Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil: Mathur Nadarajan Kathiravan , Geun Ho Gim , Jaewon Ryu , Pyung Il Kim , Chul Won Lee , Si Wouk Kim; J. Microbiol. 2015;53(11):767-775. Published online October 28, 2015; DOI: https://doi.org/10.1007/s12275-015-5454-0

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In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction
method
, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 μg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A260/A230 and A260/A280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP–purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

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Antifungal activity of violacein purified from a novel strain of Chromobacterium sp. NIIST (MTCC 5522): Anju Sasidharan , Nishanth Kumar Sasidharan , Dileepkumar Bhaskaran Nair Saraswathy Amma , Radhakrishnan Kokkuvayil Vasu , Anupama Vijaya Nataraja , Krishnakumar Bhaskaran; J. Microbiol. 2015;53(10):694-701. Published online October 2, 2015; DOI: https://doi.org/10.1007/s12275-015-5173-6

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Abstract

A novel strain of Chromobacterium sp. NIIST (MTCC 5522) producing high level of purple blue bioactive compound violacein was isolated from clay mine acidic sediment. During 24 h aerobic incubation in modified Luria Bertani medium, around 0.6 g crude violacein was produced per gram of dry weight biomass. An inexpensive method for preparing crystalline, pure violacein from crude pigment was developed (12.8 mg violacein/L) and the pure compound was characterized by different spectrometric methods. The violacein prepared was found effective against a number of plant and human pathogenic fungi and yeast species such as Cryptococcus gastricus, Trichophyton rubrum, Fusarium oxysporum, Rhizoctonia solani, Aspergillus flavus, Penicillium expansum, and Candida albicans. The best activity was recorded against Trichophyton rubrum (2 μg/ml), a human pathogen responsible for causing athlete’s foot infection. This is the first report of antifungal activity of purified violacein against pathogenic fungi and yeast.

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Identification and Characterization of an Anti-fungi Fusarium oxysporum f. sp. cucumerium Protease from the Bacillus subtilis Strain N7: Yi Luo , Lifei Sun , Zhen Zhu , Wei Ran , Qirong Shen; J. Microbiol. 2013;51(3):359-366. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2627-6

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Abstract: A newly discovered alkaline antifungal protease named P6 from Bacillus subtilis N7 was purified and partially characterized. B. subtilis N7 culture filtrates were purified by 30–60% (NH4)2SO4 precipitation, anion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of 41.38 kDa. Peptide sequence of protease P6 was determined using a 4800 Plus MALDI TOF/TOFTM Analyzer System. Self-Formed Adaptor PCR (SEFA-PCR) was used to amplify the 1,149 bp open read frame of P6. Dimensional structure prediction using Automatic Modeling Mode software showed that the protease P6 consisted of two β-barrel domains. Purified P6 strongly inhibited spore and mycelium growth of Fusarium oxysporum f. sp. cucumerium (FOC) by causing hypha lysis when the concentration was 25 μg/ml. Characterization of the purified protease indicated that it had substrate specificity for gelatin and was highly active at pH 8.0–10.6 and 70°C. The P6 protease was inhibited by EDTA (2 mmol/L), phenyl methyl sulfonyl fluoride (PMSF, 1 mmol/L), Na+, Fe3+, Cu2+, Mg2+ (5 mmol/L each) and H2O2 (2%, v/v). However, protease activity was activated by Ca2+, K+, Mn2+ (5 mmol/L each), mercaptoethanol (2%, v/v) and Tween 80 (1%, v/v). In additon, activity was also affected by organic solvents such as acetone, normal butanol and ethanol, but not hexane (25%, v/v each).

Expression, Purification, and Biochemical Properties of Arginase from Bacillus subtilis 168: Jin-Ju Yu , Ki-Bum Park , Su-Gon Kim , Suk-Heung Oh; J. Microbiol. 2013;51(2):222-228. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2669-9

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Abstract: The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The Km and Vmax values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.

Screening, Purification, and Characterization of an Extracellular Prolyl Oligopeptidase from Coprinopsis clastophylla: Jen-Tao Chen , Mei-Li Chao , Chiou-Yen Wen , Wen-Shen Chu; J. Microbiol. 2012;50(4):652-659. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2099-0

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Abstract: Culture filtrates of 22 mushrooms were screened for extracellular prolyl oligopeptidase activity. Four strains with relatively high enzyme activity were all from inky cap mushrooms. The production of Coprinopsis clastophylla prolyl oligopeptidase was associated with the growth of the fungus and the enzyme was not released by cell lysis. The enzyme was purified 285-fold to a specific activity of 52.05 U/mg. It was purified to a single band on a native polyacrylamide gel. However, the enzyme separated into three bands on a sodium dodecyl sulfate-polyacrylamide gel with mobility corresponding to molecular weights of approximately 84, 60, and 26 kDa. The results of tandem mass spectrometric analysis revealed that the 60 kDa protein was likely a degradation product of the 84 kDa protein. The isoelectric point of the purified enzyme was 5.2. The purified enzyme had an optimal pH and temperature of 8.0 and 37°C, respectively. Diisopropyl fluorophosphate (DFP), p-chloromercuribenzoaic acid (PCMB), Hg2+, and Cu2+ strongly inhibited C. clastophylla prolyl oligopeptidase. This enzyme is a serine peptidase and one or more cysteine residues of the enzyme are close to the active site.

