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All Journal HAYATI Journal of Biosciences Reaktor International Journal of Renewable Energy Development JURNAL KIMIA SAINS DAN APLIKASI Microbiology Indonesia Jurnal Matematika & Sains Journal of Engineering and Technological Sciences Indonesian Journal of Biotechnology EduChemia: Jurnal Kimia dan Pendidikan INDONESIAN JOURNAL OF PHARMACY Indonesian Journal of Chemistry Majalah Ilmiah Biologi BIOSFERA: A Scientific Journal Indonesian Journal of Biotechnology and Biodiversity International Journal of Renewable Energy Development

Enny Ratnaningsih

Biochemistry Research Division, Faculty Of Mathematics And Natural Sciences, Institut Teknologi Bandung

Author-ID : 259973

Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Control & Systems Engineering Earth & Planetary Sciences Education Electrical & Electronics Engineering Energy Engineering Environmental Science Immunology & microbiology Materials Science & Nanotechnology Mathematics Medicine & Pharmacology Other

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Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1 Enny Ratnaningsih; Idris Idris
Indonesian Journal of Biotechnology Vol 22, No 2 (2017)
Publisher : Universitas Gadjah Mada

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.

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1 Enny Ratnaningsih; Sulistiya Nirta Sunaryo; Idris Idris; Rindia Maharani Putri
Reaktor Volume 21 No. 2 June 2021
Publisher : Dept. of Chemical Engineering, Diponegoro University

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

PRODUKSI ANTIBIOTIKA OLEH Bacillus subtilis M10 DALAM MEDIA UREA-SORBITOL Supartono Supartono; Nanik Wijayati; Lina Herlina; Enny Ratnaningsih
Reaktor Volume 13, Nomor 3, Juni 2011
Publisher : Dept. of Chemical Engineering, Diponegoro University

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.

Isolasi Karakterisasi Bakteri Pendegradasi Asam Monokloroasetat dari Tanah Vina Juliana Anggraeni; Enny Ratnaningsih; Zeily Nurachman
EduChemia (Jurnal Kimia dan Pendidikan) Vol 2, No 2 (2017): Available Online in July 2017
Publisher : Department of Chemical Education Faculty of Teacher Training and Education Universitas Su

Senyawa organohalogen merupakan salah satu polutan terbesar di lingkungan. Bioremediasi merupakan salah langkah yang dapat dilakukanuntuk mengurangi polusi organohalogen. Beberapa bakteri tanah diketahui memiliki enzim dehalogenase dan berpotensi digunakan sebagai bioremediator senyawa organohalogen. Pada penelitian ini, dilakukan isolasi terhadap bakteri tanah yang mampu mendegradasi Asam Monokloroasetat (MCA) dan mengkarakaterisasi kemampuan tumbuh bakteri-bakteri tersebut pada berbagai konsentrasi MCA. Hasil isolasi didapatkan5 koloni bakteri yang mampu tumbuh pada medium dengankonsentrasi MCA yang tinggi yaitu 10mM. BakteriPG3, TJ4, PW2,CW1, dan PG2 masing-masing mampu melepaskanion klorida sebesar 95,14%; 91,89%; 89,46%; 89,46; 88,81% pada medium yang mengandung1mM MCA 29,24%; 28,17%; 28,10%; 24,31%; 26,16% pada 5mM MCA, dan 13,03%; 12,09%; 9.95%; 8,35%; 8,72% pada 10mM MCA. Terlihat bahwa degradasi organohalogen terjadi lebih efektif pada medium dengan konsentrasi MCA rendah, dan bakteri PG3 mempunyai kemampuan yang tertinggi.Pertumbuhan kelima bakteri mencapai fasa stationer pada18-24 jam denganOD600 sebesar 0.3-0.4. Senyawa organohalogen merupakan salah satu polutan terbesar di lingkungan. Bioremediasi merupakan salah langkah yang dapat dilakukanuntuk mengurangi polusi organohalogen. Beberapa bakteri tanah diketahui memiliki enzim dehalogenase dan berpotensi digunakan sebagai bioremediator senyawa organohalogen. Pada penelitian ini, dilakukan isolasi terhadap bakteri tanah yang mampu mendegradasi Asam Monokloroasetat (MCA) dan mengkarakaterisasi kemampuan tumbuh bakteri-bakteri tersebut pada berbagai konsentrasi MCA. Hasil isolasi didapatkan5 koloni bakteri yang mampu tumbuh pada medium dengankonsentrasi MCA yang tinggi yaitu 10mM. BakteriPG3, TJ4, PW2,CW1, dan PG2 masing-masing mampu melepaskanion klorida sebesar 95,14%; 91,89%; 89,46%; 89,46; 88,81% pada medium yang mengandung1mM MCA 29,24%; 28,17%; 28,10%; 24,31%; 26,16% pada 5mM MCA, dan 13,03%; 12,09%; 9.95%; 8,35%; 8,72% pada 10mM MCA. Terlihat bahwa degradasi organohalogen terjadi lebih efektif pada medium dengan konsentrasi MCA rendah, dan bakteri PG3 mempunyai kemampuan yang tertinggi.Pertumbuhan kelima bakteri mencapai fasa stationer pada18-24 jam denganOD600 sebesar 0.3-0.4.

