Biochemical Properties of Mercuric Reductase from Local Isolate of Bacillus sp for Bioremediation Agent

Mercuric reductase is the important enzyme which catalyzes a reduction of a toxic Hg2+ to non-toxic Hg0. The enzyme which has been potentially used as mercury bioremediation agent is produced by mercury resistant bacteria. These research aims are to determinate the resistance level of a local Bacillus sp to HgCl2 in media, to determine the mercuric reductase activity from the bacteria, and to determine the biochemical properties of the mercuric reductase. The Bacillus sp was grown in the Nutrient Broth media with various of 0; 20; 40; 60; 120; and 160 µM HgCl2 to know the response of the bacteria against mercury, The cell growth of Bacillus sp was measured by optical density (OD) method of at λ 600 nm. The mercuric reductase activity was assayed in the solution of MRA (Mercury Reductase Assay), then the oxidized NADPH was observed by the spectrophotometry method at λ340 nm. The result showed that the Bacillus sp has been resistant to media containing mercury at 120 µM, but the microbial growth was decreased by 50% in media containing mercury 80 µM. The Bacillus sp could produce highly the mercuric reductase enzyme at 16 hours of growth time with enzyme activity as 0.574 Unit/µg. The mercuric reductase from the bacteria has an optimum activity at pH 6 and temperature 37 °C

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RESISTANCE LEVEL OF Pseudomonas stutzeri AGAINST MERCURY AND ITS ABILITY IN PRODUCTION OF MERCURY REDUCTASE ENZYME

Molekul ◽

10.20884/1.jm.2016.11.2.256 ◽

2016 ◽

Vol 11 (2) ◽

pp. 230

Author(s):

Purkan Purkan ◽

Safita Nurmalyya ◽

Sofijan Hadi

Keyword(s):

Reductase Activity ◽

Pseudomonas Stutzeri ◽

Mercury Ion ◽

Resistant Bacteria ◽

Resistance Level ◽

Fermentation Time ◽

Optimum Activity ◽

Mercury Reductase ◽

Mercury Resistant Bacteria

Mercury reductase is an enzyme that is able to reduce Hg2+ to Hg0 non toxic. This enzyme is usually produced by mercury resistant bacteria. The research wanted to determine the resistance of indigenous Pseudomonas stutzeri isolate toward mercury and to explore the mercury reductase activity which is produced by the bacteria. The results of resistance assay of the Pseudomonas stutzeri toward mercury ion showed that the isolate could survive in media containing HgCl2 up to a concentration of 80 µM. The bacteria could produce mercury reductase optimally at the 24th of fermentation time. The enzyme showed optimum activity at pH 7 and temperature of 45 oC

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MERCURY REDUCTASE ACTIVITY OF AN INDIGENOUS MERCURY RESISTANT BACTERIAL ISOLATE (Bacillus sp. S1) FROM KALIMAS-SURABAYA AS A POTENTIAL REDUCING AGENT FOR MERCURIAL ION (HG2+)

KnE Life Sciences ◽

10.18502/kls.v2i1.190 ◽

2015 ◽

Vol 2 (1) ◽

pp. 446 ◽

Cited By ~ 1

Author(s):

Enny Zulaika ◽

Langkah Sembiring

Keyword(s):

