• Asian-Australasian Journal of Animal Sciences
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• 제27권4호
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• pp.600-607
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• 2014

The aim of this study was to assess the effect of substrate to inoculum ratio (S/I ratio) on the biochemical methane potential (BMP) and anaerobic biodegradability ($D_{deg}$) of different piggery slaughterhouse wastes, such as piggery blood, intestine residue, and digestive tract content. These wastes were sampled from a piggery slaughterhouse located in Kimje, South Korea. Cumulative methane production curves for the wastes were obtained from the anaerobic batch fermentation having different S/I ratios of 0.1, 0.5, 1.0, and 1.5. BMP and anaerobic biodegradabilities ($D_{deg}$) of the wastes were calculated from cumulative methane production data for the tested conditions. At the lowest S/I ration of 0.1, BMPs of piggery blood, intestine residue, and digestive tract content were determined to be 0.799, 0.848, and $1.076Nm^3kg^{-1}-VS_{added}$, respectively, which were above the theoretical methane potentials of 0.539, 0.644, and $0.517Nm^3kg^{-1}-VS_{added}$ for blood, intestine residue, and digestive tract content, respectively. However, BMPs obtained from the higher S/I ratios of 0.5, 1.0, and 1.5 were within the theoretical range for all three types of waste and were not significantly different for the different S/I ratios tested. Anaerobic biodegradabilities calculated from BMP data showed a similar tendency. These results imply that, for BMP assay in an anaerobic reactor, the S/I ratio of anaerobic reactor should be above 0.1 and the inoculum should be sufficiently stabilized to avoid further degradation during the assay.

• BMB Reports
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• 제30권5호
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• pp.370-377
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• 1997

A novel assay method is described for rapid quantitation of reaction rate of sterol ${\Delta}^7$-reductase (${\Delta}^7$-SR) which catalyzes reduction of the ${\Delta}^7$-double bond of sterols. Of six different organ tissues-liver, small intestine, brain, lung, kidney, and testis-. ${\Delta}^7$-SR activity was detected only in liver (2.30 nmol/min/mg protein) and testis (0.11 nmol/min/mg protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate, we assessed both kinetics ($K_m=210\;{\mu}M$, $V_{max}=1.93\;nmol/min/mg$) and inhibition of the rat hepatic ${\Delta}^7$-SR against well-studied cholesterol lowering agents such as triparanol ($IC_{50}=16\;{\mu}M$). 3-$\beta$-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) ($IC_{50}=5.2\;{\mu}M$), and trans-1.4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) ($IC_{50}=0.25\;{\mu}M$). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e., ${\Delta}^7$-SR, sterol ${\Delta}^8$-isomerase, and sterol ${\Delta}^14$-reductase). ${\Delta}^7$-SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound ($K_i=0.109\;{\mu}M$). Substrate specificity studies of the microsomal ${\Delta}^7$-SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.6-fold higher than that for lathosterol. ${\Delta}^7$-SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol (>2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin ($\simeq$5.0-fold). Finally, microsomal ${\Delta}^7$-SR was solubilized by 1.5% 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and enriched on PEG (0~10%) precipitation, which should be suitable for further purification of the enzyme.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.489-492
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• 2000

The eventual goal of this study is to elucidate roles of plant terpenoids (e.g., cymene, limonene and others) as natural substrates in the cometabolic biodegradation of PCBs and to develop an effective PCB bioremediation technology. The aim of this study was to examine how plant terpenoids, as natural substrates or inducers would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. The PCB congener degradation activities were first monitored through resting cell assay technique that could detect degradation products of the substrate. The congener removal was also confirmed by concommitant GC analysis. The PCB degraders, Pseudononas sp. P166 and Caynebacterium sp. T104 were found to grow on both biphenyl and terpenoids ((S)-(-) limonene, p-cymene and ${\alpha}-terpinene$) whereas Arthrobacter B1B could not grow on the terpenoids as a sole carbon source. The strain B1B grown on biphenyl showed a good degradation activity for 4,4'-dichlorobiphenyl (DCBp) while strains P166 and T104 gave about 25% of B1B activity. Induction of degradation by cymene, limonene and terpine was hardly detected by the resting cell assay technique. This appeared to be due to relatively lower induction effect of these terpenoids compared with biphenyl. However, a subsequent GC analysis showed that the congener could be removed up to 30% by the resting cells of T104 grown on the terpenoids. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate for PCB biodegradation.

