• Title/Summary/Keyword: Enzyme Kinetics

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Studies on Screening and Iolation of ${\alpha}-Amylase$ Inhibitors of Soil Microorganisms( II ) -Isolation and Activities of the Inhibitor of Streptomyces Strain DMC-72- (토양균의 ${\alpha}-Amylase$ 저해제 검색 및 분리에 관한 연주(제2보) -스트렙토마이세스속 DMC-72 균주의 저해 성분의 분리 및 작용-)

  • Kim, Kyung-Jae;Lee, Shung-Hee;Kim, Jung-Woo;Kim, Ha-Won;Shim, Mi-Ja;Choi, Eung-Chil;Kim, Byong-Kak
    • The Korean Journal of Mycology
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    • v.13 no.4
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    • pp.203-212
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    • 1985
  • Of 450 strains isolated from the soil microbes collected in various locations in Korea, a strain had a strong inhibitory activity against bacterial ${\alpha}-amylase$ and was named strain DMC-72 of the genus Streptomyces. The amylase inhibitory metabolite produced by this strain was purified by means of acetone precipitation, adsorption on Amberlite IRC-50 and SP-Sephadex C-25. The inhibitor was found to be a derivative of oligosaccharides by spectral and chemical data. The inhibitor was stable at the pH range of $1{\sim}13$ and at $100^{\circ}C$ for half an hour, also inhibited other amylases such as salivary ${\alpha}-amylase$, pancreatic ${\alpha}-amylase$, fungal ${\alpha}-amylase$ and glucoamylase. However, it showed no inhibitory activity against ${\alpha}-glucosidase$, ${\beta}-glucosidase$, dextranase, and ${\beta}-amylase$. The kinetic studies of the inhibitor showed that its inhibitory effects on starch hydrolysis by ${\alpha}-amylase$ were noncompetitive.

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Effect of chloride ions on the catalytic properties of human pancreatic α-amylase isozyme produced in Pichia pastoris (Pichia pastoris에서 생산된 인체 췌장 α-아밀레이스 동질효소의 촉매활성에 대한 염소이온의 영향)

  • Kim, Min-Gyu;Kim, Young-Wan
    • Korean Journal of Food Science and Technology
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    • v.48 no.4
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    • pp.341-346
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    • 2016
  • The AMY2B gene, encoding human pancreatic ${\alpha}$-amylase isozyme (HPA II), was expressed in Pichia pastoris, and the effects of chloride ions on HPA II activity toward starch substrates were investigated. As seen with chloride ion-dependent ${\alpha}$-amylases-including HPA I, the isozyme of HPA II-chloride ions increased enzyme activity and shifted the optimal pH to an alkaline pH. The activity enhancement by chloride was more significant at pH 8 than that at pH 6, suggesting that the protonation state of the general acid/base catalyst of HPA II was important for the hydrolysis of starches at an alkaline pH because of the increase in its $pK_a$ by chloride ions. The turnover values for cereal starches as the substrates markedly increased in the presence of chloride by up to 7.2-fold, whereas that for soluble starch increased by only 1.7-fold. Chloride inhibited substrate hydrolysis at high substrate concentrations, with $K_i$ values ranging from 6 to 15 mg/mL.

In vitro Acetolactate Synthase Inhibition of LGC-40863 in Rice and Barnyardgrass (시규제초제 LGC-40863의 벼와 피에 대한 Acetolactate synthase 저해 활성)

  • Bae, Y.T.;Lee, J.H.;Koo, S.J.
    • Korean Journal of Weed Science
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    • v.17 no.1
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    • pp.66-70
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    • 1997
  • LGC-40863(proposed common name ; pyribenzoxim), (benzophenone O-[2,6-bis[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzoyl]oxime) is a new rice herbicide being developed by LG Chemical Ltd. The herbicide is highly selective between rice(Oryza sativa L.) and weeds including barnyardgrass (Echinochloa crus-galli(L.) P. Beauv.), and assumed to inhibit acetolactate synthase(ALS ; EC 4.1.3.18) because other structurally related herbicides inhibit the enzyme. To know inhibitory activity and the mode of inhibition of LGC-40863, $I_{50}$(concentration inhibiting ALS activity by 50%) and inhibition kinetics were investigated using ALS extracted from rice and barnyardgrass. $I_{50}$ values of LGC-40863 were 14 and 16mM in rice and barnyardgrass, respectively. In contrast to imazapyr(2-[4,5-dihydro-4-mythyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridine-carboxylic acid) which showed an uncompetitive inhibition pattern, LGC-40863 was a noncompetitive inhibitor to ALS with respect to pyruvate similar to chlorsulfuron(2-chloro-N-((4-methoxy-6-methyl-l,3,5-triazin-2-yl) aminocarbonyl)benz-enesulfonamide) in both plants.

