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1.
Int J Syst Evol Microbiol ; 70(2): 732-737, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31702538

ABSTRACT

An obligately anaerobic, Gram-stain-positive and coccus-shaped bacterium, designated strain YH-panp20T, was isolated from pig faeces. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate belongs to the family Erysipelotrichaceae, and is most closely related to Catenisphaera adipataccumulans KCTC 15517T (93.5 % sequence similarity), followed by Faecalitalea cylindroides KCTC 5815T (92.2 %), Faecalicoccus acidiformans KCTC 15521T (90.2 %) and Holdemanella biformis KCTC 5969T (89.6 %). Average nucleotide identity values between YH-panp20T and its closest relatives were lower than 71 %. The G+C content of the isolate was 38.4 mol%, and its cell-wall peptidoglycan was found to be of A1γ type and contained meso-diaminopimelic acid. The predominant fatty acids were C18 : 1 cis 9, C18 : 0 DMA and C16 : 0. The major end-products of glucose fermentation were lactate, acetate and formate. Therefore, based on the phenotypic, phylogenetic and chemotaxonomic properties, a novel genus and species, Absicoccus porci gen. nov., sp. nov., is proposed for isolate YH-panp20T (=KCTC 15747T=JCM 32769T).


Subject(s)
Firmicutes/classification , Phylogeny , Swine/microbiology , Animals , Bacterial Typing Techniques , Base Composition , Cell Wall/chemistry , DNA, Bacterial/genetics , Diaminopimelic Acid/chemistry , Fatty Acids/chemistry , Feces/microbiology , Fermentation , Firmicutes/isolation & purification , Gram-Positive Rods/genetics , Peptidoglycan/chemistry , RNA, Ribosomal, 16S/genetics , Republic of Korea , Sequence Analysis, DNA
2.
J Food Sci ; 84(11): 3186-3193, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31589348

ABSTRACT

Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001 in Escherichia coli BL21 (DE3), forming single O-α-D-glucosyl-(1″→6') rebaudioside A with yield of 86%. O-α-D-glucosyl-(1″→6') rebaudioside A was purified using HPLC and Diaion HP-20 and its properties were characterized for possible use as a food ingredient. Almost 98% of O-α-D-glucosyl-(1″→6') rebaudioside A was dissolved after 15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O-α-D-glucosyl-(1″→6') rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus, O-α-D-glucosyl-(1″→6') rebaudioside A could be used as a highly pure and improved sweetener with high stability in commercial drinks. PRACTICAL APPLICATION: The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation. Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks. This method can be applied to obtain high value-added bioactive compounds by enzymatic modification.


Subject(s)
Bacterial Proteins/chemistry , Diterpenes, Kaurane/chemistry , Glucosyltransferases/chemistry , Leuconostoc/enzymology , Sweetening Agents/chemistry , Biocatalysis , Chromatography, High Pressure Liquid
3.
Int J Syst Evol Microbiol ; 67(11): 4352-4357, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28945536

ABSTRACT

Three bacterial isolates from human faeces, YS-aM39T, R2F3-3-3T and R2F3-5-1, were characterized as Gram-negative, strictly anaerobic, non-spore-forming, non-motile, and rod-shaped. Isolate YS-aM39T formed a distinct line of descent, showing greatest 16S rRNA gene sequence relatedness with R2F3-3-3T (97.5 %), R2F3-5-1 (97.5 %), Bacteroides ovatus (98.8 %) and Bacteroides xylanisolvens (97.2 %). Isolates R2F3-3-3T and R2F3-5-1 also formed a distinct line of descent, sharing greatest 16S rRNA gene sequence relatedness with B. ovatus (98.2 %) and B. xylanisolvens (97.2 %). The DNA G+C content of YS-aM39T was 44.8 mol%, that of R2F3-3-3T was 42.4 mol% and that of R2F3-5-1 was 42.6 mol%. The respiratory quinone of all three isolates was menaquinone MK-10. Polar lipid analysis identified phosphatidylethanolamine as the major lipid. The predominant fatty acids in all three isolates were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 3-OH and iso-C17 : 0 3-OH. The major end products of glucose fermentation were acetic acid, lactic acid and formic acid. DNA-DNA hybridization data indicated that two isolates, YS-aM39T and R2F3-3-3T, represent a species distinct from B. ovatus and B. xylanisolvens. Finally, in this study, the two isolates represented two new species in the genus Bacteroides, for which we propose the names Bacteroides koreensis sp. nov. (type strain, YS-aM39T=KCTC 15520T=JCM 31393T) and Bacteroides kribbi sp. nov. (type strain, R2F3-3-3T=KCTC 15460T=JCM 31391T).