Room Temperature ◽

Bacterial Isolate ◽

Reductase Activity ◽

Elemental Mercury ◽

Total Cell ◽

Resistant Bacteria ◽

Bacillus Sp ◽

Reduction Efficiency ◽

Mercury Reductase ◽

Mercury Resistant Bacteria

Mercury reductase activity and Hg2+ lowering capacity of a Mercury Resistant Bacteria (MRB) (Bacillus sp. S1) which was isolated from Kalimas River of Surabaya, Indonesia were studied. The activity was determined by Mercury Reductase Assay System (MRAS) in a solution mixture contained 50 mM PBS (pH ± 7.0), 0.5 mM EDTA, 200 µM MgSO4, 0.1% (v/v) ß-merchaptoethanol, 200 µM NADH2 and 25 mg/L HgCl2 and one volume of crude extract incubated at room temperature for various interval period of time. Mercury reductase activity was measured spectrophotometrically at 340 nm. One unit of reductase activity was defined as one molar of oxidized NADH2 produced per total cell per minute. Results of study showed that, the isolate could resist the concentration of HgCl2 up to 11 mg/L. At 30 minute incubation period at room temperature, the highest mercury reductase activity and the Hg2+ lowering capacity was found to be 0.006 unit/109cell and 1.48 mg/L/109cell/minute, respectively with the reduction efficiency of Hg2+ to Hg0 of 0.18% per minute. Therefore, it can be concluded that the Bacillus sp. S1 isolate could be assumed to be exellent mercury bioremediation agent since it was found to be highly mercury resistant and very efficient to reduce cationic mercury (Hg2+) to elemental mercury (Hg0). Keywords: Kalimas-Surabaya, Bacillus sp. S1, Mercury resistant, Mercury reductase

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IDENTIFIKASI BAKTERI RESISTEN MERKURI PADA KARANG GIGI, URIN DAN FESES PADA INDIVIDU KELURAHAN PAKADOODAN KOTA BITUNG

Jurnal e-Biomedik ◽

10.35790/ebm.2.1.2014.4396 ◽

2014 ◽

Vol 2 (1) ◽

Author(s):

Maichel Yorgen

Keyword(s):

Research Result ◽

Clinical Manifestations ◽

Resistant Bacteria ◽

Bacillus Sp ◽

Resistance Level ◽

The Individual ◽

Mercury Detoxification ◽

Bacteria Resistance ◽

Mercury Resistant Bacteria ◽

Phenyl Mercury

Abstract: Mercury is one of kinds of hazardous metals which is highly found in nature and spread in rocklike, ore, soil, water, and air as the inorganic and organic compounds. The metal of mercury is dangerous to human’s life because it can cause the substantially clinical manifestations started from being poisoned, nerve palsy and even cause death. One of attempts to mercury detoxification can be done by using mercury-resistant microorganism such as mercury-resistant bacteria. That would happen if the mercury spread inside the human’s body with small value in a very chronic time can make the bacteria inside the human’s body to be able to adapt even able to reduce mercury so that it becomes harmless. The research aims to identify the kind of bacteria that resists to mercury, in this case, to the kind of mercury HgCl2 and Phenyl Mercury found on tartar, urine, and feses upon the individual in Pakadoodan Village of Bitung City by using the way to check the value of bacteria resistance to mercury, afterwards the Physiology test, Biochemical, and Morphology is done to identify the kind of that bacteria. From the research result done there are 4 genus of bacteria which was successfully identified, those are, Streptococcus sp, E.Coli, Bacillus sp, and Staphylococcus sp. By the classification HgCl2 is the bacteria with the genus of Bacillus sp which is found in urine, feses and tartar, and Staphylococcus sp which is found also in urine with the resistance level of 40 ppm each and phenyl mercury is the bacteria with the genus of Streptococcus sp, which is found in tartar and E.Coli which is found in feses and urine with the resistance level 20 ppm each. Keywords: mercury, bacteria, mercury-resistant bacteria Abstrak: Merkuri adalah salah satu jenis logam berbahaya yang banyak ditemukan di alam dan tersebar dalam batu - batuan, biji tambang, tanah, air dan udara sebagai senyawa anorganik dan organik. Logam merkuri sangat berbahaya terhadap kehidupan manusia karena dapat memberikan manifestasi klinik yang cukup bermakna mulai dari keracunan, kelumpuhan saraf bahkan dapat meneyebabkan kematian. Salah satu usaha untuk detoksifikasi merkuri dapat dilakukan dengan menggunakan mikroorganisme resisten merkuri seperti bakteri resisten merkuri. Hal itu dapat terjadi jika merkuri yang terpapar dalam tubuh manusia dengan kadar yang kecil dalam waktu yang sangat kronis dapat membuat bakteri dalam tubuh manusia tersebut dapat beradaptasi bahkan dapat mampu meruduksi merkuri tersebut sehingga menjadi tidak berbahaya. Penelitian ini bertujuan untuk mengidentifikasi jenis bakteri yang resisten terhadap merkuri dalam hal ini terhadap jenis merkuri HgCl2 dan fenil merkuri yang terdapat pada karang gigi, urin dan feses pada individu kelurahan Pakadoodan Kota Bitung dengan cara memeriksa kadar resisten bakteri tersebut terhadap merkuri, setelah itu dilakukan uji fisiologi, uji biokimia dan uji morfolgi untuk mengidentifikasi jenis bakteri tersebut. Dari hasil penelitian yang dilakukan terdapat 4 genus bakteri yang berhasil diidentifikasi yaitu Streptococcus sp, E.coli, Bacillus sp, dan Staphylococcus sp. Dengan klasifikasi HgCl2 adalah bakteri dengan genus Bacillus sp yang terdapat pada urin, feses dan karang gigi, dan Staphylococcus sp yang terdapat juga pada urin dengan tingkat resistensi masing-masing 40 ppm dan fenil merkuri adalah bakteri dengan genus Streptococcus sp, yang terdapat pada karang gigi dan E.coli yang terdapat pada feses dan urin dengan tingkat resistensi masing-masing 20 ppm. Kata kunci: merkuri, bakteri, bakteri resisten merkuri

