans. The active extracts were then fractionated by coloumn chromatography.The result showed that the ethyl acetate fraction of methanol extract of P. sarmentosum roots have an antifungus activity against C. albicans. Phytochemical study showed that the active fractions of the methanol extracts of P. sarmentosum roots contained alkaloid.Key words : Piper sarmentosum, activity test, Candida albicans.

Cloning of acetyl-CoA acetyltransferase gene from Halomonas elongata BK-AG18 and in silico analysis of its gene product

Indonesian Journal of Biotechnology Vol 22, No 1 (2017)
Publisher : Universitas Gadjah Mada

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Abstract

Polyhydroxybutyrate (PHB) is a biodegradable polymer that can be used as a substitute for petrochemical plastics. Bacteria accumulate PHB in their cells as carbon and energy reserves because of unbalanced growth conditions.  This study aimed to amplify phbA from the chromosomal DNA of Halomonas elongata BK-AG18, a PHB-producing bacterium that was previously isolated from the Bledug Kuwu mud crater of Central Java, Indonesia. The obtained phbA amplicon was 1176 bp. This fragment was cloned into a pGEM-T Easy cloning vector and used to transform Eschericia coli TOP10. The recombinant colonies were selected using blue-white screening, confirmed by size screening, reconfirmed by re-PCR, and sequenced. When putative phbA sequences were aligned with H. elongata DSM2581 chromosome using BLASTN, this sequence showed 99% identity. The deduced amino acid sequences of this clone showed 100% identity to PhbA of  H. elongata DSM2581, suggesting that the obtained cloned fragment is a  phbA  gene. The 3D structure predicted by I-TASSER showed that PhbA of H. elongata  BK-AG18 had a high similarity to the acetyl CoA acetyltransferase structure of  Ralstonia eutropha H16. PhbA of H. elongata BK-AG18 possesses three catalytic residues, namely Cys88, His348, and Cys378.

Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Indonesian Journal of Biotechnology Vol 22, No 2 (2017)
Publisher : Universitas Gadjah Mada

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Abstract

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0  using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily.

Expression of haloacid dehalogenase gene and its molecular protein characterization from Klebsiella pneumoniae ITB1

Indonesian Journal of Biotechnology Vol 22, No 1 (2017)
Publisher : Universitas Gadjah Mada

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Abstract

Organohalogen compounds are widely used industrially and agriculturally, as well as in households as flame retardants and refrigerants. However, these compounds can become significant pollutants through their accidental or deliberate release into the environment in large quantities. Dehalogenase is an enzyme with the potential to be used in the removal of organohalogen contaminants. A previous study successfully subcloned a 690 bp of haloacid dehalogenase gene (hakp1) from Klebsiella pneumoniae ITB1 into a pET-30a(+) expression system to achieve high enzyme productivity. IPTG was used as an inducer to express a pET-hakp1 recombinant clone in Escherichia coli BL21 (DE3). The molecular mass of the haloacid dehalogenase Hakp1 protein was 30 kDa as determined by SDS-PAGE. Zymogram analysis showed that this recombinant protein has dehalogenase activity as shown by the formation of AgCl white precipitate. A quantitative assay of haloacid dehalogenase Hakp1 gave a specific activity of 84.29 U/mg with the optimum temperature of 40°C at pH 9. Predicted three-dimensional structure of Hakp1 showed α/β motif folding which comprised of cap and core domain. The predicted active sites of Hakp1 were Asp8, Glu10, Leu22, Phe23, Trp90, Ser125, Ser126, Lys159, and Asp184 with Asp8, Glu10, Ser126, and Lys159 act as binding residue. This recombinant haloacid dehalogenase clone provides an alternative agent for effective bioremediation of organohalogen pollutants.

MUTATION ON Bacillus subtilis BAC4 USING ACRIDINE ORANGE AS AN EFFORT FOR INCREASING ANTIBIOTIC PRODUCTION

Indonesian Journal of Chemistry Vol 8, No 2 (2008)
Publisher : Universitas Gadjah Mada

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Abstract

The efforts to get a new antibiotic require to be done continuously, because infection diseases still become the main health problems in Indonesia. A new local strain of Bacillus subtilis BAC4 has been known producing an antibiotic that inhibites Serratia marcescens ATCC 27117 growth. Nevertheless, the optimum conditions have not been studied seriously. The objective of this research was to conduct mutation on B. subtilis BAC4 in order to obtain a mutant cell that overproduct in producing antibiotic. The mutation process was performed by using acridine orange of 1 g.L-1 randomly at various volumes. The production of antibiotic was conducted using batch fermentation and antibiotic assay was performed with agar absorption method using S.  marcescens ATCC 27117 as bacteria assay. Research result provided a B. subtilis M10 mutant with overproduction of antibiotic. Characterization of B. subtilis M10 mutant showed that the mutant cell has size of (0.5-1.0) µm x (1.85-2.5) µm; spore has the form of ellipse with thick wavy wall, positive reaction for catalase, and forming acid from glucose and xylose.   Keywords: mutant, Bacillus, acridin, and antibiotics

Identifikasi Senyawa Alkaloid dari Akar Piper sarmentosum Roxb. Ex Hunter dan Uji Aktivitasnya terhadap Jamur Candida albicans

Majalah Ilmiah Biologi BIOSFERA: A Scientific Journal Vol 22, No 2 (2005)
Publisher : Fakultas Biologi | Universitas Jenderal Soedirman

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Abstract

Piper sarmentosum Roxb. Ex Hunter or “Sirih duduk” has long been used for traditional medicine to cure various diseases, such as fungus infections. The investigation of the bioactive compounds of P. sarmentosum roots has not been carried out. This research was aimed to isolate the bioactive compounds from P. sarmentosum roots. The results showed that methanol extracts of P. sarmentosum roots have an activity on Candida albicans. The separation a bioactive compounds from methanol extracts of P. sarmentosum roots was performed by column chromatography, thin layer chromatography and recrystalizations. The identifications of the bioactive compounds were carried out using ultra violet spectrometry, infrared spectrometry, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. The results indicated that from methanol extracts, an alkaloid compound of piperoylpyrol derivative was 5-hydroxy-5- (3,4-methylenedioxyphenyl)-2-pentenoyl pyrol, could be purely isolated. Examination of bioactivity at concentration 0.10-2.50 mg/ml showed that this compound had an activity on C. albicans.