Subject(s)
Bacteroides/classification , Feces/microbiology , Phylogeny , Bacterial Typing Techniques , Bacteroides/genetics , Bacteroides/isolation & purification , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Humans , Nucleic Acid Hybridization , Phosphatidylethanolamines/chemistry , RNA, Ribosomal, 16S/genetics , Republic of Korea , Sequence Analysis, DNA , Ubiquinone/chemistry
4.
AMB Express ; 7(1): 167, 2017 Aug 29.
Article in English | MEDLINE | ID: mdl-28853018

ABSTRACT

Menthol has a considerable cooling effect, but the use range of menthol is limited because of its extremely low solubility in water and inherent flavor. (-)-Menthol ß-glucoside was determined to be more soluble in water (>27 times) than (-)-menthol α-glucoside; hence, ß-anomer-selective glucosylation of menthol is necessary. The in vitro glycosylation of (-)-menthol by uridine diphosphate glycosyltransferase (BLC) from Bacillus licheniformis generated (-)-menthol ß-glucoside and new (-)-menthol ß-galactoside and (-)-menthol N-acetylglucosamine. The maximum conversion rate of menthol to (-)-menthol ß-D-glucoside by BLC was found to be 58.9%. Importantly, (-)-menthol ß-D-glucoside had a higher cooling effect and no flavor compared with menthol. In addition, (-)-menthol ß-D-glucoside was determined to be a non-sensitizer in a skin allergy test in the human cell line activation test, whereas menthol was a sensitizer.

5.
Anaerobe ; 48: 70-75, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28754474

ABSTRACT

Gram-positive, straight or slightly curved rod-shaped bacteria, designated as strains N6H1-5T and N6H1-3, were isolated from fecal samples of old dog. The analysis of 16S rRNA gene sequences indicated that the isolates belonged to the Clostridium cluster XVI and were closely related to Eubacterium dolichum KCTC 5832T, Eubacterium tortuosum DSM 3987T, Clostridium innocuum KCTC 5183T, Allobaculum stecoricanis DSM 13633T, Eubacterium limosum KCTC 3266T, and Clostridium butyricum KCTC 1871T, with 94.0%, 93.8%, 92.0%, 84.9%, 80.7%, and 80.0% sequence similarity, respectively. Chemotaxonomic data supported placement of the strains N6H1-5T and N6H1-3 in the new taxon. The strains contained m-diaminopimelic acid cell wall peptidoglycan; the major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and glycolipids (GL); and the major fatty acids were C18:1cis 9 (30.7%) and C16:0 (17.1%). The predominant metabolic end product was lactic acid. The G + C content was 35.8 mol%. The most closely related species, E. dolichum and E. tortuosum, were also assigned to the new taxon, based on the phylogenetic analysis and phenotypic data. Thus, the type strain N6H1-5T (=KCTC 15422 = JCM 30884) represents a novel genus and species, for which the name Absiella argi gen. nov., sp. nov is proposed. It is also proposed that E. dolichum KCTC 5832T and E. tortuosum DSM 3987T be transferred to this new genus, and named Absiella dolichum comb. nov. and Absiella tortuosum comb. nov., respectively.


Subject(s)
Bacterial Typing Techniques/veterinary , Clostridium/classification , Eubacterium/classification , Firmicutes/classification , Intestines/microbiology , Animals , Base Composition/genetics , Clostridium/genetics , Clostridium/isolation & purification , DNA, Bacterial/genetics , Dogs , Eubacterium/genetics , Eubacterium/isolation & purification , Fatty Acids/analysis , Firmicutes/genetics , Firmicutes/isolation & purification , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
6.
J Microbiol ; 54(4): 311-21, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27033207

ABSTRACT

2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/ß)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and ß8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.


