• Title/Summary/Keyword: npgA

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Modulation of the Regioselectivity of a Thermotoga neapolitana $\beta$-Glucosidase by Site-Directed Mutagenesis

  • Choi, Ki-Won;Park, Kyung-Min;Jun, So-Young;Park, Cheon-Seok;Park, Kwan-Hwa;Cha, Jae-Ho
    • Journal of Microbiology and Biotechnology
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    • v.18 no.5
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    • pp.901-907
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    • 2008
  • Thermotoga neapolitana $\beta$-glucosidase (BglA) was subjected to site-directed mutagenesis in an effort to increase its ability to synthesize arbutin derivatives by transglycosylation. The transglycosylation reaction of the wild-type enzyme displays major ${\beta}(1,6)$ and minor ${\beta}(1,3)$ or ${\beta}(1,4)$ regioselectivity. The three mutants, N291T, F412S, and N291T/F412S, increased the ratio of transglycosylation/hydrolysis compared with the wild-type enzyme when pNPG and arbutin were used as a substrate and an acceptor, respectively. N291T and N219T/F412S had transglycosylation/hydrolysis ratios about 3- and 8-fold higher, respectively, than that of the wild-type enzyme. This is due to the decreased hydrolytic activity of the mutant rather than increased transglycosylation activity. Interestingly, N291T showed altered regioselectivity, as well as increased transglycosylation products. TLC analysis of the transglycosylation products indicated that N291T retained its ${\beta}(1,3)$ regioselectivity, but lost its ${\beta}(1,4)$ and ${\beta}(1,6)$ regioselectivity. The altered regioselectivity of N291T using two other acceptors, esculin and salicin, was also confirmed by TLC. The major transglycosylation products of the wild type and N291T mutant were clearly different. This result suggests that Asn-291 is highly involved in the catalytic mechanism by controlling the transglycosylation reaction.

Synthesis of Poly(Styrene-co-GMA) and its Application as in situ Reactive Compatabilizer (Poly(Styrene-co-GMA)의 합성과 in situ Reactive Compatabilizer 로서의 응용)

  • Kim, Ju-Young;Suh, Kyung-Do
    • Applied Chemistry for Engineering
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    • v.3 no.3
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    • pp.499-506
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    • 1992
  • Copolymer of Styrene and GMA(glycidyl methachylate), having reactive ratios of $r_1=0.53$, $r_2=0.44$, was synthesized in dioxane using AIBN as free radical initiator. Followed by the reaction of ethylene diamine with copolymer PGS, amine groups were introduced to the PGS(NPGS). The composition of copolymer was determined by elemental analyzer. Poly(glycidyl methacrylate) (PGMA) was obtained in benzene using AIBN as free radical initiator. The NPGS-PGMA blend of 50/50 composition was prepared by mixing these polymers in THF at $65^{\circ}C$. Glass transition temperature (Tg) of NPGS-PGMA blend was measured by DSC. The blend showed a single Tg. Accordingly, it was clear that the NPGS was compatible with PGMA. An intermolecular reaction between amine groups of NPGS and epoxy groups of PGMA imparts compatibility in the NPGS-PGMA blend. When the NPGS-PGMA blend was added to the incompatible PS-PGMA blend, PS-PGMA blend showed Tg change. Scanning Electron Micrograph(SEM) showed a fine morphology in this blend. Consequently, it was apparent that the NPGS-PGMA blend acted as a compatibilizer for the PS-PGMA blend.

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Effects of Traditional Recipes and Saenghwatang on Postpartume Care (전통 산후 회복식과 한방 생화탕이 산모의 회복 정도에 미치는 효과)

  • Park, Sung-Hye
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.34 no.5
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    • pp.652-658
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    • 2005
  • This experimental was carried out to evaluate the clinical effects of Ophicephalus argus and Crubita moschate which have been traditionally applied to postpartum care in Korea, and compare them with the effect of Saenghwtang. Fifty Sprague-Dawley female rats weighing $250\~280$ g were divided into five groups: a normal saline-treated group (NSG), a Saenghuatang-treated group (STG), an Ophicephlus argus-treated group (OTG), and a Crubita moschate-treated group (CTG), also non-pregnant group (NPG). Except for the NPG, each extract was administered for one week to each group after delivery. We measured the WBC, RBC, serum levels of hemoglobin and hematocrit, the platelet count, serum levels of fibrinogen, albumin, thyroxine and urine levels of sodium and potassium. STG showed a significant (p<0.05) decrease of WBC count, fibrinogen content and urine levels of sodium and potassium and a significant (p<0.05) increase of RBC, hemoglobin, albumin and thyroxine in comparison with those of the NSG. OTG showed a significant (p<0.05) decrease of WBC count, fibrinogen and the significant (p<0.05) increase of albumin and thyroxine in comparison with those of the NSG. CTG showed a significant (p<0.05) increase of albumin and thyroxine in comparison with that of NSG. These results suggest that Saenghwatang is more effective than Ophicephalus argus and Crubita moschate for postpartum recuperation although they also have some effects on recuperation of deteriorative blood components after delivery. Therefore, these findings indicate that futher investigation for the other effects of Ophicephalus argus and Crubita moschate is necessary.

