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ABSTRACT: Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with
ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST)
in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0
∼ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from
full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led
to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GSTVpr resulted in the proteolytic degradation
of the protein and no GST fusion Vpr protein was detected.
Keywords
Bacillus subtilis
-mass spectrometry-UUG start codon-Vpr-zymography
Biotechnology and Bioprocess Engineering 04/2012; 15(3):446-452. · 1.28 Impact Factor
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ABSTRACT: A Gram-positive, endospore-forming, new Bacillus species, strain BL4-6(T), was isolated from tidal flat sediment of the Yellow Sea. Strain BL4-6(T) is a straight rod, with motility by peritrichate flagella. The cell wall contains meso-diaminopimelic acid, and the major respiratory quinone is menaquinone-7. The major fatty acids are iso-C(15:0) and summed feature 3 (containing C(16:1) ω7c/iso-C(15:0) 2OH, and/or iso-C(15:0) 2OH/C(16:1) ω7c). Cells are catalase-positive and oxidase-negative. The G+C content of the genomic DNA is 38.0 mol%. Based on a comparative 16S rRNA gene sequence analysis, the isolate belongs to the genus Bacillus, forms a clade with the Bacillus cereus group, and is closely related to Bacillus mycoides (98.5%), Bacillus cereus (98.5%), Bacillus anthracis (98.4%), Bacillus thuringiensis (98.4%), Bacillus weihenstephanensis (98.1%), and Bacillus pseudomycoides (97.5%). The isolate showed less than 85% similarity of the gyrA gene sequence and below 95% similarity of the rpoB gene sequence to the members of this group. DNA-DNA relatedness between strain BL4-6(T) and B. cereus group was found to be in a range of 22.8-42.3%, and thus BL4-6(T) represents a unique species. On the basis of these studies, strain BL4-6(T) (=KCTC 13319(T) =JCM 15802(T)) is proposed to represent the type strain of a novel species, Bacillus manliponensis sp. nov.
The Journal of Microbiology 12/2011; 49(6):1027-32. · 1.10 Impact Factor
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Min Young Jung, Joong-Su Kim,
Woon Kee Paek,
Igor Styrak,
In-Soon Park,
Yeseul Sin,
Jayoung Paek,
Keun Ae Park,
Hongik Kim,
Hong Lim Kim,
Young-Hyo Chang
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ABSTRACT: A Gram-positive, rod-shaped, endospore-forming bacterium, designated strain BLB-1(T), was isolated from samples of tidal flat sediment from the Yellow Sea. 16S rRNA gene sequence analysis demonstrated that the isolate belonged to the Bacillus rRNA group 2 and was closely related to Bacillus massiliensis CIP 108446(T) (97.4 %), Bacillus odysseyi ATCC PTA-4993(T) (96.7 %), Lysinibacillus fusiformis DSM 2898(T) (96.2 %) and Lysinibacillus boronitolerans DSM 17140(T) (95.9 %). Sequence similarities with related species in other genera, including Caryophanon, Sporosarcina and Solibacillus, were <96.1 %. Chemotaxonomic data supported the affiliation of strain BLB-1(T) with the genus Lysinibacillus. The major menaquinone was MK-7, the cell-wall sugars were glucose and xylose, the cell-wall peptidoglycan type was A4α (l-Lys-d-Asp), the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and several unknown phospholipids, and the major fatty acids were anteiso-C(15 : 0) (35.6 %), iso-C(15 : 0) (25.6 %) and anteiso-C(17 : 0) (16.5 %). The most closely related species, Bacillus massiliensis and Bacillus odysseyi, were also assigned to this genus based on phylogenetic analysis and phenotypic data. The results of DNA-DNA hybridizations and phenotypic tests supported the differentiation of all three taxa from species of the genus Lysinibacillus with validly published names. Thus, strain BLB-1(T) ( = KCTC 13296(T) = JCM 15800(T)) represents a novel species, for which the name Lysinibacillus sinduriensis sp. nov. is proposed. It is also proposed that Bacillus massiliensis CIP 108446(T) ( = 4400831(T) = CCUG49529(T) = KCTC 13178(T)) and Bacillus odysseyi NBRC 100172(T) ( = 34hs-1(T) = ATCC PTA-4993(T) = NRRL B-30641(T) = DSM 18869(T) = CIP 108263(T) = KCTC 3961(T)) be transferred to the genus Lysinibacillus as Lysinibacillus massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov., respectively.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 11/2011; 62(Pt 10):2347-55. · 2.11 Impact Factor
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ABSTRACT: A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79 % identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified by using Ni-NTA affinity chromatography and characterized. The optimum temperature and pH for DERA were 50 degrees C and 6.0, respectively. The specific activity for deoxyribose 5-phosphate (DR5P), substrate, was 62 micronmol/min/mg. The Km value for DR5P was determined to be 145 mM with the Kcat value of 3.2 times 10(2 /sec) from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).