Journal Articles

Purification and Characterization of a Novel Laccase from the Edible Mushroom Hericium coralloides: Ya-Jie Zou , He-Xiang Wang , Tzi-Bun Ng , Chen-Yang Huang , Jin-Xia Zhang; J. Microbiol. 2012;50(1):72-78. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1372-6

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Abstract: A novel laccase from the edible mushroom Hericium coralloides was purified by ion exchange chromatography on diethylaminoethyl (DEAE) cellulose, carboxymethyl (CM) cellulose, and Q-Sepharose columns followed by fast protein liquid chromatography gel filtration on a Superdex 75 column. Analysis by gel filtration and SDS-PAGE indicated that the protein is a monomer in solution with a molecular mass of 65 kDa. Its N-terminal amino acid sequence was AVGDDTPQLY, which exhibits partial sequence homology to previously isolated laccases. Optimum activity was observed at pH 2.2 and at 40°C. The enzyme showed activity toward a variety of substrates, the most sensitive of which was 2,2􍿁-azinobis [3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS). The degradation activity toward substrates was ABTS > N,N-dimethyl-1,4-phenylenediamine > catechol > 2-methylcatechol > pyrogallol. The laccase did not exert any antiproliferative activity against Hep G2 or MCF 7 tumor cell lines at a concentration of 60 μM, unlike some previously reported mushroom proteins, but showed significant activity toward human immunodeficiency virus-1 (HIV-1) reverse transcriptase with an IC50 of 0.06 μM.

A Novel Ribonuclease with Potent HIV-1 Reverse Transcriptase Inhibitory Activity from Cultured Mushroom Schizophyllum commune: Yong-Chang Zhao , Guo-Qing Zhang , Tzi-Bun Ng , He-Xiang Wang; J. Microbiol. 2011;49(5):803-808. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1098-x

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Abstract: A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 μ/mg), the second highest activity toward poly (C) (244.7 μ/mg), less activity toward poly (A) (167.4 μ/mg), and much weaker activity toward poly (G) (114.5 μ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC50 of 65 μM. No effect on [3H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 μM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

Research Support, Non-U.S. Gov'ts

Purification and Partial Characterization of a Detergent and Oxidizing Agent Stable Alkaline Protease from a Newly Isolated Bacillus subtilis VSG-4 of Tropical Soil: Sib Sankar Giri , V. Sukumaran , Shib Sankar Sen , M. Oviya , B. Nazeema Banu , Prasant Kumar Jena; J. Microbiol. 2011;49(3):455-461. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0427-4

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Abstract: An extracellular detergent tolerant protease producing strain VSG-4 was isolated from tropical soil sample and identified as Bacillus subtilis based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The VSG-4 protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and sephadex G-200 gel permeation chromatography with a 17.4 purification fold. The purified enzyme was active and stable over a broad range of pH (8.0-11.0, optimum at 9.0) and temperature (40°C to 60°C, optimum at 50°C). The thermostability of the enzyme was significantly increased by the addition CaCl2. This enzyme was strongly inhibited by PMSF and DFP, suggesting that it belongs to the serine protease superfamily. The purified VSG-4 alkaline protease showed remarkable stability in anionic (5 mM SDS) and ionic (1% Trion X-100 and 1% Tween 80) detergents. It retained 97±2% and 83.6±1.1% of its initial activity after 1 h preincubation in the presence of 1% H2O2 and 1% sodium perborate, respectively. Furthermore, the purified enzyme showed excellent stability and compatibility with some commercial laundry detergents besides its stain removal capacity. Considering these promising properties, VSG-4 protease may find tremendous application in laundry detergent formulations.

Expression, Purification, and Characterization of Recombinant Fibulin-5 in a Prokaryote Expression System: Myoung Seok Jeong , Chang Soo Kang , Yeon Soo Han , In Seok Bang; J. Microbiol. 2010;48(5):695-700. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0320-6

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Abstract

Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N2+-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.