• Biomolecules & Therapeutics
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• 제21권6호
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• pp.487-492
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• 2013

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

• 대한수의학회지
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• 제29권4호
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• pp.567-575
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• 1989

This study was conducted to evaluate the possibility of application of a microenzyme-linked immunosorbent assay(micro-ELISA) for the serodiagnosis of specific toxoplasma antibodies in swine sera and this test was performed as a microplate system by coating the polystyrene plates with toxoplasma soluble antigen, incubated serially diluted sera, then added horse radish peroxidase labelled goat anti-swine IgG(r) conjugate followed by o-phenylenediamine as substrate. The color development by enzyme-substrate reaction was determined by the photometric reading [ELISA reader at 490nm (OD)] and visual reading. The soluble antigen was prepared from the tachyzoites in mouse peritoneal cavity. A total of 1,200 swine sera from pig slaughter-house and a total of 116 swine sera from pig breeding station (S-C farm) were tested for the detection of antibodies to Toxoplasma gondii. The results obtained were summarized as follows: 1. The optimal reactions of indirect ELISA for the test sera were determined by the dilution of antigen 1:256 and 1:3,200 of horse radish peroxidase conjugate [anti-swine IgG(r)]. 2. The specific togoplasma antibody(IgG) in pigs infected with Tp artificially were detected as the serum titers of 1:64 or 1:128 at one week postinfection. 3. Of a total of 1,200 swine sera from pig slaughter-house 505 samples of sera were detected as positive (42.1%) and of a total of 116 swine sera from S-C pig breeding station 68 samples of sera as positive (58.6%). 4. The specific antibody(IgG) detection rates against a total of 1,200 test sera from pig slaughter-house were not significant between male (43.1%) and female (40.7%). 5. The indirect ELISA was proved to be a sensitive and specific procedure for the serodiagnosis of swine toxoplasmosis and also evaluated as an effective screening test for the large scale of test samples in laboratory.

• Journal of Acupuncture Research
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• 제17권3호
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• pp.176-187
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• 2000

Objectives : This study was purposed to investigate the antioxidative effects of Paeoniae radix aqua-acupuncture solution(PR) on culture liver cell system, lipid peroxidation and antioxidative enzyme activities in tert-butyl hydroperoxide(t-BHP) treatmented conditions. Methods : Cultured normal rat liver cell(Ac2F) were prepared and incubated with or without PR(at 2% volume in culture medium). After 16~18hr, cells placed in DMEM medium without serum, and then incubated with 1mM t-BHP for 2hr. Viable cells were detected by MTT assay, and the levels of lipid peroxide(LPO) were measured by TBA method. And catalase activity was measured as the decrease in hydrogen peroxide absorbance at 240nm on spectrophotometer using 30mM hydrogen peroxide. Superoxide dismutase(SOD) were assayed by recording the inhibition of nitro blue tetrazolium reduction with xanthine and xanthine oxidase. Glutathione peroxidase(GPX) activity was determined by the modified coupled assay developed by Paglia and Lawrence. The reaction was started by addition of 2.2mM hydrogen peroxide as substrate. The change in absorbance at 340nm was measured for 1min on spectrophotometer. Glutathione-S-transferase(GST) activity was assayed with CDNB as substrate and enzyme activity of GST towards the glutathione conjugation of CDNB. Results : Cell killing was significantly enhanced by addition of t-BHP compared to those of untreated group. PR pretreated cell resisted the toxic effects of t-BHP. LPO levels of t-BHP treatment group were significantly higher than other groups. This increased level was significandy reduced by PR pretreatment. The t-BHP treatment resulted in a decrease of catalase, GPX and GST activities. By contrast, PR pretreatment markedly increased compare to those of untreated groups. Conclusions : T-BHP which can produce intracellular free radical was used for inducer of the peroxidation of cellular lipids. PR protected the cell death induced by t-BHP and significantly increased cell viabiliry in the normal rat liver cell, and showed effective inhibition of lipid peroxidation, and elevations of catalase, GPX and GST activities. These results suggested that PR might play a protective role in lipid peroxidation by free radicals.

