• Title/Summary/Keyword: MgO substrate

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AT9283, 1-Cyclopropyl-3-(3-(5-(Morpholinomethyl)-1H-Benzo[d] Imidazole-2-yl)-1H-Pyrazol-4-yl) Urea, Inhibits Syk to Suppress Mast Cell-Mediated Allergic Response

  • Kim, Su Jeong;Choi, Min Yeong;Min, Keun Young;Jo, Min Geun;Kim, Jie Min;Kim, Hyung Sik;Kim, Young Mi
    • Biomolecules & Therapeutics
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    • v.30 no.6
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    • pp.520-528
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    • 2022
  • Mast cells are an effector cell that plays a pivotal role in type I hypersensitive immune responses. Mast cells exist in connective tissues, such as skin and mucosal tissue, and contain granules which contain bioactive substances such as histamine and heparin in cells. The granules of mast cells are secreted by antigen stimulation to cause the type I allergic hypersensitivity. In addition, stimulated by antigen, mast cells synthesize and secrete various eicosanoids and cytokines. While AT9283 is known to have anticancer effects, the therapeutic effect of AT9283 on allergic disorders is completely unknown. In this study, it was found that AT9283 reversibly inhibited antigen-IgE binding-induced degranulation in mast cells (IC50, approx. 0.58 μM) and suppressed the secretion of the inflammatory cytokines IL-4 (IC50, approx. 0.09 μM) and TNF-α (IC50, approx. 0.19 μM). For a mechanism of mast cell inhibition, while not inhibiting Syk phosphorylation, AT9283 suppressed the activation of LAT, a downstream substrate protein of Syk, in a dose-dependent manner. As expected, AT9283 also inhibited the activation of PLCγ1 and Akt, downstream signaling molecules of Syk/LAT, and MAP kinases such as JNK, Erk1/2, and P38. In an in vitro protein tyrosine kinase assay, AT9283 directly inhibited Syk activity. Next, AT9283 dose-dependently inhibited passive cutaneous anaphylaxis (PCA), an IgE-mediated allergic acute response, in mice (ED50, approx. 34 mg/kg, p.o.). These findings suggest that AT9283 has potential to use as a new drug for alleviating the symptoms of IgE-mediated allergic disorders.

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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Isolation and Characteristics of a Phenol-degrading Bacterium, Rhodococcus pyridinovorans P21 (페놀분해세균 Rhodococcus pyridinovorans P21의 분리 및 페놀분해 특성)

  • Cho, Kwang-Sik;Lee, Sang-Mee;Shin, Myung-Jae;Park, Soo-Yun;Lee, Ye-Ram;Jang, Eun-Young;Son, Hong-Joo
    • Journal of Life Science
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    • v.24 no.9
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    • pp.988-994
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    • 2014
  • The effluents of chemical and petroleum industries often contain non-biodegradable aromatic compounds, with phenol being one of the major organic pollutants present among a wide variety of highly toxic organic chemicals. Phenol is toxic upon ingestion, contact, or inhalation, and it is lethal to fish even at concentrations as low as 0.005 ppm. Phenol biodegradation has been studied in detail using bacterial strains. However, these microorganisms suffer from substrate inhibition at high concentrations of phenol, whereby growth is inhibited. A phenol-degrading bacterium, P21, was isolated from oil-contaminated soil. The phenotypic characteristics and a phylogenetic analysis indicated the close relationship of strain P21 to Rhodococcus pyridinovorans. Phenol biodegradation by strain P21 was studied under shaking condition. The optimal conditions for phenol biodegradation by strain P21 were 0.09% $KNO_3$, 0.1% $K_2HPO_4$, 0.3% $NaH_2PO_4$, 0.015% $MgSO_4{\cdot}7H_2O$, 0.001% $FeSO_4{\cdot}7H_2O$, initial pH 9, and $20-30^{\circ}C$, respectively. When 1,000 ppm of phenol was added to the optimal medium, the strain P21 completely degraded it within two days. Rhodococcus pyridinovorans P21 could grow in up to 1,500 ppm of phenol as the sole carbon source in a batch culture, but it could not grow in a medium containing above 2,000 ppm. Moreover, strain P21 could utilize toxic compounds, such as toluene, xylene, and hexane, as a sole carbon source. However, no growth was detected on chloroform.