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Protein Method for Investigating Mercuric Reductase Gene Expression in Aquatic Environments

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.695-702.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 695-702 ◽

Cited By ~ 18

Author(s):

O. A. Ogunseitan

Keyword(s):

Gene Expression ◽

Colorimetric Assay ◽

Reductase Activity ◽

Molecular Techniques ◽

Resistant Bacteria ◽

Specific Gene ◽

Oxidoreductase Activity ◽

Mercuric Reductase ◽

Mercuric Ion ◽

Reductase Gene

A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r 2 = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 102 CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 ± 2)% over the range of population densities investigated (102 to 108 CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.

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Indigeneous Streptomyces spp. isolated from Cyperus rotundus rhizosphere indicate high mercuric reductase activity as a pontential bioremediation agent

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220357 ◽

2021 ◽

Vol 22 (3) ◽

Author(s):

Hanum Mukti Rahayu ◽

Wahyu Aristyaning Putri ◽

Anis Uswatun Khasanah ◽

LANGKAH SEMBIRING ◽

Yekti Asih Purwestri

Keyword(s):

Specific Activity ◽

Reductase Activity ◽

Cyperus Rotundus ◽

Cell Free Extract ◽

Mercuric Reductase ◽

Streptomyces Spp ◽

Sds Page ◽

Optimum Ph ◽

Bioremediation Agent ◽

Optimum Ph And Temperature

Abstract. Rahayu HM, Putri WA, Khasanah AU, Sembiring L, Purwestri YA. 2021. Indigenous Streptomyces spp. isolated from Cyperus rotundus rhizosphere indicate high mercuric reductase activity as a potential bioremediation agent. Biodiversitas 22: 1519-1526. The purification and characterization of mercuric reductase of four indigenous Streptomyces spp. from Cyperus rotundus L. rhizosphere in mercury-contaminated area have been investigated. Cell-free extract was obtained by disrupting cells using sea sand at 4 °C followed by centrifugation. Mercuric reductase was purified by ammonium sulfate precipitation, dialysis, and chromatography column (DEAE Sepharose anion column chromatography). The determination of optimum pH and temperature of mercuric reductase activity was measured based on the number of NADPH2 oxidized to NADP per mg protein per minute using a spectrophotometer. The molecular weight of mercuric reductase was determined using SDS-PAGE. Result showed that the highest specific activity of mercuric reductase was recorded from Streptomyces spp. BR28. The optimum pH and temperature of cell-free extract enzyme mercuric reductase were 7.5 and 80 °C, respectively. The enzyme was purified to 431.87-fold with specific activity 21918.95 U/mg protein. SDS PAGE showed that the molecular weight of mercuric reductase in Streptomyces spp. BR 28 ranged from 50 kDa to 75 kDa. It can be concluded that Streptomyces isolates contain mercuric reductase and have potential as mercury bioremediation agent to overcome mercury contamination in the environment.