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Designing Modified Atmosphere Packaging for Persimmon (Diospyros kaki cv. Fuyu) Fruit Based on Respiration Modelling (단감의 최적 Modified Atmosphere포장 규격 설정)

  • Ahn, Gwang-Hwan;Choi, Seong-Jin;Lee, Dong-Sun
    • KOREAN JOURNAL OF PACKAGING SCIENCE & TECHNOLOGY
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    • v.13 no.2
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    • pp.67-73
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    • 2007
  • A respiration rate analysed by enzyme kinetics-based respiration model and gas permeability data of LDPE film were applied to design the optical modified atmosphere (MA) packaging condition of persimmon (Diospyros kaki cv. Fuyu) fruits. The fruit quality rapidly decreases due to physiology disorder such as softening and peel blackening. $O_2$ permeance ($Q_{O2}$ in $ml{\cdot}hr^{-1}{\cdot}atm^{-1}{\cdot}m^{-2}$) and $CO_2$ performance ($Q_{CO2}$ in $ml{\cdot}hr^{-1}{\cdot}atm^{-1}{\cdot}m^{-2}$) of low density polyethylene (LDPE) film samples were measured at $0^{\circ}C$ and described as function of thickness (L in ${\mu}m$) as $Q_{O2}=(2540{\times}1/L)-16$, and $Q_{CO2}=(13742{\times}1/L)-70$, respectively. MA package containing single persimmon fruit of 225g was designed and tested experimentally at $0^{\circ}C$ by using LDPE films. Package atmospheres predicted from the relationship of $O_2$, $CO_2$ and $N_2$ balances on the packages was in good agreement with those obtained experimentally. Physiology disorder occurrence was the lowest at 52 ${\mu}m$ package that attained optimum gas condition ($O_2$ 2.8% and $CO_2$ 5.4%). The computer simulation was found to be effective to help to design the optimum MA packaging condition of individual persimmon fruit.

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The effect of immobilization of heparin and bone morphogenic protein-2 to bovine bone substitute on osteoblast-like cell's function

  • Huh, Jung-Bo;Kim, Sung-Eun;Song, Se-Kyung;Yun, Mi-Jung;Shim, Ji-Suk;Lee, Jeong-Yo;Shin, Sang-Wan
    • The Journal of Advanced Prosthodontics
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    • v.3 no.3
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    • pp.145-151
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    • 2011
  • PURPOSE. This study was performed to investigate the ability of recombinant human-bone morphogenic protein-2 immobilized on a heparin-grafted bone substrate to enhance the osteoblastic functions. MATERIALS AND METHODS. The Bio-$Oss^{(R)}$, not coated with any material, was used as a control group. In rhBMP-2-Bio-$Oss^{(R)}$ group, rhBMP-2 was coated with Bio-$Oss^{(R)}$ using only deep and dry methods (50 ng/mL, 24 h). In heparinized rhBMP-2-Bio-$Oss^{(R)}$ group, dopamine was anchored to the surface of Bio-$Oss^{(R)}$, and coated with heparin. rhBMP-2 was immobilized onto the heparinized- Bio-$Oss^{(R)}$ surface. The release kinetics of the rhBMP-2-Bio-$Oss^{(R)}$ and heparinized rhBMP-2-Bio-$Oss^{(R)}$ were analyzed using an enzyme-linked immunosorbent assay. The biological activities of the MG63 cells on the three groups were investigated via cytotoxicity assay, cell proliferation assay, alkaline phosphatase (ALP) measurement, and calcium deposition determination. Statistical comparisons were carried out by one-way ANOVA test. Differences were considered statistically significant at $^*$P<.05 and $^{**}$P<.001. RESULTS. The heparinized rhBMP-2-Bio-$Oss^{(R)}$ showed more sustained release compared to the rhBMP-2-Bio-$Oss^{(R)}$ over an extended time. In the measurement of the ALP activity, the heparinized group showed a significantly higher ALP activity when compared with the non-heparinized groups (P<.05). The MG63 cells cultivated in the group with rhBMP-2 showed increased calcium deposition, and the MG63 cells from the heparinized group increased more than those that were cultivated in the non-heparinized groups. CONCLUSION. Heparin increased the rhBMP-2 release amount and made sustained release possible, and heparinized Bio-$Oss^{(R)}$ with rhBMP-2 successfully improved the osteoblastic functions.

