Journal of Microbiology

Protocol

Protocol for efficient recovery of high-quality DNA from microbiome of marine invertebrates: Yeong-Jun Park, Jae Kyu Lim, Yeon-Ju Lee, Kae Kyoung Kwon; J. Microbiol. 2025;63(9):e2507003. Published online September 30, 2025; DOI: https://doi.org/10.71150/jm.2507003

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Abstract PDF: Marine organisms often form symbiotic relationships with various microorganisms to adapt and thrive in harsh environments. These symbiotic microbes contribute to host survival by providing nutrition, modulating the hosts’ immune system, and supporting overall physiological stability. Advances in high-throughput sequencing technologies have enabled a deeper understanding of the structure and function of symbiotic microbial communities, as well as host-microbe interactions. Notably, symbiotic bacteria associated with marine invertebrates such as corals and sponges are recognized as a potential source of useful bioactive compounds, including antibiotics and enzymes. However, obtaining high-quality microbial DNA from host tissues still remains a technical challenge due to the presence of unknown substances. This study focuses on optimizing sample preparation and DNA extraction procedures and additional purification to improve the recovery of microbial DNA while minimizing host DNA contamination. Comparison between several methods was conducted using sponge samples to evaluate DNA quality and microbial recovery. A sample designated as 2110BU-001 was collected from the east coast of the Republic of Korea and used for culture-independent microbial cell isolation. Total bacterial DNA was extracted by using a manual Phenol-Chloroform protocol and three commercial kits. DNA extracted using the standard manual method showed both the highest yield and the largest fragment size. However, PCR (Polymerase chain reaction) test showed that quality of manually extracted DNA was not enough for sequencing. Therefore, the quality of DNA was improved through additional purification steps. Briefly, host eukaryotic cells were removed by mechanical process and almost only bacterial DNA was successfully obtained by combination of manual extraction method and further purification processes. The established protocol was successfully introduced to extraction of metagenomic DNA from mussel and jellyfish microbiomes, indicating that it can be widely applied to various marine organisms.

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 PDF

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).

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Abstract PDF: 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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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.

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Characterization and crystal structure of prolyl endopeptidase from abalone (Haliotis discus hannai)
Wan-Yu Li, Yue Li, Yu-Lei Chen, Jian-Jian Hu, Hylemariam Mihiretie Mengist, Guang-Ming Liu, Tengchuan Jin, Min-Jie Cao
Food Chemistry.2020; 333: 127452. CrossRef
A prolyl endopeptidase from Flammulina velutipes for the possible degradation of celiac disease provoking toxic peptides in cereal proteins
Kathrin Schulz, Lucienne Giesler, Diana Linke, Ralf G. Berger
Process Biochemistry.2018; 73: 47. CrossRef
The MSDIN family in amanitin-producing mushrooms and evolution of the prolyl oligopeptidase genes
Hong Luo, Qing Cai, Yunjiao Lüli, Xuan Li, Rohita Sinha, Heather E. Hallen-Adams, Zhu L. Yang
IMA Fungus.2018; 9(2): 225. CrossRef
Prolyl-specific peptidases for applications in food protein hydrolysis
Nicole Mika, Holger Zorn, Martin Rühl
Applied Microbiology and Biotechnology.2015; 99(19): 7837. CrossRef

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 PDF: 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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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.