Effects of Radix Sophora Flavescentis Extract on Insulin Secretion in RIN-m5F Cells and $\alpha$-glucosidase Inhibition (고삼(苦蔘)이 RIN-m5F세포의 인슐린 분비와 $\alpha$-glucosidase 활성 억제에 미치는 영향)

  • An, So-Hyun;Cho, Chung-Sik;Kim, Cheol-Jung
    • The Journal of Internal Korean Medicine
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    • v.30 no.3
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    • pp.481-494
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    • 2009
  • Background : Radix Sophora Flavescentis (SF) is used for the treatment of diabetes mellitus in Traditional Korean Medicine. However, little is known about the effects of Radix Sophora Flavescentis extract (SFE) on the hypoglycemic mechanism. Objective : We performed a series of experiments to verify the effects of SFE on the proliferation of RIN-m5F, the secretion and synthetic processes of insulin with glucose stimulation and inhibition of $\alpha$-glucosidase. Methods : Various amounts of SFE were added to the RIN-m5F cell culture to identify the effects on the cell proliferation, total amounts of insulin secretion, and related gene expression at the molecular level. Also to identify the inhibitory effect on the $\alpha$-glucosidase activities, ${\rho}NPG$ assay was done with various SFE concentrations followed by comparison with control. Results : SFE did not show considerable effects on RIN-m5F cells proliferation, insulin secretion or insulin mRNA expression, whichever phenomena did not depend on the glucose concentration. However, SFE significantly inhibited $\alpha$-glucosidase activity in a dose dependent manner compared to control. Conclusions : This study showed that SFE has potent $\alpha$-glucosidase inhibitory activity. Thus, SF may by used for the improvement of overall glycemic control. Further mechanism studies on the lipid toxicity and oxidation stress of SF seem to be necessary.

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Comparison of on Rat Intestinal Digestive Enzyme Inhibitory Activity and Antioxidant Enzyme Activity of Korean and Chinese Schizandra chinensis

  • Chae Hee-Jun;Hwang Hyun-Ik;Lee In-Soon;Moon Hae-Yeon
    • Biomedical Science Letters
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    • v.11 no.4
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    • pp.517-523
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    • 2005
  • The purpose of this study was to determine the effect of rat intestinal a-glucosidase inhibitor; methanol $(80\%)$, ethanol $(80\%)$ and water extract of Schizandra chinensis in Korea (KS: Schizandra chinensis in Korea) and China (CS: Schizandra chinensis in China). When the final concentration was 1 mg/ml for each sample (KS and CS), methanol extract of KS ($IC_{50}$ 1.62 mg/ml) showed $46.8\%$, ethanol extract of KS ($IC_{50}$ 1.48 mg/ml) showed $47.4\%$, water extract of KS ($IC_{50}$ 1.72 mg/ml) showed $46.3\%$ and methanol extract of CS ($IC_{50}$ 8.35 mg/ml) showed $13.3\%$, ethanol extract of CS ($IC_{50}$ 8.05 mg/lml) showed $16\%$, water extract of CS ($IC_{50}$ 8.37 mg/ml) showed $11.54\%$ of inhibitor for p-nitrophenyl $\alpha-D-glucopyranoside$ (pNPG) $\alpha-glcosidase$ activity, respectively. And the contents of total phenol, flavonoid of Schizandra chinensis were measured. When the final concentration was 1mg/ml for each sample (KS and CS), total phenol and flavonoid in KS were higher than CS, respectively. The order superoxide dismutase (SOD) activity $IC_{50}$ values of each solvent extracts of KS were: 2.006 mg/ml methanol extract, 2.304 mg/ml ethanol extract and 2.5 mg/ml water extract, which were higher than that of each solvent extracts CS as: 2.881 mg/ml methanol extract, 3.085 mg/ml ethanol extract and 3.190 mg/ml water extract.