Subject(s)
Aldehyde-Lyases/chemistry , Aldehyde-Lyases/metabolism , Streptococcus suis/enzymology , Aldehyde-Lyases/genetics , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Escherichia coli/genetics , Models, Molecular , Molecular Sequence Data , Molecular Weight , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Subunits/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribosemonophosphates/metabolism , Streptococcus suis/genetics , Substrate Specificity
7.
J Microbiol Biotechnol ; 26(3): 493-7, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26809800

ABSTRACT

Glucosyl rubusosides were synthesized by two dextransucrases. LcDexT was obtained from Leuconosotoc citreum, that LlDexT was obtained from Leuconostoc lactis. LcDexT and LlDexT regioselectively transferred a glucosyl residue to the 13-O-glucosyl moiety of rubusoside with high yield of 59-66% as analyzed by TLC and HPLC. Evaluation of the sweetness of these glucosyl rubusosides showed that their quality of taste, in particular, was superior to that of rubusoside. These results indicate that transglucosylation at the 13-O-glucosyl moiety of rubusoside by different regioselective dextransucrases can be applicable for increasing its sweetness and quality of taste.


Subject(s)
Bacterial Proteins/chemistry , Diterpenes, Kaurane/chemistry , Flavoring Agents/chemistry , Glucosides/chemistry , Glucosyltransferases/chemistry , Leuconostoc/enzymology , Bacterial Proteins/metabolism , Biocatalysis , Diterpenes, Kaurane/metabolism , Flavoring Agents/metabolism , Glucosides/metabolism , Glucosyltransferases/metabolism , Glycosylation , Humans , Taste
8.
PLoS One ; 9(11): e112256, 2014.
Article in English | MEDLINE | ID: mdl-25396430

ABSTRACT

Silver materials have been widely used in diverse fields. However, their toxicity and their mechanism, especially in different forms, have not been studied sufficiently. Thus, cytotoxicity, apoptosis, and interleukin-1beta (IL-1ß) production were investigated using macrophage-like THP-1 cells in the presence of Ag microparticles (AgMPs, 2.7 µm), Ag submicroparticles (AgSMPs, 150 nm), and Ag wires (AgWs, 274 nm×5.3 µm). The levels of cytotoxicity, apoptosis, and IL-1ß production by AgWs were higher than those by the other two AgSMPs and AgMPs. This trend was also observed with each step of the signaling mechanism for IL-1ß production, which is a single pathway affiliated with ROS generation or lysosomal rupture or both, cathepsin B, caspase-1 (NALP3 inflammasome), and finally IL-1ß production in THP-1 cells. All these results suggest that, for development of safe and effective silver materials, the shape or form of silver materials should be considered, especially for macrophage cell lines because epithelial cell lines are not overly sensitive to silver materials.


Subject(s)
Interleukin-1beta/biosynthesis , Monocytes/metabolism , Nanoparticles/chemistry , Nanowires/chemistry , Signal Transduction/drug effects , Silver/pharmacology , Carrier Proteins/metabolism , Caspase 1/metabolism , Cathepsin B/metabolism , Cell Death/drug effects , Cell Line , Cell Shape/drug effects , Enzyme Activation/drug effects , Humans , Inflammasomes/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Monocytes/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein , Nanoparticles/toxicity , Nanoparticles/ultrastructure , Nanowires/ultrastructure , Reactive Oxygen Species/metabolism , Tetradecanoylphorbol Acetate/pharmacology
9.
Appl Environ Microbiol ; 80(23): 7235-43, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25239890

ABSTRACT

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-ß-d-glucoside, resveratrol 4'-O-ß-d-glucoside, resveratrol 3,5-O-ß-d-diglucoside, and resveratrol 3,5,4'-O-ß-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-ß-d-2-deoxyglucoside and resveratrol 3,5-O-ß-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4'-O-ß-d-galactoside, resveratrol 4'-O-ß-d-viosaminoside, resveratrol 3-O-ß-l-rhamnoside, and resveratrol 3-O-ß-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.