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Schizophyllum commune, an underrated edible and medicinal mushroom: farm to industry
Disha Dasgupta, Pranjali Basu, Anamika Paul, Krishnendu Acharya, Nilanjan Chakraborty
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Investigation of the inhibition of respiratory bacterial pathogens and HIV-1 enzymes by twenty-one South African mushroom species
Jenske Didloff, Gerhardt J. Boukes, Maryna van de Venter, Bennie Viljoen, Michael Lee, Candice Blom, Rebecca A. Dwyer, Sharlene Govender
South African Journal of Botany.2024; 166: 375. CrossRef
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Ana Sofia Sousa, Helena Araújo-Rodrigues, Manuela Estevez Pintado
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Evidence for the Presence of Hyphae and Fruiting Body Calcium Oxalate Crystallites in the Split Gill Medicinal Mushroom Schizophyllum commune (Agaricomycetes)
Xiangyue Xiao, Tianji Huang, Jingyi Zhang, Wei Liu, Hong Ji, Nemat O. Keyhani, Hui Chen, Weijie Wu, Chi Song
International Journal of Medicinal Mushrooms.2022; 24(10): 83. CrossRef
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Yu Zhang, Guoying Zhang, Jianya Ling
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Hitesh Chopra, Awdhesh Kumar Mishra, Atif Amin Baig, Tapan Kumar Mohanta, Yugal Kishore Mohanta, Kwang-Hyun Baek
Journal of Fungi.2021; 7(9): 728. CrossRef
Lectins purified from medicinal and edible mushrooms: Insights into their antiviral activity against pathogenic viruses
Yousra A. El-Maradny, Esmail M. El-Fakharany, Marwa M. Abu-Serie, Mona H. Hashish, Heba S. Selim
International Journal of Biological Macromolecules.2021; 179: 239. CrossRef
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Rong Zhou, Zhao Kun Liu, Ye Ni Zhang, Jack Ho Wong, Tzi Bun Ng, Fang Liu
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A novel ribonuclease with antiproliferative activity toward leukemia and lymphoma cells and HIV-1 reverse transcriptase inhibitory activity from the mushroom, Hohenbuehelia serotina
RUI ZHANG, LIYAN ZHAO, HEXIANG WANG, TZI BUN NG
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A Novel Ribonuclease with HIV-1 Reverse Transcriptase Inhibitory Activity from the Edible Mushroom Hygrophorus russula
Mengjuan Zhu, Lijing Xu, Xiao Chen, Zengqiang Ma, Hexiang Wang, T. B. Ng
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Purification and characterization of a novel acid phosphatase from the split gill mushroom Schizophyllum commune
Guo‐Qing Zhang, Qing‐Jun Chen, Jian Sun, He‐Xiang Wang, Chun‐Hua Han
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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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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.

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Vikram H. Raval, Rupal H. Joshi, Hitarth B. Bhatt, Satya P. Singh
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Cloning, expression, purification and characterisation of serine alkaline protease from Bacillus subtilis RD7
Yewande Suberu, Idowu Akande, Titilola Samuel, Adekunle Lawal, Ademola Olaniran
Biocatalysis and Agricultural Biotechnology.2019; 20: 101264. CrossRef
Alkaline serine protease from the new halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R: purification and properties
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Journal of Basic Microbiology.2018; 58(1): 88. CrossRef
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Enzyme and Microbial Technology.2017; 106: 97. CrossRef
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Thirumalai Maruthiah, Grasian Immanuel, Arunachalam Palavesam
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Vikram H. Raval, Sumitha Pillai, Chirantan M. Rawal, Satya P. Singh
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Tropical Animal Health and Production.2013; 45(4): 987. CrossRef
Fermentation and Quality Characteristics of Cheonggukjang Fermented with Bacillus subtilis BC-P1
Sung-Yong Park, Mi-Ae Bang, Boung-Jun Oh, Jeong-Hoon Park, Won-Seob Song, Kyung-Min Choi, Eui-Su Choung, Hee-Ock Boo, Seung-Sik Cho
The Korean Journal of Microbiology.2013; 49(3): 262. CrossRef
IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROMBacillus subtilisVSG-4 AND ITS APPLICATION AS CO2SEQUESTERER
M. Oviya, Sib Sankar Giri, V. Sukumaran, P. Natarajan
Preparative Biochemistry and Biotechnology.2012; 42(5): 462. CrossRef

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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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.

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Impact of UV pre-treatment on the Longissimus thoracis et lumborum muscle proteomes of dry-aged beef cuts: A characterisation within two sampling locations
Sara Álvarez, Carlos Álvarez, Anne Maria Mullen, Eileen O'Neill, Mohammed Gagaoua
Meat Science.2025; 221: 109729. CrossRef
Genome-wide association study on reproductive traits in Jinghai Yellow Chicken
G.X. Zhang, Q.C. Fan, J.Y. Wang, T. Zhang, Q. Xue, H.Q. Shi
Animal Reproduction Science.2015; 163: 30. CrossRef
Production and on-column re-folding of human vascular endothelial growth factor 165 in Escherichia coli
Sun Kwon Bang, Young Sik Kim, Byung Soo Chang, Cheol Beom Park, In Seok Bang
Biotechnology and Bioprocess Engineering.2013; 18(5): 835. CrossRef

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 PDF: 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 PDF: 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 PDF: 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 PDF: 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 PDF: 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'ts

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 PDF: 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.