Journal of Microbiology and Biotechnology 06/2010; 20(6):995-1000. · 1.38 Impact Factor
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ABSTRACT: A gene encoding glucansucrase was identified in Leuconostoc lactis EG001 isolated from lactic acid bacteria (LAB) in Kimchi, a traditional Korean fermented food. The L. lactis EG001 glucansucrase gene consists of 4503 bp open reading frame (ORF) and encodes an enzyme of 1500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (75%) to that of dextransucrase of L. mesenteroides. The gene was cloned and over-expressed in Escherichia coli strain. The recombinant enzyme was purified via Ni-NTA affinity chromatography and its enzymatic properties were characterized. The enzyme exhibited optimum activity at 30 degrees C and pH 5.0. In addition, the enzyme was able to catalyze the glycosylation of l-ascorbic acid to l-ascorbic acid 2-glucoside. The glycosylated product via EG001 glucansucrase has the potential as an antioxidant in industrial applications.
Microbiological Research 10/2009; 165(5):384-91. · 2.31 Impact Factor
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ABSTRACT: A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine-sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP-CGM). The OP-CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS-polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.
Analytical Biochemistry 09/2009; 397(2):259-61. · 3.00 Impact Factor
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ABSTRACT: Two novel spore-forming lactic acid bacteria, strains SL213T and SL1213, were isolated from vineyard soils in Korea. Cells of both isolates were rod-shaped bacilli and contained subterminal, ellipsoidal spores. Strains were facultatively anaerobic, catalase-positive, oxidase-negative and motile with single flagella. meso-Diaminopimelic acid, glucose and galactose were detected in whole-cell hydrolysates. Major fatty acids found in the strains were anteiso-C15:0, iso-C15:0, iso-C16:0, C16:0 and anteiso-C17:0. The G+C contents of the DNA were 46.1 and 46.3 mol%. 16S rRNA gene sequences from the two strains were almost identical (99.9%) and placed them in the genus Bacillus, according to phylogenetic analysis. The type strains most closely related to SL213T were Bacillus coagulans ATCC 7050T and Bacillus badius ATCC 14574T, with 16S rRNA gene sequence similarities of 96.9 and 95.9%, respectively. Levels of DNA-DNA relatedness between strain SL213T and strain SL1213, B. coagulans ATCC 7050T and B. badius ATCC 14574T were 92.5, 49.0 and 27.5%, respectively. On the basis of 16S rRNA gene sequences and chemotaxonomic and phenotypic evidence given in this study, we report that SL213T represents a novel species, for which the name Bacillus acidiproducens sp. nov. is proposed. The type strain is SL213T (=KCTC 13078T=JCM 14638T).
International journal of systematic and evolutionary microbiology 08/2009; 59(Pt 9):2226-31. · 2.27 Impact Factor
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ABSTRACT: A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific activity was 137 micromol/min/mg. The Michaelis constant (k(m) value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 degrees C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.
Protein Expression and Purification 07/2009; 68(2):196-200. · 1.59 Impact Factor
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ABSTRACT: A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.
Electrophoresis 07/2009; 30(12):2234-7. · 3.30 Impact Factor
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Jun Hyuck Lee,
Jungdon Bae,
Dooil Kim,
Yongseok Choi,
Young Jun Im,
Sukhoon Koh, Joong Su Kim,
Mun-Kyoung Kim,
Gil Bu Kang,
Suk-In Hong,
Dae-Sil Lee,
Soo Hyun Eom
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ABSTRACT: It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.