Characteristics on the Removal of Emulsified Vegetable Oil in Wastewater using Bio logical Fluidized Bed (생물학적(生物學的) 유동층(流動層)을 이용(利用)한 수중(水中)의 식물성유(植物性油) 제거특성(除去特性))

  • Kim, Hwan Gi;Park, Ro Sam
    • KSCE Journal of Civil and Environmental Engineering Research
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    • v.10 no.3
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    • pp.127-136
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    • 1990
  • In this paper, the experimental study was carried out for the removal of olive oil in wastewater by the use of Biological Fluidized Bed(BFB) with the reticulated polypropylene sheets as media. The nonbiodegradable olive oil, one of the animal and vegitable oil, was used bacause of the relative simplicity of constitution. Biological degraciability and removal characteristics of emulsified olive oil were studied by batch and continuous experiments respectively. From the results of batch experiments, it was observed that the emulsified olive oli used in BFB reactor was absorbed by media and sludge in about 12 hours, and degradation of the absorbed olive oli was mostly completed for 24 hours. The functional relationship of Michaelis-Menten's Enzyme reaction equation exists between oil concentration and maximum specific rate of olive oil. From the continuous experiments for the removal of olive oil using BFB reactor, it was proved that the substrate removal rate coefficient $k=0.004d^{-1}$, which is the first order kinetics. It was apperared that oxygen utlization coefficients for synthesis(a') and endogeneous respiration(b') of microorganisms in the reactor are respectively 0.85mg $O_2/mg$ $COD_{cr}$ and 0.011mg $O_2/mg$ BVS. day.

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Catalytic properties of wheat phytase that favorably degrades long-chain inorganic polyphosphate

  • An, Jeongmin;Cho, Jaiesoon
    • Asian-Australasian Journal of Animal Sciences
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    • v.33 no.1
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    • pp.127-131
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    • 2020
  • Objective: This study was conducted to determine catalytic properties of wheat phytase with exopolyphosphatase activity toward medium-chain and long-chain inorganic polyphosphate (polyP) substrates for comparative purpose. Methods: Exopolyphosphatase assay of wheat phytase toward polyP75 (medium-chain polyP with average 75 phosphate residues) and polyP1150 (long-chain polyP with average 1150 phosphate residues) was performed at pH 5.2 and pH 7.5. Its activity toward these substrates was investigated in the presence of Mg2+, Ni2+, Co2+, Mn2+, or ethylenediaminetetraacetic acid (EDTA). Michaelis constant (Km) and maximum reaction velocity (Vmax) were determined from Lineweaver-Burk plot with polyP75 or polyP1150. Monophosphate esterase activity toward p-nitrophenyl phosphate (pNPP) was assayed in the presence of polyP75 or polyP1150. Results: Wheat phytase dephosphorylated polyP75 and polyP1150 at pH 7.5 more effectively than that at pH 5.2. Its exopolyphosphatase activity toward polyP75 at pH 5.2 was 1.4-fold higher than that toward polyP1150 whereas its activity toward polyP75 at pH 7.5 was 1.4-fold lower than that toward polyP1150. Regarding enzyme kinetics, Km for polyP75 was 1.4-fold lower than that for polyP1150 while Vmax for polyP1150 was 2-fold higher than that for polyP75. The presence of Mg2+, Ni2+, Co2+, Mn2+, or EDTA (1 or 5 mM) exhibited no inhibitory effect on its activity toward polyP75. Its activity toward polyP1150 was inhibited by 1 mM of Ni2+ or Co2+ and 5 mM of Ni2+, Co2+, or Mg2+. Ni2+ inhibited its activity toward polyP1150 the most strongly among tested additives. Both polyP75 and polyP1150 inhibited the monophosphate esterase activity of wheat phytase toward pNPP in a dose-dependent manner. Conclusion: Wheat phytase with an unexpected exopolyphosphatase activity has potential as a therapeutic tool and a next-generational feed additive for controlling long-chain polyP-induced inappropriate inflammation from Campylobacter jejuni and Salmonella typhimurium infection in public health and animal husbandry.

