• Title/Summary/Keyword: esterase assay

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Dual Application of p-Nitrophenol Alkanoate-Based Assay for Soil Selection and Screening of Microbial Strains for Bioplastic Degradation

  • Nara Shin;Jinok Oh;Suwon Kim;Yeda Lee;Yuni Shin;Suhye Choi;Shashi Kant Bhatia;Yung-Hun Yang
    • Journal of Microbiology and Biotechnology
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    • v.34 no.7
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    • pp.1530-1543
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    • 2024
  • With an increase in the commercialization of bioplastics, the importance of screening for plastic-degrading strains and microbes has emerged. Conventional methods for screening such strains are time-consuming and labor-intensive. Therefore, we suggest a method for quickly and effectively screening plastic-degrading microbial strains through dual esterase assays for soil and isolated strains, using p-nitrophenyl alkanoates as substrates. To select microbe-abundant soil, the total amount of phospholipid fatty acids (PLFAs) included in each soil sample was analyzed, and esterase assays were performed for each soil sample to compare the esterase activity of each soil. In addition, by analyzing the correlation coefficients and sensitivity between the amount of PLFAs and the degree of esterase activity according to the substrate, it was confirmed that substrate pNP-C2 is the most useful index for soil containing several microbes having esterase activity. In addition, esterase assays of the isolated strains allowed us to select the most active strain as the degrading strain, and 16S rRNA results confirmed that it was Bacillus sp. N04 showed the highest degradation activity for polybutylene succinate (PBS) as measured in liquid culture for 7 days, with a degradation yield of 99%. Furthermore, Bacillus sp. N04 showed degradation activity against various bioplastics. We propose the dual application of p-nitrophenyl alkanoates as an efficient method to first select the appropriate soil and then to screen for plastic-degrading strains in it, and conclude that pNP-C2 in particular, is a useful indicator.

Study on the Memory Enhancement of the Extract of Gongjadaesungjichimjung-bang(GDJB) (공자대성지침중방(孔子大聖智枕中方)의 기억증진(記憶增進) 효과(效果)에 관(關)한 실험적(實驗的) 연구(硏究))

  • Kang, Yeon-Sug;Chang, Mi-Kyung;Kim, Geun-Woo;Koo, Byung-Soo
    • Journal of Oriental Neuropsychiatry
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    • v.14 no.1
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    • pp.75-84
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    • 2003
  • Object : The present experiments were designed to study on the memory enhancement of the extract of Gongjadaesungjichimjung-bang(GDJB). Methods : The water extract of GCJB has been tested for its activities on memory enhancement by passive avoidance task in vivo and for its inhibitory effect on the acetylcholine esterase activity. Results : GDJB water extract significantly enhanced the memory at a concentration of 50mg/kg, but this effect did not proportionally increased at a dose of l00mg/kg and significantly inhibited the acetylcholine esterase activity in a dose-dependent manner in in vitro assay with IC50 value of 1.57mg/ml and also in in vivo assay. Conclusion : The extract of GDJB showed a memory enhancement as well as the inhibitory effect on acetylcholine esterase activity, which suggest that this prescription may be applied for the treatment of memory impairment.

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Synergistic Effect of Acetylalginate Esterase and Alginate Lyase on the Degradation of Acetylalginate from Pseudomonas aeruginosa ATCC 39324 (P. aeruginosa ATCC 39324 생산 아세틸알긴산의 분해반응에서 아세틸알긴산 아세틸분해효소와 알긴산 분해효소의 상승효과)

  • Kim, Hee Sook
    • Journal of Life Science
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    • v.23 no.12
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    • pp.1420-1427
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    • 2013
  • A novel acetylalginate esterase (AcAlgE) gene was previously cloned and characterized from Sphingomonas sp. MJ-3. In this study, the synergistic effects of MJ-3 AcAlgE, and KS-408 alginate lyase on the degradation of acetylalginate from Pseudomonas aeruginosa were investigated by using high-field 1H-NMR and an FPLC-equipped peptide column. The alginate lyase coupled assay of AcAlgE showed that degradation of high molecular weight acetylalginate was more difficult than degradation of acid hydrolyzed acetylalginate. The degradation of acetylalginate by alginate lyase was easier after AcAlgE was used to remove the acetyl group from acetylalginate. This result showed that the recombinant AcAlgE enhanced the degradation of acetylalginate by alginate lyase.