Biochemical and Biophysical Research Communications 10/2006; 347(3):616-25. · 2.48 Impact Factor
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ChemBioChem 12/2005; 6(11):1963-6. · 3.94 Impact Factor
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Jungdon Bae,
Dooil Kim,
Yongseok Choi,
Sukhoon Koh,
Jung Eun Park, Joong Su Kim,
Seong Hoon Moon,
Bo-Hyun Park,
Miri Park,
Hye-Eun Song,
Suk-In Hong,
Dae-Sil Lee
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ABSTRACT: A recombinant thermophilic Thermus caldophilus GK24 hexokinase, one of the ROK-type (repressor protein, open reading frames, and sugar kinase) proteins, exists uniquely as a 120 kDa molecule with four subunits (31 kDa), in contrast to eukaryotic and bacterial sugar kinases which are monomers or dimers. The optimal temperature and pH for the enzyme reaction are 70-80 degrees C and 7.5, respectively. This enzyme shows broad specificity toward glucose, mannose, glucosamine, allose, 2-deoxyglucose, and fructose. To understand the sugar specificity at a structural level, the enzyme-ATP/Mg2+-sugar binding complex models have been constructed. It has been shown that the sugar specificity is probably dependent on the interaction energy occurred by the positional proximity of sugars bound in the active site of the enzyme, which exhibits a tolerance to modification at C2 or C3 of glucose.
Biochemical and Biophysical Research Communications 10/2005; 334(3):754-63. · 2.48 Impact Factor
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ABSTRACT: A thermostable DNA polymerase from Thermus caldophilus GK24 was purified to near homogeneity by chromatographic methods, including ion-exchange, gel-filtration and affinity chromatography. The purified enzyme had a specific activity of 8400 U/mg at 75°C and a molecular mass of 95 kDa, estimated by SDS/PAGE and Superose-12 gel filtration. Reaction conditions were investigated in terms of pH, metal-ion concentration and temperature. Experimental results showed that T. caldophilus (Tca) DNA polymerase had a maximum activity near pH 8.7 at 75°C. The N-terminal sequence of the enzyme was highly similar to that of Thermus aquaticus (Taq) DNA polymerase, which was consistent with the fact that the enzyme had 5′-to-3′ exonuclease activity and no 3′-to-5′ exonuclease activity. Gene amplification using Tca DNA polymerase resulted in longer products than amplification using Taq DNA polymerase.
European Journal of Biochemistry. 03/2005; 214(1):135 - 140.
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ABSTRACT: Purified trehalose synthase from Thermus caldophilus GK24 produced 18–86% trehalose from 10 mM–1 M maltose. The enzyme also catalyzed the conversion of ,-trehalose into maltose but did not act on other disaccharides. The yield of trehalose from maltose by this enzyme increased 30% more at 40C than at 80C and was independent of the substrate concentration. The maximum yield of ,-trehalose from 10 mM maltose reached 86% at 40C. In addition, ,-trehalose was also formed from maltose or ,-trehalose at 3.5% yield at 80C. Rapid Science Ltd. 1998
Biotechnology Letters 07/1998; 20(8):757-761. · 1.68 Impact Factor
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ABSTRACT: Silver nanoparticles have antimicrobial activity against many pathogenic microbes. Here, the preparation of a nanosized Ag–silica hybrid complex (NSS) prepared by γ-irradiation is described. The effects of both NSS and reduced Ag nanoparticles (Ag0) on the growth of the model plant Arabidopsis thaliana were tested. The application of 1–10 ppm NSS complex improved Arabidopsis growth in soil, whereas 100 ppm NSS resulted in weakly curled leaves. In addition, supplementation of Murashige and Skoog (MS) growth medium with 1 ppm NSS promoted the root growth of Arabidopsis seedlings, but root growth was inhibited by supplementation with 10 ppm NSS. To investigate whether the NSS complex could induce plant defense responses, the expression of pathogenesis-related (PR) genes that are implicated in systemic acquired resistance (SAR) in Arabidopsis plants was examined. PR1, PR2 and PR5 were significantly up-regulated by each application of 10 ppm NSS complex or Ag0 to the rosette leaves. Furthermore, pretreatment with the NSS complex induced more pathogen resistance to the virulent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) compared to water treatment in Arabidopsis plants.Research highlights► We describe the preparation of silver nanoparticles using γ-irradiation technique. ► We examine the effects of silver nanoparticles on the growth of Arabidopsis. ► Silver nanoparticles induced the expression of SAR marker genes. ► Silver nanoparticles exhibited enhanced disease resistance to the bacterial pathogen.
Radiation Physics and Chemistry. 81(2):180-184.