Purification and Characterization of Laccase from the White Rot Fungus Trametes versicolor: Moon-Jeong Han , Hyoung-Tae Choi , Hong-Gyu Song; J. Microbiol. 2005;43(6):555-560.; DOI: https://doi.org/2290 [pii]

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Abstract PDF: Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2''-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50oC. The Km value of the enzyme for substrate ABTS is 12.8 M and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

Purification and Characterization of NADPH-Dependent Cr(VI) Reductase from Escherichia coli ATCC 33456: Woo-Chul Bae , Han-Ki Lee , Young-Chool Choe , Deok-Jin Jahng , Sang-Hee Lee , Sang-Jin Kim , Jung-Hyun Lee , Byeong-Chul Jeong; J. Microbiol. 2005;43(1):21-27.; DOI: https://doi.org/2143 [pii]

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Abstract PDF: A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37^oC. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61% higher activity than NADH. The K_m values for NADPH and NADH were determined to be 47.5 and 17.2 umol, and the V_max values 322.2 and 130.7 umol Cr(VI) min^-1mg^-1 protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag^2+, Cd^2+, Hg^2+, and Zn^2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.

Purification of Filamentous Bacteriophage M13 by Expanded Bed Anion Exchange Chromatography: Tau Chuan Ling , Chee Kin Loong , Wen Siang Tan , Beng Ti Tey , Wan Mohammad Wan Abdullah , Arbakariya Ariff; J. Microbiol. 2004;42(3):228-232.; DOI: https://doi.org/2084 [pii]

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Abstract PDF: In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine^TM 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (H_o=15cm) of STREAMLINE DEAE (r=1.2 g/cm^3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.

Purification and Characterization of Thermostable β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris Grown on Microcrystalline Cellulose: Jeong-Jun Yoon , Ki-Yeon Kim , Chang-Jun Cha; J. Microbiol. 2008;46(1):51-55.; DOI: https://doi.org/10.1007/s12275-007-0230-4

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An extracellular β-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC- MS/MS suggested that the protein has high homology with fungal β-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-β-D-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki= 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified β-glucosidase was observed at pH 4.5 and 70°C. The F. palustris protein exhibited half-lives of 97 h at 55°C and 15 h at 65°C, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-β-lactoside, p-nitrophenyl-β-xyloside, p-nitrophenyl-α-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the β-glucosidase from F. palustris can be classified as an aryl-β-glucosidase with cellobiase activity.

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Purification and characterization of a xylanase from alkalophilic cephalosporium sp. RYM-202: Kang, Myoung Kyu , Kwon, Tae Ik , Yang, Young Ki , Rhee, Young Ha; J. Microbiol. 1995;33(2):109-114.

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Abstract PDF: Alkalophilic Cephalosporium sp. RYM-202 produced multiple xylanases extracellularly. One of these xylanases was purified to electrophoretical homogeneity by chromatography with DEAE-Sephadex A-50, Sephacryl S-200 HR and Superose 12 HR. The purified xylanase differed from most other microbial xylanases in that it had low-molecular weight and acidic isoelectric point. The molecular weight of the xylanase in that it had low-molecular weight and acidic isoelectric point. The molecular weight of the xylanase was 23 kDa by SDS-polyacrylamide electrophoresis and 24 kDa by gel permeation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity permentation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity permeation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity at pH 8.0 and 50℃. It was stable over a wide range of pH and retained more than 80% of its original activity after 24 h of incubation even at pH 12. The Km values of this enzyme on birchwood xylan and oat spelts xylan were 2.33 and 3.45 mg/ml, respectively. The complete inhibition of the enzyme of n-bromosuccinimide suggests the involvement of tryptophan in the active site. The sylanase lacked activity towards crystalline cellulose and carboxymethyl cellulose.

Purification and Characterization of an Extracellular Protease from Culture Filtrate of Salmonella schttmulleri: Na, Byoung Kuk , Song, Chul Yong; J. Microbiol. 1995;33(3):244-251.

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Abstract PDF: An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca^2+, Zn^2+, Fe^2+, Mg^2+ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40℃. It was stable at least for 1 week at 40℃ and maintained its activity for 24 hours at 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.

Characteristics of trypsin-like protease and metalloprotease associated with mycelium differentiation of streptomyces albidoflavus SMF301: Kang, Sung Gyun , Kim, In Seop , Jeong, Byung Cheol , Ryu, Jae Gon , Rho, Yong Taik , Lee, Kye Joon; J. Microbiol. 1995;33(4):307-314.