Fractionation and activity assay of the root extract of Piper sarmentosum Roxb. ex Hunter against Candida albicans Hartiwi Diastuti; Sadijah Achmad; Enny Ratnaningsih
Indonesian Journal of Pharmacy Vol 15 No 2, 2004
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

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.

Improving the Effectiveness of Crude-Oil Hydrocarbon Biodegradation Employing Azotobacter chroococcum as Co-Inoculant PUJAWATI SURYATMANA PARNADI; EDWAN KARDENA; ENNY RATNANINGSIH; . WISJNUPRAPTO
Microbiology Indonesia Vol. 1 No. 1 (2007): April 2007
Publisher : Indonesian Society for microbiology

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.

MUTATION ON Bacillus subtilis BAC4 USING ACRIDINE ORANGE AS AN EFFORT FOR INCREASING ANTIBIOTIC PRODUCTION Supartono Supartono; Nanik Wijayati; Lina Herlina; Enny Ratnaningsih
Indonesian Journal of Chemistry Vol 8, No 2 (2008)
Publisher : Universitas Gadjah Mada

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.

Levan Produced by the Halophilic Bacterium Bacillus licheniformis BK1 as a Nanoparticle for Protein Immobilization Ira Oktavia; Aidah Nur Fithriah; Nur Umriani Permatasari; Enny Ratnaningsih; Rukman Hertadi
Indonesian Journal of Chemistry Vol 20, No 3 (2020)
Publisher : Universitas Gadjah Mada

This study examined the potential of levan from the halophilic bacterium Bacillus licheniformis BK1 as a nanoparticle system for protein immobilization. Levan produced by B. licheniformis BK1 was obtained by incubating the bacterium in the optimized Belghith medium, containing 15% (w/v) sucrose, 7.5% (w/v) NaCl and pH 8, in a rotary shaker at 150 rpm for 16 h, at 40 °C. The structure of the levan produced was verified by FTIR and NMR. It appeared that the levan had the same structure as that from Erwinia herbicola. The obtained levan was then used as a nanoparticle system to immobilize BSA and lysozyme proteins. The BSA-nanoparticle had a non-spherical shape with a surface charge of about -4.72 mV and a size distribution in the range of 83–298 nm. In contrast, the lysozyme-nanoparticle exhibited more spherical shapes with a surface charge of -2.57 mV and 206–285 nm size distribution. The efficiency of immobilization was about 74.26% and 81.77% for BSA and lysozyme, respectively. The study thus shows that levan produced by B. licheniformis BK1 can be used as a nanoparticle system for protein immobilization.