Growth and Nutrient Contents of Tomato Plug Seedlings as Influenced by Pre-planting Fertilizer Levels and Initiation Time of Fertigation (토마토 플러그육묘시 기비수준 및 관비 시작일이 묘 생육과 무기성분 함량에 미치는 영향)

  • Choi, Jong-Myung;Kim, Byoung-Gon
    • Journal of Bio-Environment Control
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    • v.16 no.4
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    • pp.379-387
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    • 2007
  • This research was conducted to evaluate the influence of pre-planting fertilizer levels (PFL) and initiation time of fertigation (ITF) on growth and nutrient contents of tomato plug seedlings. The pre-planting fertilizer levels in a coir+peatmoss+perlite (4:4:2, v/v/v) substrate were adjusted to 0.5X, 1.0X and 1.0X, and initiation time of fertigation was set to 7, 14,21 and 28 days after sowing. Elevated PFL in same ITF increased plant growth such as fresh and dry weights at 35 and 70 days after sowing. Plugs with early feeding among treatments of equal amount of PFL also showed better growth as compared to those of later feeding. In each ITF, 0.5X treatment had the higher tissue $P_2O_5$ contents than 1.0X and 1.5X treatments. Elevated PFL resulted in the decrease of tissue K, Mg and Fe contents and increase of tissue Ca contents. The pH in soil solution of all root substrates except 0.5X treatment at 35 and 70 days after sowing were greater than 7.0, which is too high. This suggests that the amounts and kinds of Ca containing fertilizers should be altered to decrease the pH. The results of this research indicated that fertilizer levels should be increased to 1.5X except Ca fertilizer, and fertigation immediately after moving plug trays from germination room to greenhouse is required to increase crop growth and decrease cropping time.

Kinetic Studies of Lactic Acid Fermentation (Part 3) Effect of Phenol Derivatives on Fermentation (유산균발효에 관한 동력학적 연구 (제3보) 발효에 미치는 Phenol 유도체의 영향)

  • LEE Keun-Tai;YANG Hyeun-Suk
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.14 no.4
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    • pp.212-216
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    • 1981
  • The growth of Lactobacillus bulgaricus treated with vanillin, ortho-vanillin and guaiaco1 was studied on synthetic medium in mechanically agitated chemostat culture, The exponential-phase growth rate exhibited a maximum at the cells treated with 50 ppm vanillin. That stimulation, however, appears to be an effect on growth rate rather than total cell growth. And the others were inhibited by the chemicals. Much greater inhibition in growth of the cells treated with 100 ppm of each chemical than oars treated with 50 ppm was observed after 25 hour fomentation. For aerobic microbes, the alcohol dehydrogenase reaction is enhanced for the reproduction of NAD, which consequently cause to stimulate fermentation. For micro-aerophilic microbes , however, the same effect was not observed at the present study at least in the case of cell concentration. However except f or one treated with 50 ppm vanillin the same effect was observed in the case of growth is to. From the result using the glucose as a substrate, it was found that the cell concentrations measured in terms of ultimate optical density (UOB/ml), were 0.96 and 0.92, when treated with 50 and 100 ppm vanillin; 0.40 and 0.45 when treated with ortho-vanillin 50 and 100 ppm: 0.49 and 0.47, when treated with guaiacol 50 and 100 ppm. The specific growth rates were 0.44, 0.15, 0.25, 0.29, 0.37, and 0.34; the specific production rates wire 0.33, 0.15, 0.16, 0.22, 0.28, and 0.26 and the glucose concentrations (g/1) after 25 hour fermentation were 23.5, 32.8, 31.5, 29.5, 28.0 and 28.8, these all in the same sequences as the first.

