• Korean Journal of Food Science and Technology
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• v.38 no.3
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• pp.419-426
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• 2006

A thermophilic microorganism, strain JE 375, which produces a thermostable protease, was isolated from soil and compost in Korea. This gram-positive, rod-shaped, catalase positive, motility positive, and hemolysis ${\beta}$ containing organism was implicated in glucose fermentation, mannitol fermentation, xylose oxidation, aerobic activity and spore formation. The color of the colony was yellowish white. The temperature range for growth at pH 6.5 was between 55 and $70^{\circ}C$, with an optimum growth temperature of $65^{\circ}C$. This result confirmed the strain JE 375 as a thermophilic microorganism. The enzyme was produced aerobically at $65^{\circ}C$ during 20 hr in a medium (pH 6.5) containing 1% trypton. 1% maltose, 0.5% yeast extract and 1% NaCl. The 16S rDNA of strain JE 375 had 97.6% sequence similarity with the 16S rDNA of Bacillus caldoxyloyticus. On the basis of biochemical and physiological properties and phylogenetic analysis, we named the isolated strain as Bacillus sp. JE 375. The thermostable protease from Bacillus sp. JE 375 had been partially purified and characterized. The molecular weight of the enzyme was deduced from SDS-PAGE and gel chromatography as 55 kDa and its optimal temperature was $60^{\circ}C$. The enzyme showed its highest activity at pH 7.5 and was stable from pH 7.0 to 8.0.

• Applied Biological Chemistry
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• v.40 no.5
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• pp.375-379
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• 1997

Two ribosome-inactivating proteins, PRIP 1 and PRIP 2 have been isolated from the leaves of $Cucurbita\;moschata\;D_{UCHESNE}$. Crude extracts were purified through ammonium sulfate precipitation and column chromatography using DE-52 cellulose, S-Sepharose, FPLC Suprose 12 HR and FPLC Mono-S. The molecular weights of PRIP 1 and PRIP 2 were 31,000 and 30,500, respectively. PRIP 2 was thermostabe and maintained its activity even after the incubation of the protein at $50^{\circ}C$ for 30 min. In a cell free in vitro translation system using rabbit reticulocyte lysate, protein synthesis was inhibited by the addition of PRIP 1 and PRIP 2. The $IC_{50}$ of PRIP 1 and PRIP 2 were 0.82 nM and 0.79 nM, respectively. The comparison of N-terminal amino acid sequences of the PRIP 1 and PRIP 2 with known RIPs revealed that PRIP 1 shows sequence similarity with Luffin B from Luffa cylindrica and Trichokirin from Trichosanthes kirilowii Maximowicz and PRH) 2 has sequence similarity with Momordin II and MAP 30 from Momordica charantia.

• The Korean Journal of Zoology
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• v.27 no.1
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• pp.25-34
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• 1984

Two species of fishes of Genus Liobagrus that had been collected from three separate river systems in Korea were compared by their morphology and gel electrophoretic patterns of water soluble proteins. In morphology L. andersoni from Han River was distinctly different from L. mediadiposalis collected from both Gum River and Nagdong River. But L. andersoni collected from Gum River not only showed partial similarity for both groups of fishes above in their classifying characteristics but also had a unique ratio of body width/standard length. Comparable similarity and difference among the three groups were also noted in their protein patterns of SDS polyacrylamide gel electrophoresis of various tissues. The difference was clearer and more distinct in the gel run with muscle proteins. A couple of more distinctly different low molecular polypeptides were detected by two dimensional gel electrophoresis. Since the protein patterns shown in this study are not only agreeable with the morphological results but also provide detailed comparisons, fishes of Liobagrus from various water sources can be classified reliably by gel electrophoresis. On the bases of tghe findings above, L. andersoni like fishes collected from Gum River should no longer be classified as L. andersoni. Before naming them as an independent species, the possibility of natural hybrid between the two defined species, or of a varient of L. andersoni by geograpic isolation should be tested.

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.636-643
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• 2001

Bacillus sp. AIR-5, a strain from soil, produced an extracellular maltopentaose-forming amylase from amylose and soluble starch. This bacterium produced 8.9 g/l of maltopentaose from 40 g/l of soluble starch in a batch fermentation and the maltopentaose made up 90 % of the maltooligosaccharides produced (from maltose to maltoheptaose). The culture supernatant was concentrated using a 30 K molecular weight cut-off membrane and purified by DEAE-Cellulose and Sephadex G-150 column chromatographies. The purified protein showed one band on a native-PAGE and its molecular mass was estimated as 250 kDa. The 250-kDa protein was composed of tetramers of a 63-kDa protein. the isoelectric point of the purified protein was pH 6.9, and the optimum temperature for the enzyme activity was $45^{\circ}C$. The enzyme was quickly inactivated above $55^{\circ}C$, and showed a maximum activity at pH 8.5 and over 90% stability between a pH of 6 to 10. The putative N-terminal amino acid sequence of AIR-5 amylase, ATINNGTLMQYFEWYVPNDG, showed a 96% sequence similarity with that of BLA, a general liquefying amylase.

• Journal of Life Science
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• v.14 no.6 s.67
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• pp.986-990
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• 2004

Five strains of Vibrio vulnificus, which cause serious septicemia in human worldwidely, was isolated from marine environments of Korea costal area from May to July of 2002. The isolated strains were identified by API 20E kit and partial 16S rDNA sequence analysis. 16S rDNA sequence of the isolates showed $99\%$ similarity with V. vulnificus ATCC 27562. The proteins of V. vulnificus isolates were examined by analyzing patterns of the cell lysates and outer membrane proteins (OMP). The OMP separated from cell lysates showed the common protein band. Therefore OMP profiles might be useful for the identification of V. vulnificus sp.

• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.24-32
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• 2009

The KNUC25 strain isolated from over-fermented whole Chinese cabbage kimchi was examined for its physiological characteristics using API 50 CHL system assay and identified as Lactobacillus paraplantarum by analysis of whole-cell protein SDS-PAGE pattern assay and similarity of 16S rDNA sequence. L. paraplantarum KNUC25 had a broad antimicrobial activity spectrum from Gram positive to Gram negative bacteria. Scanning electron micrograph analysis showed that KNUC25 might attack to cell surface of indicator cells and destruction can lead to inhibition of the cell growth. The antimicrobial substance of the KNUC25 strain was stable to various degrading enzymes and at high temperature and not a plasmid-born matter. Resistance to proteolytic enzymes showed that an antimicrobial activity of KNUC25 might not be caused by proteinous substance. Maximum production of antimicrobial substance was the exponential growth phase at $30^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.522-529
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• 2019

A Bacillus strain, SJ4, exhibiting strong fibrinolytic activity was isolated from saeu (shrimp, Acetes chinensis) jeotgal, a Korean traditional fermented food and was identified as B. subtilis. The B. subtilis SJ4 strain can grow at a NaCl concentration of up to 15% (w/v). The fibrinolytic activity of B. subtilis SJ4 (152.0 U/ml) cultured in Luria-Bertani (LB) broth for 48 h at 37℃ with aeration was higher than that of B. subtilis SJ4 cultured in TSB (124.5 U/ml) under same culture conditions. The major proteins in the LB culture supernatant of B. subtilis SJ4 were analyzed by SDS-PAGE, which revealed three major bands (23, 25, and 28 kDa). The band (23 kDa) with strong fibrinolytic activity, analyzed on fibrin zymogram, was observed at 60-96 h of cultivation. The aprESJ4 gene encoding the major fibrinolytic enzyme, AprESJ4, was cloned by PCR. The aprESJ4 gene sequence exhibited high similarities with the fibrinolytic gene sequences of other Bacillus species. The amino acid sequence of AprESJ4 exhibited 98.9 and 98.4% similarity with subtilisin NAT and AprE2 of B. subtilis, respectively. Hence, B. subtilis SJ4 can be a potential starter culture for jeotgal products.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1054-1059
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• 2005

An extracellular protease was identified from Bacillus subtilis KCCM 10257 by N-terminal sequencing and mass spectral analysis. The molecular mass of the extracellular protease was estimated to be 28 kDa by SDS-PAGE. Sequencing of the N-terminal of the protease revealed the sequence of A(G,S,R)QXVPYG(A)V(P,L)SQ. The N-terminal sequence exhibited close similarity to the sequence of other proteases from Bacillus sp. A mass list of the monoisotopic peaks in the MALDI-TOF spectrum was searched after peptide fragmentation of the protease. Six peptide sequences exhibiting monoisotopic masses of 1,276.61, 1,513.67, 1,652.81, 1,661.83, 1,252.61, and 1,033.46 were observed from the fragmented protease. These monisotopic masses corresponded to the lytic enzyme L27 from Bacillus subtilis 168, and the Mowse score was found to be 75. A doubly charged Top product (MS) at a m/z of 517.3 exhibiting a molecular mass of 1034.6 was further analyzed by de novo sequencing using a PE Sciex QSTAR Hybrid Quadropole-TOF (MS/MS) mass spectrometer. MS/MS spectra of the Top product (MS) at a m/z of 517.3 obtained from the fragmented peptide mixture of protease with Q-star contained the b-ion series of 114.2, 171.2, 286.2, 357.2, 504.2, 667.4, 830.1, and 887.1 and y-ion series of 147.5, 204.2, 367.2, 530.3, 677.4, 748.4, 863.4, and 920.5. The sequence of analyzed peptide ion was identified as LGDAFYYG from the b- and y-ion series by de novo sequencing and corresponded to the results from the MALDI-TOF spectrum. From these results the extracellular protease from Bacillus subtilis KCCM 10257 was successfully identified with the lytic enzyme L27 from Bacillus subtilis 168.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1753-1759
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• 2006

Paenibacillus sp. HY-8 isolated from the digestive tracts of the longicorn beetle, Moechotypa diphysis, produced an extracellular endoxylanase with a molecular weight of 20 kDa estimated by SDS-PAGE. The xylanase was purified to near electrophoretic homogeneity from the culture supernatant after ammonium sulfate precipitation, gel filtration, and ionexchange chromatography. The purified xylanase exhibited the highest activities at pH 6.0 and $50^{\circ}C$. The $K_m\;and\;V_{max}$ values were 7.2 mg/ml and 16.3 U/mg, respectively, for birchwood xylan as the substrate. Nucleotide sequence of the PCR-cloned gene was determined to have the open reading frame encoding a polypeptide of 212 amino acids. The N-terminal amino acid sequence and the nucleotide sequence analyses predicted that the precursor xylanase contained a signal peptide composed of 28 amino acids and a catalytically active 19.9-kDa peptide fragment. The deduced amino acid sequence shared extensive similarity with those of the glycoside hydrolase family 11 of xylanases from other bacteria. The predicted amino acid sequence contained two glutamate residues, previously identified as essential and conserved for active sites in other xylanases of the glycoside hydrolase family 11.

• Toxicological Research
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• v.26 no.1
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• pp.47-52
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• 2010

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.