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Identification and Transferability of Tetracycline Resistance in Streptococcus thermophilus during Milk Fermentation, Storage, and Gastrointestinal Transit

Fermentation ◽

10.3390/fermentation7020065 ◽

2021 ◽

Vol 7 (2) ◽

pp. 65

Author(s):

Armin Tarrah ◽

Shadi Pakroo ◽

Viviana Corich ◽

Alessio Giacomini

Keyword(s):

Efflux Pump ◽

Acquired Resistance ◽

Gastrointestinal Transit ◽

Tetracycline Resistance ◽

Lactobacillus Delbrueckii ◽

Genomic Islands ◽

Resistant Bacteria ◽

Human Pathogens ◽

Resistance Level ◽

Milk Fermentation

The existence of antibiotic-resistant bacteria in food products, particularly those carrying acquired resistance genes, has increased concerns about the transmission of these genes from beneficial microbes to human pathogens. In this study, we evaluated the antibiotic resistance-susceptibility patterns of 16 antibiotics in eight S. thermophilus strains, whose genome sequence is available, using phenotypic and genomic approaches. The minimal inhibitory concentration values collected revealed intermediate resistance to aminoglycosides, whereas susceptibility was detected for different classes of β-lactams, quinolones, glycopeptide, macrolides, and sulfonamides in all strains. A high tetracycline resistance level has been detected in strain M17PTZA496, whose genome analysis indicated the presence of the tet(S) gene and the multidrug and toxic compound extrusion (MATE) family efflux pump. Moreover, an in-depth genomic analysis revealed genomic islands and an integrative and mobilizable element (IME) in the proximity of the gene tet(S). However, despite the presence of a prophage, genomic islands, and IME, no horizontal gene transfer was detected to Lactobacillus delbrueckii subsp. lactis DSM 20355 and Lactobacillusrhamnosus GG during 24 h of skim milk fermentation, 2 weeks of refrigerated storage, and 4 h of simulated gastrointestinal transit.

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Production and Properties of a Bacterial Thermostable Exo-inulinase

Zeitschrift für Naturforschung C ◽

10.1515/znc-2001-11-1220 ◽

2001 ◽

Vol 56 (11-12) ◽

pp. 1022-1028 ◽

Cited By ~ 7

Author(s):

Kristina Uzunova ◽

Anna Vassileva ◽

Margarita Kambourova ◽

Viara Ivanova ◽

Dimitrina Spasova ◽

...

Keyword(s):

Enzyme Production ◽

Enzyme Preparation ◽

Batch Cultivation ◽

Crude Enzyme ◽

Growth Time ◽

Bacillus Sp ◽

High Thermostability ◽

Crude Enzyme Preparation ◽

Ph Range

Abstract Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activi ties after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 °C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and inver tase activities were 92-98% after pretreatment at 65 °C for 60 min in the presence of substrate inulin.

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UJI RESISTENSI BAKTERI BACILLUS SP YANG DIISOLASI DARI PLAK GIGI TERHADAP MERKURI DAN ERITROMISIN

Jurnal e-Biomedik ◽

10.35790/ebm.3.1.2015.6610 ◽

2015 ◽

Vol 3 (1) ◽

Author(s):

Terence Kanzil ◽

Fatimawali . ◽

Aaltje Manampiring

Keyword(s):