Inhibitory Activities of Basidiomycetes on Prolyl Endopeptidase, Acetylcholine Esterase and Coagulation (담자균 추출물의 Prolyl Endopeptidase, Acetylcholine Esterase 저해 및 항혈전 응고활성)

  • Lee, Hyun-Jin;Kim, Jong-Sik;Heo, Gun-Young;Lee, Kyung-Bok;Rhee, In-Koo;Song, Kyung-Sik
    • Applied Biological Chemistry
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    • v.42 no.4
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    • pp.336-343
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    • 1999
  • Fifty six mycelial cultured Basidiomycetes were screened for their inhibitory effects against prolyl endopeptidase(PEP), acetylcholine esterase(AChE) and thrombus coagulation. Out of them, methanolic extract of mycelium and/or ethylacetate(EtOAc) soluble fraction from culture broth of Peniophora quercina, Amanita aspera, Phellinus chrysoloma, Grifola frondosa, Wolfiporia extensa, Clavicorona pyxidata and Phanerochaete sordida inhibited more than 90% of PEP activity at 40 ppm. The extracts of Lenzites betulina, Phellinus chrysoloma, Wolfiporia extensa, Phanerochaete sorrlida, Hypocrea nigricans, Coriolus azureus, Flammulina velutipes, Phlebiopsis gigantea and Bondarzewia montana exhibited about 40% of inhibitory activity against AChE at 40 ppm. In thrombin times assay, the extracts of Amanita aspera, Oxyporus latemarginata, Peniophora quercina, Fomes fomenfarius, Trametes versicolor, and Phlebiopsis gigantea delayed coagulation of thrombus about two to three times over control at ca 550 ppm. In activated partial thromboplastin times assay, none of the tested Basidiomycetes showed significant effect.

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Determination and Characterization of Thermostable Esterolytic Activity from a Novel Thermophilic Bacterium Anoxybacillus gonensis A4

  • Faiz, Ozlem;Colak, Ahmet;Saglam, Nagihan;Canakci, Sabriye;Belduz, Ali Osman
    • BMB Reports
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    • v.40 no.4
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    • pp.588-594
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    • 2007
  • A novel hot spring thermophile, Anoxybacillus gonensis A4 (A. gonensis A4) was investigated in terms of capability of tributyrin degradation and characterization of its thermostable esterase activity by the hydrolysis of p-nitrophenyl butyrate (PNPB). It was observed that A. gonensis A4 has an esterase with a molecular weight of 62 kDa. The extracellular crude preparation was characterized in terms of substrate specificity, pH and temperature optima and stability, kinetic parameters and inhibition/activation behaviour towards some chemicals and metal ions. Tributyrin agar assay showed that A. gonensis A4 secreted an esterase and $V_{max}$ and $K_m$ values of its activity were found to be 800 U/L and 176.5 ${\mu}M$, respectively in the presence of PNPB substrate. The optimum temperature and pH, for A. gonensis A4 esterase was $60-80^{\circ}C$ and 5.5, respectively. Although the enzyme activity was not significantly changed by incubating crude extract solution at $30-70^{\circ}C$ for 1 h, the enzyme activity was fully lost at $80^{\circ}C$ for same incubation period. The pH-stability profile showed that original crude esterase activity increased nearly 2-fold at pH 6.0. The effect of some chemicals on crude esterase activity indicated that A. gonensis A4 produce an esterase having serine residue in active site and -SH groups were essential for its activity.

Development of Membrane Strip Assay System for Lipoprotein Cholesterol (Membrane strip을 이용한 지질단백질 Cholesterol 측정시스템의 개발)

  • 신인수;백세환
    • KSBB Journal
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    • v.11 no.2
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    • pp.140-150
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    • 1996
  • To develop a home-version assay system for plasma lipoprotein cholesterol, variables that can control the assay performance were optimized. The system was constructcd by using two major components: nitrocellulose membrane strip with immobilized enzymes (cholesterol esterase, cholesterol oxidase, and horseradish peroxidase); and sample carrier solution containing non-ionic detergent (Triton X-100) and chromogen (3,3'-diaminobenzidine). Once a sample combined with the carrier was absorbed from the bottom of the strip, cholesterol was delivered by capillary action to the immobilized enzymes and a sequential reactions took place. In the final reaction, the chromogen was oxidized and then generated a color as signal that was proportional to the concentration of cholesterol. The signal intensity was enhanced by optimizing conditions for the immobilization of enzymes and the chemical composition of carriel. Under these conditions, a dose-response curve was obtained and revealed a high sensitivity enough to measure the cholesterol in blood.