• Fisheries and Aquatic Sciences
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• 제27권5호
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• pp.314-328
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• 2024

The mechanism for the elimination of xenobiotics undergoes three different phases of reactions in organisms. Among these, glutathione S-transferases (GSTs) are classified as phase II detoxification enzymes, catalyzing the conjugation of electrophilic substrates to glutathione or reduced hydroperoxides. This study aimed to investigate the molecular characteristics, detoxification functions, and immune responses of GST omega (LhGSTO1) and kappa (LhGSTK1) in redlip mullet. The open reading frames of LhGSTO1 (720 bp) and LhGSTK1 (687 bp) encoded proteins of 239 and 228 amino acids, respectively. Sequence analysis revealed that LhGSTO1 and LhGSTK1 possessed GSH-binding sites in their N-terminal domains. Substrate-binding sites in the C-terminal domain were exclusively identified in LhGSTO1. In the tissue-specific transcription profile analysis, both LhGSTO1 and LhGSTK1 were ubiquitously expressed in all tissues of healthy mullets. Temporal expression analysis of LhGSTO1 and LhGSTK1 in the blood showed that their expression was significantly modulated by polyinosinic:polycytidylic (poly I:C), lipopolysaccharide (LPS), and Lactococcus garvieae. Different chemical and cellular assays were performed to assess the detoxification and cellular protective abilities of the two proteins. A substrate specificity test using the recombinant proteins revealed that both proteins possessed specific activity toward 1-chloro-2,4-dinitrobenzene (CDNB). In the disk diffusion assay, the smallest clearance zones were observed for LhGSTO1 and LGSTK1 against CdCl₂. In the cell protection assay, both LhGSTO1 and LhGSTK1 showed significant Cd detoxification ability compared to the control. Collectively, these results demonstrate that GST omega and kappa are involved in host defense against immune stimulants and xenobiotics in redlip mullet.

• Journal of Microbiology and Biotechnology
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• 제34권7호
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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.

• 한국물환경학회지
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• 제26권4호
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• pp.574-579
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• 2010

In this study the enzyme inhibition method using dehydrogenase which has been popularly used to estimate ecotoxicity was optimized. When three bacterial strains, Escherichia coli HB101, Enterobacter asburiae KCAD-4, and Aeromonas media KCAD-13, were compared, KCAD-4 was considered as the adequate strain to estimate toxicity because of its sensitivity and reproducibility. The optimal bacterial density was estimated as $5.4{\times}10^9CFU/mL$, at which the maximum sensitivity was observed. The phosphate buffer was suitable for the reaction solution. When the reaction times required for inhibition of enzyme activity by contact of toxicants and for reaction of damaged bacteria and substrate were tested, the optimal value was estimated as 20 min and 2 hrs, respectively. It is expected that the optimized conditions can be used to develop the standardized kits to estimate ecotoxicity of heavy metals in effluent from the industrial wastewater treatment facilities.

• Journal of Microbiology
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• 제42권3호
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• pp.194-199
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• 2004

Investigation of the expression of the riboflavin (rib) genes, which are found immediately downstream of luxG in the lux operon in Photobacterium phosphoreum, provides more information relevant to the evolution of bioluminescence, as well as to the regulation of supply of flavin substrate for bacterial bioluminescence reactions. In order to answer the question of whether or not the transcriptions of lux and rib genes are integrated, a transcriptional termination assay was performed with P. phoxphoreum DNA, containing the possible stem-loop structures, located in the intergenic region of luxF and luxE ($\Omega$$\_$A/), of luxG and ribE ($\Omega$$\_$B/), and downstream of ribA ($\Omega$$\_$c/). The expression of the CAT (Chloram-phenicol Acetyl Transferase) reporter gene was remarkably decreased upon the insertion of the stem-loop structure ($\Omega$$\_$c/) into the strong lux promoter and the reporter gene. However, the insertion of the structure ($\Omega$$\_$B/) into the intergenic region of the lux and the rib genes caused no significant change in expression from the CAT gene. In addition, the single stranded DNA in the same region was protected by the P. phosphoreum mRNA from the Sl nuclease protection assay. These results suggest that lux genes and rib genes are part of the same operon in P. phosphoreum.