Subject(s)
Bacillus/enzymology , Glucuronosyltransferase/metabolism , Glycosides/metabolism , Stilbenes/metabolism , Bacillus/genetics , Carbohydrates/analysis , Cloning, Molecular , Cytosol/chemistry , Gene Expression , Glucuronosyltransferase/genetics , Glucuronosyltransferase/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Resveratrol
10.
Anaerobe ; 30: 30-4, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25094054

ABSTRACT

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the nasal cavity and paranasal sinus related to bacterial infection. A previous study suggested that a specific bacterial group may have an important role in the course of CRS. In this study, bacteria isolated from CRS patients were characterized. A total of 15 strains were identified as Gram-positive anaerobic cocci (GPAC), which were able to utilize peptone as a sole carbon source. Sequencing of the 16S ribosomal RNA gene revealed that the isolates were closely related to members of the genus Peptoniphilus (>97% similarity) within the Clostridiales Family XI. Incertae Sedis. Genotypic and phenotypic characterization suggests that these isolates represent a novel species of the genus Peptoniphilus associated with CRS. The type strain of Peptoniphilus rhinitidis is 1-13T (=KCTC 5985T=JCM 17448T).


Subject(s)
Gram-Positive Bacteria/classification , Gram-Positive Bacteria/isolation & purification , Gram-Positive Bacterial Infections/microbiology , Rhinitis/microbiology , Sinusitis/microbiology , Anti-Bacterial Agents/pharmacology , Bacterial Typing Techniques , Chronic Disease , Cluster Analysis , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Fatty Acids/analysis , Humans , Male , Microbial Sensitivity Tests , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
11.
Protein J ; 33(1): 110-7, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24399319

ABSTRACT

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 µmol min⁻¹ mg⁻¹, V max for CMC was 1.44 µmol mg⁻¹ min⁻¹, and K m values was 25.6 mg mL⁻¹. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²âº and Co²âº. This is the first report about thermostable endoglucanase from Xanthomonas sp.


Subject(s)
Cellulase/chemistry , Cellulase/genetics , Enzyme Stability , Xanthomonas/enzymology , Cellulase/metabolism , Cloning, Molecular , Escherichia coli/genetics , Kinetics , Sequence Homology, Amino Acid , Substrate Specificity , Temperature
12.
Metab Eng ; 18: 44-52, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23563322

ABSTRACT

Most current metabolic engineering applications rely on the inactivation of unwanted reactions and the amplification of product-oriented reactions. All of the biochemical reactions involved with cellular metabolism are tightly coordinated with the electron flow, which depends on the cellular energy status. Thus, the cellular metabolic flux can be controlled either by modulation of the electron flow or the regulation of redox reactions. This study analyzed the genome-wide anaerobic fermentation products of 472 Escherichia coli single gene knockouts, which comprised mainly of dehydrogenases, oxidoreductases, and redox-related proteins. Many metabolic pathways that were located far from anaerobic mixed-acid fermentation significantly affected the profiles of lactic acid, succinic acid, acetic acid, formic acid, and ethanol. Unexpectedly, D-lactate overproduction was determined by a single gene deletion in dehydrogenases (e.g., guaB, pyrD, and serA) involved with nucleotide and amino acid metabolism. Furthermore, the combined knockouts of guaB, pyrD, serA, fnr, arcA, or arcB genes, which are involved with anaerobic transcription regulation, enhanced D-lactate overproduction. These results suggest that the anaerobic fermentation profiles of E. coli can be tuned via the disruption of peripheral dehydrogenases in anaerobic conditions.


Subject(s)
Escherichia coli K12/metabolism , Lactic Acid/biosynthesis , Metabolic Engineering , Anaerobiosis/physiology , Escherichia coli K12/genetics , Escherichia coli K12/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Deletion , Genome-Wide Association Study , Oxidoreductases/genetics , Oxidoreductases/metabolism
13.
Appl Environ Microbiol ; 79(11): 3516-21, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23542617

ABSTRACT

A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli.


Subject(s)
Bacillus/enzymology , Escherichia coli Proteins/metabolism , Glycosyltransferases/metabolism , Phloretin/metabolism , Chromatography, High Pressure Liquid , Escherichia coli , Genetic Engineering , Glycosylation , Magnetic Resonance Spectroscopy , Molecular Structure , Tandem Mass Spectrometry , Uridine Diphosphate Sugars/metabolism
14.
Appl Environ Microbiol ; 78(21): 7680-6, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22923401