Heavy Metal ◽

Toxic Effects ◽

Resistant Bacteria ◽

Bacillus Sp ◽

Gram Positive ◽

Toxic Heavy Metal ◽

Mer Operon ◽

Dental Fillings ◽

Gram Positive Bacteria ◽

Mercury Resistant Bacteria

Abstract: Mercury is a toxic heavy metal that is used for dental fillings in the form of amalgam. To reduce the toxic effects produced by mercury, mercury resistant bacteria can be used. Bacillus sp is a gram-positive bacteria that is resistant to mercury. Besides having the mer operon genes that can transform Hg2+ to Hg0 that is less toxic, Bacillus sp bacteria also produce esterase that cause these bacteria resistant to erythromycin antibiotic. Erythromycin is a macrolide class of antibiotic used for the treatment of diseases caused by Gram-positive bacteria, especially Staphylococcus and Diphtheroids. To determine the resistance of Bacillus sp bacteria against mercury and erythromycin antibiotic. This study used a descriptive exploratory method with samples of bacteria and mercury are already available in the Laboratory of Pharmaceutical Microbiology. Based on the research that has been conducted, showed that the Bacillus sp bacteria is resistant to mercury and erythromycin.Keywords: bacteria, bacillus sp, resistant, mercury, erythromycinAbstrak: Merkuri merupakan logam berat bersifat toksik yang digunakan untuk penambalan gigi dalam bentuk amalgam. Untuk mengurangi efek toksik yang dihasilkan oleh merkuri, dapat digunakan bakteri resisten merkuri. Bakteri Bacillus sp merupakan bakteri gram positif yang resisten terhadap merkuri. Selain memiliki gen mer operon yang dapat mengubah Hg2+ menjadi Hg0 yang kurang toksik, bakteri Bacillus sp juga membentuk esterase yang menyebabkan terjadinya resisten bakteri ini terhadap antibiotik eritromisin. Eritromisin adalah antibiotik golongan makrolid yang digunakan untuk pengobatan penyakit akibat bakteri Gram positif khususnya Staphylococcus dan Diphtheroids. Untuk mengetahui resistensi bakteri Bacillus sp terhadap merkuri dan antibiotik eritromisin. Penelitian ini menggunakan metode deskriptif eksploratif dengan sampel bakteri dan merkuri yang sudah tersedia di Laboratorium Mikrobiologi Farmasi. Berdasarkan hasil penelitian yang telah dilakukan, diperoleh bahwa bakteri Bacillus sp resisten terhadap merkuri dan eritromisin.Kata Kunci: bakteri, bacillus sp, resisten, merkuri, eritromisin

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Antibacterial Activity of Volatile Organic Compounds Produced by the Octocoral-Associated Bacteria Bacillus sp. BO53 and Pseudoalteromonas sp. GA327

Antibiotics ◽

10.3390/antibiotics9120923 ◽

2020 ◽

Vol 9 (12) ◽

pp. 923

Author(s):

Anette Garrido ◽

Librada A. Atencio ◽

Rita Bethancourt ◽

Ariadna Bethancourt ◽

Héctor Guzmán ◽

...

Keyword(s):

Antibacterial Activity ◽

Volatile Organic Compounds ◽

Organic Compounds ◽

Solid Phase ◽

Resistant Bacteria ◽

Bacillus Sp ◽

Antibiotic Resistant Bacteria ◽

Antibiotic Resistant ◽

Associated Bacteria ◽

Volatile Organic

The present research aimed to evaluate the antibacterial activity of volatile organic compounds (VOCs) produced by octocoral-associated bacteria Bacillus sp. BO53 and Pseudoalteromonas sp. GA327. The volatilome bioactivity of both bacteria species was evaluated against human pathogenic antibiotic-resistant bacteria, methicillin-resistant Staphylococcus aureus, Acinetobacter baumanni, and Pseudomonas aeruginosa. In this regard, the in vitro tests showed that Bacillus sp. BO53 VOCs inhibited the growth of P. aeruginosa and reduced the growth of S. aureus and A. baumanni. Furthermore, Pseudoalteromonas sp. GA327 strongly inhibited the growth of A. baumanni, and P. aeruginosa. VOCs were analyzed by headspace solid-phase microextraction (HS-SPME) joined to gas chromatography-mass spectrometry (GC-MS) methodology. Nineteen VOCs were identified, where 5-acetyl-2-methylpyridine, 2-butanone, and 2-nonanone were the major compounds identified on Bacillus sp. BO53 VOCs; while 1-pentanol, 2-butanone, and butyl formate were the primary volatile compounds detected in Pseudoalteromonas sp. GA327. We proposed that the observed bioactivity is mainly due to the efficient inhibitory biochemical mechanisms of alcohols and ketones upon antibiotic-resistant bacteria. This is the first report which describes the antibacterial activity of VOCs emitted by octocoral-associated bacteria.

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