Comparison of in vitro digestibility and chemical composition among four crop straws treated by Pleurotus ostreatus

  • Nie, Haitao;Wang, Ziyu;You, Jihao;Zhu, Gang;Wang, Hengchang;Wang, Feng
    • Asian-Australasian Journal of Animal Sciences
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    • v.33 no.1
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    • pp.24-34
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    • 2020
  • Objective: The effects of Pleurotus ostreatus on the feed utilization of broad bean stalks (BBS), rape straw (RS), paddy straw (PS), and corn stalk (CS) was examined. Methods: The four roughages were co-cultured with Pleurotus ostreatus. The chemical composition; enzyme activities of laccase, carboxymethylcellulase (CMCase) and xylanase; carbohydrate and protein fractions (based on The Cornell Net Carbohydrate and Protein System [CNCPS]) were assessed at different days after inoculation (7, 14, 21, 28 d) and un-inoculated roughages (control, 0 d). The digestibility of nutrient components and the gas production of roughage with various incubation times were monitored at 0, 2, 4, 6, 9, 12, 24, 36, 48, 60, and 72 h using an in vitro ruminal fermentation method. Results: A higher CMCase activity (0.1039 U/mL) and earlier time to peak (14 d) were detected in Pleurotus ostreatus cultured with CS (p<0.05). Significantly, the incubation length-dependent responses of cumulative gas production were observed from 24 to 72 hours post fermentation (p<0.05), and these incubation length-dependent effects on cumulative gas production of PS and CS appeared earlier (24 h) for PS and CS than those (48 h) for BBS and RS (p<0.05). The fast-degradable carbohydrate (CA) content for all four roughages significantly increased over time (p<0.05). Nonetheless, increased degradation efficiency for CA treated with Pleurotus ostreatus was detected at both 21 and 28 days of incubation (p<0.05). With the exception of PS (p<0.05), there were no significant difference among the roughages (p>0.05) in slowly-degradable carbohydrate (CB2) at different incubation times (p<0.05). Conclusion: Assessment of the alterations in chemical composition, CNCPS system fractions, and the fermentation kinetics after biological pretreatment may yield a valuable database for evaluating the biological pretreatment of Pleurotus ostreatus in ruminant feed.

BIOLOGICALLY-BASED DOSE-RESPONSE MODEL FOR NEUROTOXICITY RISK ASSESSMENT

  • Slikker, William Jr.;Gaylor, David W.
    • Toxicological Research
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    • v.6 no.2
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    • pp.205-213
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    • 1990
  • The regulation of neurotoxicants has usually been based upon setting reference doses by dividing a no observed adverse effect level (NOAEL) by uncertainty factors that theoretically account for interspecies and intraspecies extraploation of experimental results in animals to humans. Recently, we have proposed a four-step alternative procedure which provides quantitative estimates of risk as a function of dose. The first step is to establish a mathematical relationship between a biological effect or biomarker and the dose of chemical administered. The second step is to determine the distribution (variability) of individual measurements of biological effects or their biomarkers about the dose response curve. The third step is to define an adverse or abnormal level of a biological effect or biomarker in an untreated population. The fourth and final step is to combine the information from the first three steps to estimate the risk (proportion of individuals exceeding on adverse or abnormal level of a biological effect or biomarker) as a function of dose. The primary purpose of this report is to enhance the certainty of the first step of this procedure by improving our understanding of the relationship between a biomarker and dose of administered chemical. Several factors which need to be considered include: 1) the pharmacokinetics of the parent chemical, 2) the target tissue concentrations of the parent chemical or its bioactivated proximate toxicant, 3) the uptake kinetics of the parent chemical or metabolite into the target cell(s) and/or membrane interactions, and 4) the interaction of the chemical or metabolite with presumed receptor site(s). Because these theoretical factors each contain a saturable step due to definitive amounts of required enzyme, reuptake or receptor site(s), a nonlinear, saturable dose-response curve would be predicted. In order to exemplify this process, effects of the neurotoxicant, methlenedioxymethamphetamine (MDMA), were reviewed and analyzed. Our results and those of others indicate that: 1) peak concentrations of MDMA and metabolites are ochieved in rat brain by 30 min and are negligible by 24 hr, 2) a metabolite of MDMA is probably responsible for its neurotoxic effects, and 3) pretreatment with monoamine uptake blockers prevents MDMA neurotoxicity. When data generated from rats administerde MDMA were plotted as bilolgical effect (decreases in hippocampal serotonin concentrations) versus dose, a saturation curve best described the observed relationship. These results support the hypothesis that at least one saturable step is involved in MDMA neurotoxicity. We conclude that the mathematical relationship between biological effect and dose of MDMA, the first step of our quantitative neurotoxicity risk assessment procedure, should reflect this biological model information generated from the whole of the dose-response curve.