ABSTRACT

Geldanamycin (GM) is a naturally occurring anticancer agent isolated from several strains of Streptomyces hygroscopicus. However, its potential clinical utility is compromised by its severe toxicity and poor water solubility. For this reason, considerable efforts are under way to make new derivatives that have both good clinical efficacy and high water solubility. On the other hand, glycosylation is often a step that improves the water solubility and/or biological activity in many natural products of biosynthesis. Here, we report the facile production of glucose-conjugated nonbenzoquinone GM analogs using the Bacillus UDP-glycosyltransferase BL-C. Five aglycon substrates containing nonbenzoquinone aromatic rings were chosen to validate the in vitro glycosylation reaction. Putative glucoside compounds were determined through the presence of a product peak(s) and were also verified using LC/MS analyses. Further, the chemical structures of new glucoside compounds 6 and 7 were elucidated using spectroscopy data. These glucoside compounds showed a dramatic improvement in water solubility compared with that of the original aglycon, nonbenzoquinone GM.


Subject(s)
Antibiotics, Antineoplastic/metabolism , Bacillus/enzymology , Benzoquinones/metabolism , Glycosyltransferases/metabolism , Lactams, Macrocyclic/metabolism , Bacillus/genetics , Drug Design , Escherichia coli/genetics , Escherichia coli/metabolism , Glucose/metabolism , Glycosylation , Glycosyltransferases/genetics , Molecular Sequence Data , Solubility
15.
J Microbiol Biotechnol ; 22(9): 1224-9, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22814496

ABSTRACT

Puerarin (P), an isoflavone derived from kudzu roots, has strong biological activities, but its bioavailability is often limited by its low water solubility. To increase its solubility, P was glucosylated by three dextransucrases from Leuconostoc or Streptococcus species. Leuconostoc lactis EG001 dextransucrase exhibited the highest productivity of puerarin glucosides (P-Gs) among the three tested enzymes, and it primarily produced two P-Gs with a 53% yield. Their structures were identified as alpha-D-glucosyl-(1-->6)-P (P-G) by using LC-MS or (1)H- or (13)C-NMR spectroscopies and alpha-D-isomaltosyl-(1-->6)-P (P-IG2) by using specific enzymatic hydrolysis, and their solubilities were 15- and 202-fold higher than that of P, respectively. P-G and P-IG2 are easily applicable in the food and pharmaceutical industries as alternative functional materials.


Subject(s)
Glucosides/biosynthesis , Glucosyltransferases/metabolism , Isoflavones/biosynthesis , Leuconostoc/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Glucosides/chemistry , Glucosyltransferases/chemistry , Glucosyltransferases/genetics , Glucosyltransferases/isolation & purification , Glycosylation , Isoflavones/chemistry , Leuconostoc/genetics , Solubility
16.
J Agric Food Chem ; 60(24): 6210-6, 2012 Jun 20.
Article in English | MEDLINE | ID: mdl-22530920

ABSTRACT

Rubusoside (R) is a natural sweetener and a solubilizing agent with antiangiogenic and antiallergic properties. However, currently, its production is quite expensive, and therefore, we have investigated nine commercially available glycosidases to optimize an economically viable R-production method. A stevioside (ST)-specific ß-glucosidase (SSGase) was selected and purified 7-fold from Aspergillus aculeatus Viscozyme L by a two-step column chromatography procedure. The 79 kDa protein was stable from pH 3.0 to pH 7.0 at 50-60 °C. Hydrolysis of ST by SSGase produced R and steviol monoglucosyl ester as determined by (1)H and (13)C nuclear magnetic resonance (NMR). Importantly, SSGase showed higher activity toward ST than other ß-linked glucobioses. The optimal conditions for R production were 280 mM ST and 16.6 µL of SSGase at pH 5.1 and 63 °C. This is the first discussion detailing the production of R by enzymatic hydrolysis of ST and is useful for the food additive and pharmaceutical industries.


Subject(s)
Aspergillus/enzymology , Cellulases/metabolism , Diterpenes, Kaurane/biosynthesis , Diterpenes, Kaurane/metabolism , Glucosides/biosynthesis , Glucosides/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Substrate Specificity
17.
Int J Syst Evol Microbiol ; 62(Pt 10): 2347-2355, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22140163

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.