Identifikasi Senyawa Alkaloid dari Akar Piper sarmentosum Roxb. Ex Hunter dan Uji Aktivitasnya terhadap Jamur Candida albicans Hartiwi Diastuti; Sadiah Achmad; Enny Ratnaningsih
Majalah Ilmiah Biologi BIOSFERA: A Scientific Journal Vol 22, No 2 (2005)
Publisher : Fakultas Biologi | Universitas Jenderal Soedirman

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.20884/1.mib.2005.22.2.96

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.

Molecular Cloning and Expression of Haloacid Dehalogenase Gene from a Local Pseudomonas aeruginosa ITB1 Strain and Tertiary Structure Prediction of the Produced Enzyme Enny Ratnaningsih; Lousiana Dwinta Utami; Nurlaida Nurlaida; Rindia Maharani Putri
Jurnal Kimia Sains dan Aplikasi Vol 24, No 5 (2021): Volume 24 Issue 5 Year 2021
Publisher : Chemistry Department, Faculty of Sciences and Mathematics, Diponegoro University

Organohalogens are widely utilized as pesticides, herbicides, solvents, and for many other industrial purposes. However, the use of these compounds caused some negative impacts to the environment due to their toxicity and persistency. In the light of this, some microbes have been identified and employed to perform dehalogenation, converting halogenated organic compounds to non-toxic materials. In this research, we successfully cloned and sequenced the haloacid dehalogenase gene from a local Pseudomonas aeruginosa ITB1 strain, which is involved in the degradation of monochloroacetate. First, the haloacid dehalogenase gene was amplified by PCR using a pair of primers designed from the same gene sequences of other P. aeruginosa strains available in the GenBank. The cloned gene in pGEM-T in E. coli TOP10 was sequenced, analyzed, and then sub-cloned into pET-30a(+) for expression in E. coli BL21 (DE3). To facilitate direct sub-cloning, restriction sequences of EcoRI (G/AATTC) and HindIII (A/AGCTT) were added to the forward and reversed primers, respectively. The expressed protein in E. coli BL21 (DE3) appeared as a 26-kDa protein in SDS-PAGE analysis, which is in good agreement with the size predicted by ExPASy Protparam. We obtained that the best expression in LB liquid medium was achieved with 0.01 mM IPTG induction at 30°C incubation for 3 hours. We also found that the enzyme is more concentrated in the pellet cells as inclusion bodies. Furthermore, the in-silico analysis revealed that this enzyme consists of 233 amino acid residues. This enzyme’s predicted tertiary structure shows six β-sheets flanked by α-helixes and thus belongs to Group II haloacid dehalogenase. Based on the structural prediction, amino acid residues of Asp7, Ser121, and Asn122 are present in the active site and might play essential roles in catalysis. The presented study laid the foundation for recombinant haloacid dehalogenase production from P. aeruginosa local strains. It provided an insight into the utilization of recombinant local strains to remediate environmental problems caused by organohalogens.

Co-Authors . WISJNUPRAPTO Ade, Rachmad Aidah Nur Fithriah Anggraeni, Vina Juliana Anita K. Wardani DEBBIE SOEFIE RETNONINGRUM Debbie Sofie Retnoningrum DESSY NATALIA EDWAN KARDENA Endang Purwantini Hartiwi Diastuti Hartiwi Diastuti I Gede Wenten Idris Idris Idris Idris Idris Idris Idris Idris Ira Oktavia Kadja, Grandprix Thomryes Marth Khoiruddin Khoiruddin Lina Herlina Lina Herlina Lousiana Dwinta Utami Muhaimin Muhaimin Nanik Wijayati Ni Putu Yuliastri Nicholaus Prasetya Nora, Adri Nur Umriani Permatasari Nurlaida Nurlaida Nurul F. Himma Oei Ban Liang OEI BAN LIANG Pujawati Suryatmana Putri, Rindia Maharani Putu T.P. Aryanti Rachmad Ade Ridani Rino Anggoro Rindia Maharani Putri Rindia Maharani Putri Rindia Maharani Putri RUKMAN HERTADI Sadiah Achmad Sadijah Achmad Sadijah Achmad Sulistiya Nirta Sunaryo Supartono - ZEILY NURACHMAN Zuhdina Sabiqoh

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