Citations

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Purification and Characterization of the α-Glucosidase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI 756: Ana Flávia Azevedo Carvalho , Maurício Boscolo , Roberto da Silva , Henrique Ferreira , Eleni Gomes; J. Microbiol. 2010;48(4):452-459. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-9319-2

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Abstract: Αn α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0-9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na+, Ba2+, Co2+, Ni2+, Mg2+, Mn2+, Al3+, Zn2+, Ca2+ this enzyme maintained 90-105% of its maximum activity and was inhibited by Cr3+, Ag+, and Hg2+. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The Km measured for the α-glucosidase was 0.07 μM, with a Vmax of 318.0 μmol/min/mg.

Nitroreductase II Involved in 2,4,6-Trinitrotoluene Degradation: Purification and Characterization from Klebsiella sp. C1: Jung-Hye Shin , Hong-Gyu Song; J. Microbiol. 2009;47(5):536-541. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-008-0171-6

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Abstract: Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. C1 have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygen- insensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreductase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-aminodinitrotoluene).

Development of a Simple Cell Lysis Method for Recombinant DNA Using Bacteriophage Lambda Lysis Genes: Boyun Jang , Yuna Jung , Dongbin Lim; J. Microbiol. 2007;45(6):593-596.; DOI: https://doi.org/2602 [pii]

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Abstract: In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

Purification and Characterization of an Intracellular NADH: Quinone Reductase from Trametes versicolor: Sang-Soo Lee , Dong-Soo Moon , Hyoung T. Choi , Hong-Gyu Song; J. Microbiol. 2007;45(4):333-338.; DOI: https://doi.org/2564 [pii]

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Abstract: Intracellular NADH:quinone reductase involved in degradation of aromatic compounds including lignin was purified and characterized from white rot fungus Trametes versicolor. The activity of quinone reductase was maximal after 3 days of incubation in fungal culture, and the enzyme was purified to homogeneity using ion-exchange, hydrophobic interaction, and gel filtration chromatographies. The purified enzyme has a molecular mass of 41 kDa as determined by SDS-PAGE, and exhibits a broad temperature optimum between 20-40°C, with a pH optimum of 6.0. The enzyme preferred FAD as a cofactor and NADH rather than NADPH as an electron donor. Among quinone compounds tested as substrate, menadione showed the highest enzyme activity followed by 1,4-benzoquinone. The enzyme activity was inhibited by CuSO4, HgCl2, MgSO4, MnSO4, AgNO3, dicumarol, KCN, NaN3, and EDTA. Its Km and Vmax with NADH as an electron donor were 23 μM and 101 mM/mg per min, respectively, and showed a high substrate affinity. Purified quinone reductase could reduce 1,4-benzoquinone to hydroquinone, and induction of this enzyme was higher by 1,4-benzoquinone than those of other quinone compounds.

Journal Article

Meroparamycin Production by Newly Isolated Streptomyces sp. Strain MAR01: Taxonomy, Fermentation, Purification and Structural Elucidation: Moustafa Y. El-Naggar , Samy A. El-Assar , Sahar M. Abdul-Gawad; J. Microbiol. 2006;44(4):432-438.; DOI: https://doi.org/2409 [pii]

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Abstract: Twelve actinomycete strains were isolated from Egyptian soil. The isolated actinomycete strains were then screened with regard to their potential to generate antibiotics. The most potent of the producer strains was selected and identified. The cultural and physiological characteristics of the strain identified the strain as a member of the genus Streptomyces. The nucleotide sequence of the 16S rRNA gene (1.5 kb) of the most potent strain evidenced a 99% similarity with Streptomyces spp. and S. aureofaciens 16S rRNA genes, and the isolated strain was ultimately identified as Streptomyces sp. MAR01. The extraction of the fermentation broth of this strain resulted in the isolation of one major compound, which was active in vitro against gram-positive, gram-negative representatives and Candida albicans. The chemical structure of this bioactive compound was elucidated based on the spectroscopic data obtained from the application of MS, IR, UV, 1H NMR, 13C NMR, and elemental analysis techniques. Via comparison to the reference data in the relevant literature and in the database search, this antibiotic, which had a molecular formula of C19H29NO2 and a molecular weight of 303.44, was determined to differ from those produced by this genus as well as the available known antibiotics. Therefore, this antibiotic was designated Meroparamycin.

Research Support, Non-U.S. Gov't

Optimization and High-level Expression of a Functional GST-tagged rHLT-B in Escherichia coli and GM1 Binding Ability of Purified rHLT-B: Xingyuan Ma , Wenyun Zheng , Tianwen Wang , Dongzhi Wei; J. Microbiol. 2006;44(3):293-300.; DOI: https://doi.org/2383 [pii]

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Abstract: The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathion S-transferase) in E. coli BL21 (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GM1-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92% purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.

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