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Rapid Statistical Optimization of Cultural Conditions for Mass Production of Carboxymethylcellulase by a Newly Isolated Marine Bacterium, Bacillus velezensis A-68 from Rice Hulls (통계학적 방법을 사용한 해양미생물 Bacillus velezensis A-68균주의 섬유소 분해효소 생산 조건 최적화)

  • Kim, Bo-Kyung;Kim, Hye-Jin;Lee, Jin-Woo
    • Journal of Life Science
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    • v.23 no.6
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    • pp.757-769
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    • 2013
  • A microorganism producing carboxymethylcellulase (CMCase) was isolated from seawater, identified as Bacillus velezensis by analyses of 16S rDNA and partial sequences of the gyrA, and designated as B. velezensis A-68. The optimal conditions for production of CMCase by B. velezensis A-68 were established using response surface methodology (RSM). The optimal concentrations of rice hulls and yeast extract, and initial pH of the medium for cell growth were 60.2 g/l, 7.38 g/l, and 7.18, respectively, whereas those for production of CMCase were 50.0 g/l, 5.00 g/l, and 7.30. The analysis of variance (ANOVA) implied that the most significant factor for cell growth as well as production of CMCase was yeast extract. The optimal concentrations of $K_2HPO_4$, NaCl, $MgSO_4{\cdot}7H_2O$, and $(NH_4)_2SO_4$ in the medium for cell growth were 7.50, 1.00, 0.10, and 0.80 g/l, respectively, which were the same as those for production of CMCase. The optimal temperatures for cell growth and production of CMCase were 30 and $35^{\circ}C$, respectively. The maximal production of CMCase under optimized conditions was 83.8 U/ml, which was 3.3 times higher than that before optimization. In this study, rice hulls, agro-byproduct, were developed as a substrate for production of CMCase and time for production of CMCase was reduced to 3 days using a newly isolated marine bacterium.

Isolation, Purification and Some Properties of Polyphenol Oxidase from Pear (배과실(果實)의 Polyphenol Oxidase의 분리(分離) 정제(精製) 및 그 특성(特性))

  • Kang, Yoon Han;Sohn, Tae Hwa;Choi, Jong Uck
    • Current Research on Agriculture and Life Sciences
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    • v.4
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    • pp.55-64
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    • 1986
  • Polyphenol oxidase in japanese pear (Pyrus communis var. mansamkil) was isolated, partially purified and its some properties were investigated. Polyacrylamide disc gel electrophoresis indicated two bands with polyphenol oxidase activity in the extract from acetone dry powder of par flesh. These two polyphenol oxidases (PPO A and PPO B) were purified through acetone precipitation and diethylaminoethyl cellulose column chromatography. PPO A and B were purified 7.8 fold and 8.7 fold by the present procedure, respectively. The Rm values of partially purified PPO A and B were estimated to be 0.58 and 0.68, respectively. The optimum temp, and pH of PPO A activity were $33^{\circ}C$ and pH 7.0, while those of PPO B were $30^{\circ}C$ and pH 4.2, respectively. Two PPO were unstable over the temperature of $60^{\circ}C$. The substrate specificity of pear PPO showed high affinity toward o-diphenolic compounds, especially catechol in PPO A and chlorogenic acid in PPO B, but inactive toward m-diphenol, p-diphenol and monophenols. PPO A showed affinity toward the trihydroxyphenolic compound. $Zn^{{+}{+}}$ activated the PPO A activity but $Fe^{{+}{+}}$ inhibited PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity but $K^+$, $Mg^{{+}{+}}$, $Ca^{{+}{+}}$ and $Hg^{{+}{+}}$ inhibited at 10mM concentration. $Cu^{{+}{+}}$ activated the enzyme action at low concentrations but inhibited at high concentration. Inhibition studies indicated that L-ascorbic acid, L-cysteine and thiourea were most potent. The Km values of PPO A and PPO B for catechol were 20mM and 14.3mM, respectively.

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Optimization of Endoglucanase Production from Fomitopsis pinicola Mycelia (Fomitopsis pinicola 균사체로부터 Endoglucanase의 최적생산)

