• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1536-1542
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• 2020

Dekkera anomala YAE-1 strain separated from "airag" (Mongolian fermented mare's milk) produces β-glucosidase, which can convert ginsenoside Rb₁ from Panax ginseng. Ginseng- derived bioactive components such as ginsenoside Rb₁ have various immunological and anticancer activities. Airag was collected from five different mare milk farms located near Ulaanbaatar, Mongolia. YAE-1 strains were isolated from airag to examine the hydrolytic activities of β-glucosidase on Korean Panax ginseng using an API ZYM kit. Supernatants of selected cultures having β-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb₁ at 40℃, pH 5.0. The YAE-1 strain was found to be nearly identical at 99.9% homology with Dekkera anomala DB-7B, and was thus named Dekkera anomala YAE-1. This strain exerted higher β-glucosidase activity than other enzymes. Reaction mixtures from Dekkera anomala YAE-1 showed great capacity for converting ginsenoside Rb₁ to ginsenoside Rd. The β-glucosidase produced by Dekkera anomala YAE-1 was able to hydrolyze ginsenoside Rb₁ and convert it to Rd during fermentation of the ginseng. The amount of ginsenoside Rd was highly increased from 0 to 1.404 mg/ml in fermented 20% ginseng root at 7 days.

• Journal of Ginseng Research
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• v.44 no.6
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• pp.784-789
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• 2020

Background: The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb₃ from ginseng extracts is limited by the co-existence of its isomer Rb₂. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb₃ from a mixture of isomers. Methods: To isolate Rb₃ from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb₂ into ginsenoside Rd. Ginsenoside Rb₃ was then efficiently separated from the mixture using a traditional chromatographic method. Results: Chromatographic purification of Rb₃ was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb₃ can be used in further pharmaceutical studies. Conclusions: Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb₃. This method can also be applied to purify other isomeric glycoconjugates in mixtures.

• Korean Journal of Materials Research
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• v.20 no.9
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• pp.451-456
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• 2010

A promising candidate material for a $H_2$ permeable membrane is SiC due to its many unique properties. A hydrogen-selective SiC membrane was successfully fabricated on the outer surface of an intermediate multilayer $\gamma-Al_2O_3$ with a graded structure. The $\gamma-Al_2O_3$ multilayer was formed on top of a macroporous $\alpha-Al_2O_3$ support by consecutively dipping into a set of successive solutions containing boehmite sols of different particle sizes and then calcining. The boehmite sols were prepared from an aluminum isopropoxide precursor and heated to $80^{\circ}C$ with high speed stirring for 24 hrs to hydrolyze the precursor. Then the solutions were refluxed at $92^{\circ}C$ for 20 hrs to form a boehmite precipitate. The particle size of the boehmite sols was controlled according to various experimental parameters, such as acid types and acid concentrations. The topmost SiC layer was formed on top of the intermediate $\gamma-Al_2O_3$ by pyrolysis of a SiC precursor, polycarbosilane, in an Ar atmosphere. The resulting amorphous SiC-on-$Al_2O_3$ composite membrane pyrolyzed at $900^{\circ}C$ possessed a high $H_2$ permeability of $3.61\times10^{-7}$ $mol{\cdot}m^{-2}{\cdot}s^{-1}{\cdot}Pa^{-1}$ and the $H_2/CO_2$ selectivity was much higher than the theoretical value of 4.69 in all permeation temperature ranges. Gas permeabilities through a SiC membrane are affected by Knudsen diffusion and a surface diffusion mechanism, which are based on the molecular weight of gas species and movement of adsorbed gas molecules on the surface of the pores.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.141-146
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• 2012

A gene encoding the xylanase (XynA) predicted from partial genomic sequence of Paenibacillus woosongensis was cloned into Escherichia coli by PCR. This xynA gene consisted of 633 nucleotides, encoding a polypeptide of 211 amino acid residues. The deduced amino acid sequence exhibited 85-89% identity with those of several Paenibacillus xylanases, belonging to the glycosyl hydrolase family 11. As a results of expression of the structural gene by T7 promoter of a pET23a(+) expression vector, xylanase activity was higher in cell-free extract than culture filtrate of a recombinant Escherichia coli BL21(DE3) CodonPlus. However, the expression level of xylanase was not sufficient be detected by SDS-PAGE. The cell-free extract showed maximal xylanase activity at $60^{\circ}C$ and pH 5.5. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylbiose.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.307-311
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• 2005