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Abstract PDF: Trypsin like protease (TLP) and metalloprotease (MTP) were induced in associated with the mycelium differentiation in Streptomyces albidoflavus SMF301. TLP and MTP were purified and characterized from the culture. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The optimum pH and temperature of TLP were 10 and 40℃. Those of MTP were 8 and 55 ℃. TLP was stable at alkaline pH (6-9) and unstable above 45℃ and MTP was stable at alkaline pH and unstable above 80℃. Km and Vmax values with benzoyl-arginyl p-nitroanilide of TLP were 139 uM, and 10 nmole of nitroanilide released per min per ㎍ protein, respectively. Km, and Vmax values with a synthetic substrate, leucine p-nitroanilide, or MTP were 58.9 uM, 3.47 nmol of nitroanilide released per min per/㎍ protein, respectively. TLP was inhibited competitively by leupeptin; the inhibition constant was 0.0031 uM. MTP was inhibited by EDTA, phenonthroline and bestatin.

Purification and Characterization of α-amylase from Aspergillus sp. JP-1: Park, Hyung Nam , Yoo, Jin Cheol , Yang, Young Ki; J. Microbiol. 1995;33(1):80-84.

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Abstract PDF: The α-amylase was purified from Aspergillus sp. JP-1 and some enzyme characteristics were studied. The enzyme waw approzimately purified 80-fold and an overall yield was 16.5% from the culture medium by ammonium sulfate fractionation, Sephadex G-150 gel permeation chromatography, and DEAE-Sephadex A-50 ion exchange column chromatography in order. The molecular weight of the purified α-amylase has been estimated to be 56 KDa on SDS-polyacrulamide gel electrophoresis and Sephadex G-150 chromatography. The purigied enzyme functions optimally at pH5.5 and 40℃, respectively. The Km value for soluble starch was 2.5 mg/ml. The enzymatic activity increased in the presence of Ca^2+, Co^2+, EDTA, Mg^2+, Mn^2+ and Zn^2+ and was inhibited by adding Cu^2+, Fe^2+, and Ni^2+.

Purification and characterization of purine nucleoside phosphorylase (PNP) in micrococcus luteus: Choi , Hye Seon; J. Microbiol. 1996;34(1):82-89.

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Abstract PDF: Purine nucleoside phosphorylase (PNP) was purified in Micrococcus luteus (M. luteus) using streptomycin sulfate and amomonium sulfate fractionation, three times by a Sephadex G-100 gel filtration and a DEAE-Sephadex A-50 ion exchange chromatography. The enzyme was purified 72 folds with a 11% recovery and showed a single band in a nondenaturing gel electrophoresis. The M. W. of PNP turned out to be 1.35 × 10^5 dalton in G-150 gel filtration chromatography. The stability of the enzyme was increased by treatment with both substrates, MgCI₂or CaCI₂, but not significantly kcal/mol. M. luteus PNP catalyzed the phosphorolysis of inosine, deoxyinosine, guanosine and deoxyguanosine with the Km value of 1.5 × 10^-3 M, 3.0 × 10^-3 M, 5.0 × 10^-4 M, respectively. The enzyme was reacted with adenosine, 1-methylnosine and 1-methylguanosine as substrates, which were shown to be poor substrates for mammalian enzyme.

Purification of carbosymethyl cellulase from hybrid between aspergillus niger and penicillium verruculosum: Yang, Young Ki , Lee, Jung Sup , Park, Hyung Nam , Moon, Myung Nim , Kim, Hong Sub , Kim, Jong Se , Lim, Chae Young , Rhee, Young Ha; J. Microbiol. 1996;34(1):90-94.

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Abstract PDF: The carboxymethyl cellulase (CMCase) was purified from the induced culture filtrate of hybrid TAPW15703 between Aspergillus niger and penicillium verruculosum made by nuclear transfer. The enzyme was purified 80 fold with an overall yield 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and DEAE-ion exchange column chromatography. The molecular weight of the CMCase has estimated to be 32,000 daltons on SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel permeation chromatography. The purified enzyme functions optimally at pH 4.0 and 40℃. The Km value for carbosymethyl cellulose was 68 mM. The enzyme activity was increased by the presence of Mg^2+ and Mn^2+.

Purification and charactedrization of cysteine desulfhydrase from streptomyces albidoflavus SMF301: Ryu, Jae Gon , Kang, Sung Gyun , Kim, In Seop , Rho, Young Taik , Lee, Sang Hee , Lee, Kye Joon; J. Microbiol. 1997;35(2):97-102.

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Abstract PDF: Cysteine desulfhydrase (EC 4, 4, 1. 1. ) was purified from the culture supernatant of Streptomyces albidoflavus SMF301 by hydroxyapatite, gel filtration and Resource Q ion-exchange chromatography with a purification fold of six identical subunits. The enzyme was stabilized by dithiothreitol and pyridoxal 5'-phosphate during the purification procedures. The optimum pH and temperature were pH 8.6 and 35℃, respectively. The N-terminal amino acid sequence was identified as A-P-L-P-T-A-D-V-R-S-D-P-G-Y-E-W-L-G-E-A-V. The purified cystein desulfhydrase had a high substrate specificity toward cysteine, and exhibited no cystahionine λ-lyase activity. The K_m value for cysteine was determined to be 0.37 mM.