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Development of Total Cholesterol Detection System by Fluorescence Chromatography (형광 크로마토그래피에 의한 콜레스테롤 측정법의 개발)

  • Oh, Sang-Wook
    • Journal of Food Hygiene and Safety
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    • v.24 no.2
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    • pp.148-153
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    • 2009
  • In this study, we have developed a fluorescence chromatographic assay for the quantification of total cholesterol in serum, which is a well-known risk predictor for cardiovascular diseases. The new assay system consists of a chromatographic strip in a cartridge, enzyme buffer containing cholesterol esterase, cholesterol oxidase, horseradish peroxidase, and color developer AEC, and a laser fluorescence scanner. The correlation coefficient (r) between cholesterol concentration and relative fluorescence units was 0.968 in the new assay, showing a reliable linearity through the tested range of cholesterol. Recovery test and comparability with a Hitachi 747 instrument showed 106.5-94% and r = 0.939 (p<0.001), respectively. The new assay system for cholesterol was developed as a pre-POCT platform conducted in clinics since it is fast (8 min) and uses a small volume of sample ($5\;{\mu}l$), and it may be applied for on-site diagnostics to replace expensive automated biochemical analyzer.

Model Checking for Joint Modelling of Mean and Dispersion (평균과 산포의 동시 모형화에 대한 모형검토)

  • Ha, Il-Do;Lee, Woo-Dong;Cho, Geon-Ho
    • Journal of the Korean Data and Information Science Society
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    • v.8 no.2
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    • pp.195-209
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    • 1997
  • The joint modelling of mean and dispersion in quasi-likelihood models which greatly extend the scope of generalized linear models, is required in case that the dispersion parameter, the variance component of response variables, is not constant but changes by depending on any covariates. In this paper, by using statistical package GENSTAT(release 5.3.2, 1996) which makes a easily analyze real data through this joint modelling, we mention necessities that must consider this joint modelling rather than existing mean models through model checking based on graphic methods for esterase assay data introduced by Carrol and Ruppert(1987, pp.46-47), and then study methods finding reasonable joint model of mean and dispersion for this data.

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Regression Diagnostics on Joint Modelling of Mean and Dispersion (평균과 분산의 동시모형에 따른 회귀진단법에 관한 연구)

  • 강위창;이영조;송문섭
    • The Korean Journal of Applied Statistics
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    • v.13 no.2
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    • pp.407-414
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    • 2000
  • Carroll and Ruppert(1988) analyzed the esterase assay data with regression model based on quasi-likelihood. Jung and Lee(1997) introduced a goodness-of-fit test for testing the adequacy of the quasi-likelihood and claimed that there is no gross inadequacy with the model because their test was not rejected. However, Lee and Xelder(199S)'s residual plots revealed that the model did not sufficiently reflect the increase of the variance with that of the mean. In this paper, we re-analyze the esterase assay data with the joint modelling of mean and dispersion in Lee and l\elder(1998) and evaluate the validity of the fitted model by applying the residual plots. And it is illustrated that Lee and Nelder(199S)'s restricted likelihood is more efficient in goodness-of-fit test for the dispersion model.

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Fluorescence-based Assay System for Endocannabinoid Degradation Enzyme, Fatty Acid Amide Hydrolase

  • Kim, Dae-Woong;Kim, Gun-Joong;Kim, Hae-Jo;Ghil, Sung-Ho
    • Biomedical Science Letters
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    • v.16 no.4
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    • pp.279-285
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    • 2010
  • Endogenous cannabinoids (endocannabinoids) display various pharmacological effects including pain control, anti-inflammation, and neuroprotection. The synthesis and release of endocannabinoids are regulated under both physiological and pathological conditions. The main degrading enzyme of endocannabinoid is fatty acid amide hydrolase (FAAH). Therefore we have developed the fluorescence-based assay system for FAAH. We established stable CosM6 cell lines expressing human FAAH. We also synthesized 2-oxo-2H-chromen-7-yl decanoate (DAEC) as a fluorogenic substrate for FAAH. When crude membrane extracts stably expressing FAAH was incubated with DAEC at $25^{\circ}C$, FAAH reacted specifically to DAEC and catalyzes the hydrolysis of DAEC into decanoic acid and highly fluorescent coumarin. Furthermore, the serin hydrolase inhibitor, phenylmethanesulfonylfluoride, inhibited the coumarin release to the reaction buffer in concentration dependent manner. This assay system is suitable for high-throughput screening since this system has simple experimental procedure and measurement method.