  • Gu, Ji-Min;Park, Sang-Shin
    • Microbiology and Biotechnology Letters
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    • v.41 no.2
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    • pp.145-152
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    • 2013
  • The culture conditions to maximize the production of endoglucanase (EC 3.2.1.4) from the brown rot fungus Fomitopsis pinicola MKACC 54347 mycelia were investigated. Among the tested media for endoglucanase production, Mandel's mineral salts medium (MSM; 1% cellulose, 0.1% peptone, 0.14% $(NH_4)_2SO_4$, 0.03% urea, 0.2% $KH_2PO_4$, 0.03% $MgSO_4{\cdot}7H_2O$, 0.03% $CaCl_2$, and 0.1% trace metal solution (19.8 mM $FeSO_4$, 13.0 mM $MnSO_4$, 12.2 mM $ZnSO_4$, and 15.4 mM $CoCl_2$)) produced the highest activity of the enzyme. To optimize the medium composition for enzyme activity, the effects of various carbon, nitrogen, phosphorus, and inorganic sources were investigated in MSM. Maximal enzyme production was accomplished using a medium containing 2% carboxymethyl cellulose (CMC), 2% yeast extract, 0.2% $KH_2PO_4$, 0.03% $MnSO_4$, and 0.3% trace metal solution. Different physiological conditions, like incubation period and temperature, were also examined to assess their influence on enzyme production. Enzyme production from F. pinicola reached its highest level after cultivation for 8 days at $25^{\circ}C$. Nondenaturing polyacrylamide gel electrophoresis (PAGE), followed by the endoglucanase activity staining using CMC as the substrate, was performed to identify the endoglucanase under the culture conditions studied. Zymogram analysis of the culture supernatant revealed an endoglucanase band with a molecular mass of 52 kDa. The optimum pH and temperature for enzyme activity were $55^{\circ}C$ and pH 5.0, respectively.

Characteristics of Adhesive Disks in Parthenocissus tricuspidata during Attachment (착생에 따른 담쟁이덩굴 흡착근의 부착 특성)

  • Lee, Myung-Hui;Kim, In-Sun
    • Applied Microscopy
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    • v.41 no.2
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    • pp.139-145
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    • 2011
  • Parthenocissus tricuspidata is an epiphyte that lacks a main axial stem, but develops adhesive disks along the stem for climbing support. In this study, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) were utilized to examine the brick wall surface and the adhesive disks of P. tricuspidata that attached to the surface successfully. The study was mainly focused the outermost layers of both structures before and after adhesion to find out whether there has been some structural and/or physical interactions between the two. The adhesive disks adhered firmly to the brick wall by secreting adhesive materials that help them for a tight attachment to the surface. The rough wall surface appeared facilitating better attachment of the adhesive disks by infiltrating the materials into those spaces leading to some degree of interactions at the interface. EDS analysis on the outermost layers of the adhesive disks that were separated from the substrates was also consistent with the SEM data on the interaction between the adhesive disks and the substrate surface. EDS analysis of the brick wall surface and the adhesive disks demonstrated similar elements of O, Si, Fe, Al, K, Mg, and Na in their components.

Inhibitory Substance Produced by Aspergillus sp. on the Snake Venom Proteinase - Isolation of Microorganism and Biological Activities of the Inhibitor - (Aspergillus 속 균주가 생성되는 사독 Proteinase에 대한 저해물질 - 균의 분리 및 저해물질의 생물학적 작용상 -)

  • Hyun, Nam-Joo;Seu, Jung-Hwn
    • Microbiology and Biotechnology Letters
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    • v.15 no.2
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    • pp.129-134
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    • 1987
  • Aspergillus sp. (MK-24) producing a biological active substance that inhibited the venom proteinase activity was isolated from soil. The substance also inhibited the activity of trypsin and coagulation of blood, but did not inhibit papain, $\alpha$-chymotrypsin and pepsin. The substance was partially purified from culture filtrate by precipitaion with acetone, and by chromatography of DEAE-Sepadex A-50 column and Amberlite IRC-50 ion exchange. The inhibitory substance was stable in the wide pH range from 2.0 to 12.0 at 37$^{\circ}C$, but not stable at $65^{\circ}C$ in the alkaline pH. Only 12% of the activity was decreased by the heat treatment at 10$0^{\circ}C$ for two hours. The inhibition on venom proteinase (Agkistrodon bromohoffi brevicaudus) was a mixed type. The inhibitory activity depended on the preincubation time and completely depressed by cupric, zinc and cobalt ions. The inhibition on the venom proteinase was appeared strongly on casein but not on ovalbumin or hemoglobin as a substrate.

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