Subject(s)
Bacillaceae/classification , Peptidoglycan/analysis , Phylogeny , Soil Microbiology , Bacillaceae/genetics , Bacillaceae/isolation & purification , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/analysis , Geologic Sediments/microbiology , Molecular Sequence Data , Nucleic Acid Hybridization , Phospholipids/analysis , RNA, Ribosomal, 16S/genetics , Republic of Korea , Sequence Analysis, DNA
18.
J Microbiol ; 49(6): 1027-32, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22203569

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.


Subject(s)
Bacillus cereus/isolation & purification , Geologic Sediments/microbiology , Bacillus cereus/classification , Bacillus cereus/genetics , Bacillus cereus/metabolism , Base Composition , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Seawater
19.
J Microbiol Biotechnol ; 20(6): 995-1000, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20622498

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).


Subject(s)
Aldehyde-Lyases/chemistry , Bacterial Proteins/chemistry , Gene Expression , Paenibacillus/enzymology , Aldehyde-Lyases/genetics , Aldehyde-Lyases/isolation & purification , Aldehyde-Lyases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cloning, Molecular , Enzyme Stability , Kinetics , Molecular Sequence Data , Paenibacillus/classification , Paenibacillus/genetics , Paenibacillus/isolation & purification , Phylogeny , Sequence Homology, Amino Acid , Soil Microbiology
20.
Mol Cancer ; 9: 97, 2010 Apr 30.
Article in English | MEDLINE | ID: mdl-20433747

ABSTRACT

BACKGROUND: Myc is a well known driver of lymphomagenesis, and Myc-activating chromosomal translocation is the recognized hallmark of Burkitt lymphoma, an aggressive form of non-Hodgkin's lymphoma. We developed a model that mimics this translocation event by inserting a mouse Myc cDNA gene into the immunoglobulin heavy chain locus, just upstream of the intronic Emu enhancer. These mice, designated iMyc E mu, readily develop B-cell lymphoma. To study the mechanism of Myc-induced lymphoma, we analyzed signaling pathways in lymphoblastic B-cell lymphomas (LBLs) from iMyc E mu mice, and an LBL-derived cell line, iMyc E mu-1. RESULTS: Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) were constitutively activated in iMyc E mu mice, not only in LBLs but also in the splenic B-lymphocytes of young animals months before tumors developed. Moreover, inhibition of either transcription factor in iMyc E mu-1 cells suppressed growth and caused apoptosis, and the abrogation of NF-kappaB activity reduced DNA binding by both STAT3 and Myc, as well as Myc expression. Inhibition of STAT3 signaling eliminated the activity of both NF-kappaB and Myc, and resulted in a corresponding decrease in the level of Myc. Thus, in iMyc E mu-1 cells NF-kappaB and STAT3 are co-dependent and can both regulate Myc. Consistent with this, NF-kappaB and phosphorylated STAT3 were physically associated with one another. In addition, LBLs and iMyc E mu-1 cells also showed constitutive AKT phosphorylation. Blocking AKT activation by inhibiting PI3K reduced iMyc E mu-1 cell proliferation and caused apoptosis, via downregulation of NF-kappaB and STAT3 activity and a reduction of Myc levels. Co-treatment with NF-kappaB, STAT3 or/and PI3K inhibitors led to additive inhibition of iMyc E mu-1 cell proliferation, suggesting that these signaling pathways converge. CONCLUSIONS: Our findings support the notion that constitutive activation of NF-kappaB and STAT3 depends on upstream signaling through PI3K, and that this activation is important for cell survival and proliferation, as well as for maintaining the level of Myc. Together, these data implicate crosstalk among NF-kappaB, STAT3 and PI3K in the development of iMyc E mu B-cell lymphomas.


Subject(s)
Lymphoma, B-Cell/metabolism , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptor Cross-Talk/physiology , STAT3 Transcription Factor/metabolism , Signal Transduction/genetics , Animals , Apoptosis/physiology , Blotting, Western , Cell Line, Tumor , DNA Fragmentation , Disease Models, Animal , Electrophoretic Mobility Shift Assay , Enhancer Elements, Genetic , Enzyme Activation/physiology , Enzyme-Linked Immunosorbent Assay , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Immunoglobulin Heavy Chain , Genes, myc , Immunoprecipitation , Lymphoma, B-Cell/genetics , Mice , NF-kappa B/genetics , Phosphatidylinositol 3-Kinases/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/genetics
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