Marine microorganisms to produce functional biopolymers were isolated from the seashore of the Kyungsang province. Microorganisms to hydrolyze carboxy-methyl cellulose(CMC) were cultured in marin broth and the other liquid medium that contained 2.0% (w/v) glucose, 0.25% yeast extract, 0.5% $K_2HPO_4$, 1% NaCl, 0.02% $MgSO_4{\cdot}7H_2O$ and 0.06% $(NH_4)_2SO_4$ to investigate the ability to produce carboxymethyl cellululase (CMCase) under aerobic conditions. Twelve microorganisms among them showed higher activities of CMCase than B. amyloliquefaciens DL-3, which was known as a cellulase-producing strain. The microorganism showing highest activity of CMCase in this study was identified as Bacillus subtilis subsp. subtilis with 16S rDNA partial sequencing and gyrase A partial sequencing and named as B. subtilis subsp. subtilis A-53.

• Biomedical Science Letters
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• v.14 no.3
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• pp.179-186
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• 2008

Matrix metalloproteinases (MMPs), also designated matrixins, hydrolyze components of the extracellular matrix. These proteinases playa central role in many biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration. In previous data, disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, cyclooxygenase (COX)-2 expression, and prostaglandin $E_2$ production in chondrocytes cultured on plastic or during cartilage explants culture. In this study, we investigated the effects of the actin cytoskeleton architecture on MMP-2 expression and dedifferentiation by CD in rabbit articular chondrocytes. Rabbit articular chondrocytes were prepared from cartilage slices of 2-weeks-old New Zealand white rabbits by enzymatic digestion. CD was used as a disruptor of actin cytoskeleton. In this experiments measuring CD dose response, primary chondrocytes were treated with various concentrations of CD for 24h. The actin disruption was determined by immunostaining. MMP-2 expression levels were determined by immunoblot analysis and Reverse transcriptase-Polymerase chain reaction (RT-PCR) and MMP-2 activity was determined by gelatin zymography. We found that cell morphological change and up-regulation of MMP-2 expression by CD as determined via immunostaining, gelatin zymography and immunoblotting. Moreover, CD induced MMP-2 transcription was detected by RT-PCR. Also, CD-induced type II collagen expression was inhibited by MMP-2 inhibitor I treatment. Our results indicate that CD up-regulated MMP-2 activation causes dedifferentiation of articular chondrocyte.

• Molecules and Cells
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• v.40 no.9
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• pp.643-654
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• 2017

Autophagy is a degradation pathway in eukaryotic cells in which aging proteins and organelles are sequestered into double-membrane vesicles, termed autophagosomes, which fuse with vacuoles to hydrolyze cargo. The key step in autophagy is the formation of autophagosomes, which requires different kinds of vesicles, including COPII vesicles and Atg9-containing vesicles, to transport lipid double-membranes to the phagophore assembly site (PAS). In yeast, the cis-Golgi localized t-SNARE protein Sed5 plays a role in endoplasmic reticulum (ER)-Golgi and intra-Golgi vesicular transport. We report that during autophagy, sed5-1 mutant cells could not properly transport Atg8 to the PAS, resulting in multiple Atg8 dots being dispersed into the cytoplasm. Some dots were trapped in the Golgi apparatus. Sed5 regulates the anterograde trafficking of Atg9-containing vesicles to the PAS by participating in the localization of Atg23 and Atg27 to the Golgi apparatus. Furthermore, we found that overexpression of SFT1 or SFT2 (suppressor of sed5 ts) rescued the autophagy defects in sed5-1 mutant cells. Our data suggest that Sed5 plays a novel role in autophagy, by regulating the formation of Atg9-containing vesicles in the Golgi apparatus, and the genetic interaction between Sft1/2 and Sed5 is essential for autophagy.