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Phylogenetic Analysis of Oligotrophic Bacteria Found in Potable Groundwater (음용 지하수중에 분포하는 저영양세균의 계통학적 해석)

  • ;Tomoyoshi Hashimoto
    • Korean Journal of Microbiology
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    • v.38 no.4
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    • pp.293-298
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    • 2002
  • In order to investigate the ecological aspect of bacteria on groundwater, water samples were collected from various regions. Total of 318 strains were isolated from diluted nutrient broth (DNB) agar medium, and investigated their growth pattern on nutrient broth (NB) medium. As a result, all the isolated strains were divided into two groups, NB and DNB organisms. Growth of DNB organisms were suppressed in full strength NB medium but not in DNB medium, which were called oligotrophic bacteria in this study. Proportion of DNB organisms occurred in the frequency of 50-98% in potable groundwaters (CW, CJ, DPG, CJG1), however, it was 23,46% in polluted site (TJ, NPG1). One hundred and two strains were identified as oligotrophic bacteria and their phylogenetic characteristics were determined by using 16S rDNA sequencing. Based on the phylogenetic analysis, they were found to fall into three major phylogenetic groups: belonging to the Proteobacteria $\alpha$-(49 strains), $\beta$-(50 strains), $\gamma$ -(3 strains) subdivisions. The phylogenetic analysis suggested that microbial diversity of potable groundwater is more complex than that obtained in the past investigation.

Discrimination of Echinochloa colona (L.) Link from other Echinochloa Species using DNA Barcode (국내에 유입되는 열대피(Echinochloa colona) 동정: DNA 바코드 중심)

  • Lee, Jeongran;Kim, Chang-Seok;Lee, In-Yong
    • Weed & Turfgrass Science
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    • v.4 no.3
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    • pp.225-229
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    • 2015
  • Echinochloa colona is one of the most problematic weeds in the paddy fields of the world. In recent years, this species is likely to be introduced in Korea due to global warming, the expansion of international trade including agricultural products, and increasing tourists. We tried to identify the species from Korean Echinochloa crus-galli and E. oryzicola in order to establish the control measures in case of the initial influx. For this study, Echinochloa colona collected from the National Plant Germplasm System, USA were examined and E. crus-galli and E. oryzicola were collected in Korea. It is, however, very difficult to identify for Echinochloa species using morphological characters because of numerous interspecific and intraspecific types found in nature. Thus, we barcoded the species using rbcL, matK, and ITS. All three markers identified E. colona very well from the others. ITS alone may be enough as a DNA barcode for E. colona identification, when considering cost and effectiveness. The barcode sequences were deposited to the National Center for Biotechnology Information database for public use.

Molecular Cloning and Expression of a Novel Protease-resistant GH-36 $\alpha$-Galactosidase from Rhizopus sp. F78 ACCC 30795

  • Yanan, Cao;Wang, Yaru;Luo, Huiying;Shi, Pengjun;Meng, Kun;Zhou, Zhigang;Zhang, Zhifang;Yao, Bin
    • Journal of Microbiology and Biotechnology
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    • v.19 no.11
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    • pp.1295-1300
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    • 2009
  • A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.

Partial purification and Properties of Alkaline Cellulase from Pseudomonas sp. AC-711 (Pseudomonas sp. AC-711이 생산하는 알칼리성 Cellulase의 부분정제 및 효소적 성질)

  • Yoon, Min-Ho;Lim, Sang-Ho;Choi, Woo-Young
    • Korean Journal of Agricultural Science
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    • v.25 no.1
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    • pp.131-137
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    • 1998
  • The cellulase components were partially purified from the culture filtrate of the alkalophilic bacterium Pseudomonas sp. AC-711 and its enzymatic properties were characterized. The specific activity of the purified major enzyme component was 3.5 units/mg protein as carboxymethyl cellulase and the yield was 23% of the total activity of the culture broth. The molecular weight of the component was 46,000 and the Km and Vmax on CMC were determined as $15.4mg\;mL^{-1}$ and $4.17{\mu}moles\;mL^{-1}\;min^{-1}$, respectively. The enzyme was stable at the temperatures below $60^{\circ}C$ and at the pH range of 4.0~11.0, and the optimal temperature and pH were $60^{\circ}C$ and pH 8.0, respectively. The enzyme activity was not significantly affected by the common surfactants (concentration: 0.05%) such as ${\alpha}$-olefin sulfonate, linear alkylbenzene sulfonate, sodium dodecyl sulfonate, hexadecyltrimethylammonium bromide and Tween 80. The enzyme was activated by the metal ions such as $Ca^{2+}$, $Cu^{2+}$, $Co^{2+}$, whereas inhibited by $Hg^{2+}$ and $Zn^{2+}$. The enzyme exhibited relatively high activity toward amorphous CMC as compared with crystalline substrates such as filter paper and avicel.

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