Purification and characterization of extracellular aspartic proteinase of candida albicans: Na, Byoung-Kuk , Lee, Seong Il , Kim, Sin Ok , Park, Young Kil , Bai, Gill Han , Kim, Sang Jae , Song, Chul Yong; J. Microbiol. 1997;35(2):109-116.

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Abstract PDF: An extracellular proteinase of Candida albicans was purified by a combination of 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45℃. The addition of divalent cations, Ca^2+, Zn^2+ and Mg^2+, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe^2+, Ag^2+ and Cu^2+. With BSA as substrate, an apparent K_m was determined to be 7 × 10^7 M and K_I, using pepstatin A as an inhibitor, was 8.05 × 10^8 M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P₁position, but the enzyme activity was highly reduced when the P₂position was Phe or Pro. This enzyme showed antigenicity against sera of patients with candidiasis.

Rapid and Simple Purification of Biologically Active Human Hepatitis B virus Transactivator-X Proteins: Poo, Ha Ryoung , Kim, Sun Ok , Sohn, Mi Jin , Lee, Sook , Lee, Young Ik; J. Microbiol. 1998;36(1):55-58.

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Abstract PDF: The human hepatitis B virus-X(HBV-X) was cloned into an expression vector, pET3d, containing a T7 promoter and direct expression was induced in Escherichia coli. The expressed HBV-V protein was purified to homogeneity by centrifugation, ion-exchange chromatography and gel filtration. After gel filtration, renaturation of HBV-X were performed using dialysis against serially diluted urea buffer. The biological activity of refolded HBV-X protein was confirmed by enhancement of protein/DNA complexes using gel-shift analysis.

DNA Sequencing and Expression of the Circumsporozoite Protein of Plasmodium vivax Korean Isolate in Escherichia coli: Hyeong Woo Lee , Jong Soo Lee , Won Ja Lee , Ho Sa Lee; J. Microbiol. 1999;37(4):234-242.

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Abstract PDF: To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyunggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no corss reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagonis of malaria patients and seroepidemiology.

Purification and Characterization of Chitinase from a Marine Bacterium, Vibrio sp. 98CJ11027: Shin Hye Park , Jung-Hyun Lee , Hong Kum Lee; J. Microbiol. 2000;38(4):224-229.

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Abstract PDF: Chitin-degrading marine bacterial strain 98CJ11027 was isolated from bryozoa from the coastal area of Cheju Island, Korea, and identified as a member of the genus Vibrio. The molecular mass of the main extracellular chitinase (chitinase I), purified from strain 98CJ11027, was estimated to be 98 kDa. The optimal condition for chitinase I activity is pH 6.0 and 45 C. The activity was inhibited by Fe^+2 and Cu^+2. Chitinase I displayed the hydrolysis type of chitobiosidase and catalyzed reversed hydrolysis leading to the synthesis of tetraacetylchitotetraose.

Purification and Characterization of Extracellular Temperature-Stable Serine Protease from Aeromonas hydrophila: Soo-Jin Cho , Jong-Ho Park , Seong Joo Park , Jong-Soon Lim , Eung Ho Kim , Yeon-Jae Cho , Kwang-Soo Shin; J. Microbiol. 2003;41(3):207-211.

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Abstract PDF: Extracellular protease, from Aeromonas hydrophila Ni 39, was purified 16.7-fold to electrophoretic homogeneity with an overall yield of 19.9%, through a purification procedure of acetone precipitation, and Q Sepharose and Sephacryl S-200 chromatographies. The isoelectric point of the enzyme was 6.0 and the molecular mass, as determined by Sephacryl S-200 HR chromatography, was found to be about 102 kDa. SDS/PAGE revealed that the enzyme consisted of two subunits, with molecular masses of 65.9 kDa. Under standard assay conditions, the apparent K_m value of the enzyme toward casein was 0.32 mg/ml. About 90% of the proteolytic activity remained after heating at 60 ℃ for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60℃, and the optimum pH of the enzyme was 9.0. The enzyme was inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), by about 87.9%, but not by E64, EDTA, pepstatin or 1,10-phenanthroline. The enzyme activity was inhibited slightly by Ca_2^+, Mg_2^+ and Zn_2^+ ions.

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