• Archives of Pharmacal Research
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• v.26 no.4
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• pp.258-263
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• 2003

Colon-specific delivery of glucocorticoids is highly desirable for the efficient treatment of inflammatory bowel disease. We synthesized prednisolone 21-sulfate sodium (PDS) as a colon-specific prodrug of prednisolone (PD) and investigated its properties using rats as test animals. We expected that introduction of sulfate ester as a sodium salt might increase the hydrophilicity and restrict the absorption in the GI tract. If PDS is stable and nonabsorbable in the upper intestine, it will be delivered to the colon as an intact form, where it hydrolyze by the sulfatase to release PD. Compared with PD, the solubility of PDS increased and the apparent partition coefficient decreased greatly. PDS was stable on incubation with pH 1.2 and 6.8 buffer solutions and with the contents of the stomach and small intestine. On incubation with the cecal contents, PDS decreased to 9.6％ of the dose in 10 h producing PD. The amount of PD increased to give a maximum 54％ of the dose and decreased. As a control, when PD was incubated with the cecal contents, it decreased to 29％ of the dose in 8 h, which implied that reduction of PD proceeded under such conditions. These results suggested that hydrolysis of PDS took place to produce and accumulate PD, which decreased by reduction as the incubation period extended. Our results suggested that PDS can be a promising colon-specific prodrug of PD, and sulfate ester group might serve as a potential colon-specific promoiety, especially for the drugs which are resistant to reduction in the colon.

• Proceedings of the Korean Society of Crop Science Conference
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• 2007.04a
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• pp.65-73
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• 2007

The objective of this study is to understand how regulatory mechanisms respond to sugar status for more efficient carbon utilization and source-sink regulation in plants. So, we need to identify and characterize many components of sugar-response pathways for a better understanding of sugar responses. For this end, genes responding change of sugar status were screened using Arabidpsis cDNA arrays, and confirmed thirty-six genes to be regulated by sucrose supply in detached leaves by RNA blot analysis. Eleven of them encoding proteins for amino acid metabolism and carbohydrate metabolism were repressed by sugars. The remaining genes induced by sugar supply were for protein synthesis including ribosomal proteins and elongation factors. Among them, I focused on three hydrolase genes encoding putative $\beta$-galactosidase, $\beta$-xylosidase, and $\beta$-glucosidase that were transcriptionally induced in sugar starvation. Homology search indicated that these enzymes were involved in hydrolysis of cell wall polysaccharides. In addition to my results, recent transcriptome analysis suggested multiple genes for cell wall degradation were induced by sugar starvation. Thus, I hypothesized that enzyme for cell wall degradation were synthesized and secreted to hydrolyze cell wall polysaccharides producing carbon source under sugar-starved conditions. In fact, the enzymatic activities of these three enzymes increased in culture medium of Arabidopsis suspension cells under sugar starvation. The $\beta$-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved condition with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. Further, contents of cell wall polysaccharides especially pectin and hemicellulose were markedly decreased associating with sugar starvation in detached leaves. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These results supported my idea that cell wall has one of function to supply carbon source in addition to determination of cell shape and physical support of plant bodies.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.501-510
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• 2007

The present study was conducted to establish primary bovine muscle satellite cell (MSC) culture conditions and to investigate the effects of various steroid hormones on transcription of the genes involved in muscle cell proliferation and differentiation. Of three different types of proteases (type II collagenase, pronase and trypsin-EDTA) used to hydrolyze the myogenic satellite cells from muscle tissues, trypsin-EDTA treatment yielded the highest number of cells. The cells separated by hydrolysis with type II collagenase and incubated on gelatin-coated plates showed an enhanced cell attachment onto the culture plate and cell proliferation at an initial stage of cell growth. In this study, the bovine MSCs were maintained in vitro up to passage 16 without revealing any significant morphological change, and even to when the cells died at passage 21 with decreased or almost no cell growth or deformities. When the cells were incubated in a steroid-depleted environment (DMEM(-)/10% CDFBS (charcoal-dextran stripped FBS)), they grew slowly initially, and were widened and deformed. In addition, when the cells were transferred to an incubation medium containing steroid (DMEM(+)/10% FBS), the deformed cells resumed their growth and returned to a normal morphology, suggesting that steroid hormones are crucial in maintaining normal MSC morphology and growth. The results demonstrated that treatments with 19-nortestosterone and testosterone significantly increased AR gene expression (p<0.05), implying that both testosterone and 19-nortestosterone bind with AR and that the hormone bound-AR complex up-regulates the genes of its own receptor (AR) plus other genes involved in satellite cell growth and differentiation in bovine muscle.