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Inhibition of Enzymatic Degradation of Leucine Enkephalin and $[D-Ala^2]$-Leucine Enkephalinamide in Various Rabbit Mucosal Extracts by Inhibitors (효소 억제제에 의한 토끼의 점막 추출액중 로이신엔케팔린 및 [D-알라$^2$-로이신엔케팔린아미드의 분해 억제)

  • Chun, In-Koo;Park, In-Sook;Hyun, Jeen
    • Journal of Pharmaceutical Investigation
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    • v.26 no.3
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    • pp.175-185
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    • 1996
  • To inhibit the enzymatic degradation of leucine enkephalin (Leu-Enk) and its synthetic analog. $[D-ala^2]$-leucine enkephalinamide (YAGFL), in the nasal, rectal and vaginal mucosal and serosal extracts of rabbits, effects of enzyme inhibitors such as amastatin (AM), puromycin (PM), thiorphan (TP), thimerosal (TM), EDTA, N-carboxymethyl-Phe-Leu (CPL), phenylethyl alcohol (PEA), phenylmercuric acetate (PMA), benzalkonium chloride (BC) and modified cyclodextrins, alone or in combination, were observed by assaying the pentapeptides staying intact during incubation. Mucosa extracts were prepared by exposing freshly-excised mucosal specimens mounted on Valia-Chien cells to isotonic phosphate buffer while stirring. The degradation of Leu-Enk and YAGFL followed the apparent first-order kinetics. The half-lives (mean) in the nasal, rectal and vaginal mucosal extracts were found to be 1.07, 0.33 and 1.14 hr for Leu-Enk, and 16.9, 6.2 and 6.8 hr for YAGFL, respectively. AM or PM, which is an aminopeptidase inhibitor, did not show a sufficient inhibition of Leu-Enk $(50\;{\mu}g/ml)$ degradation in all kinds of extracts. $Dimethyl-{\beta}-cyclodextrin\;(DM-{\beta}-CyD)$ decreased the degradation rate constants of Leu-Enk about 2 or 3 times, comparing with no additive. However, the use of mixed inhibitors of AM $(50\;{\mu}M)$/TM (0.25 mM)/EDTA (5 mM) resulted in a full stabilization of Leu-Enk by decreasing the degradation rate constants 67.3, 161.3 and 113.8 times far the nasal, rectal and vaginal mucosal extracts, respectively, comparing with no inhibitor. With mixed inhibitors, Leu-Enk remained intact more than 90% after 6 hr-incubation. In the stabilization of YAGFL, hM, TP or CPL alone showed little efffct, and some additives demonstrated a considerable inhibition of YAGFL degradation in the rank order of TM > BC > EDTA. However, the addition of mixed inhibitors such as TM (0.5 mM) and EDTA (5 mM) into the extracts protected YAGFL from the degradation by more than 85% even after 24 hr-incubation, suggesting almost complete inhibition of YAGFL degradation in the extract. On the other hand, $DM-{\beta}-CyD\;or\;hydroxypropyl-{\beta}-cyclodextrin$ (10%) were also found to retard enzymatic degradation rates of YAGFL markedly, and resulted in staying intact more than 80% of YAGFL in the nasal and vaginal mucosal extracts, and more than 60% in the rectal mucosal extract after 16 hr-incubation.

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