• Journal of Microbiology
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• v.42 no.2
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• pp.99-102
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• 2004

Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the puri-fication of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4$^{\circ}C$; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5 ％ glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD$\sub$600/ of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.

• BMB Reports
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• v.44 no.7
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• pp.458-461
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• 2011

Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys ($D_4K$). The assay for enteropeptidase has utilized $GD_4K$-conjugated 2-naphthylamine ($GD_4K$-NA) as a fluorogenic probe over the last 30 years. However, no other $D_4K$-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of $GD_4K$-conjugated 7-amino-4-methylcoumarin ($GD_4K$-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a $K_M$ of 0.025 mM and a $k_{cat}$ of 65 $sec^{-1}$ for $GD_4K$-AMC, whereas it has a $K_M$ of 0.5 to 0.6 mM and a $k_{cat}$ of 25 $sec^{-1}$ for $GD_4K$-NA. The optimum pH of $GD_4K$-AMC hydrolysis was pH 8.0. Our data indicate that $GD_4K$-AMC is more suitable as a substrate for enteropeptidase than $GD_4K$-NA.

• Korean Chemical Engineering Research
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• v.54 no.3
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• pp.380-386
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• 2016

In this study, we described a paper-based neuraminidase assay sensor (PNAS) which can be applied to detect the infection by influenza viruses. The PNAS was designed and manufactured to quantitatively identify the levels of neuraminidase in the sample, which is based on colorimetric analysis using the X-Neu5Ac substrate. The limit of detection of the PNAS was determined as 0.004 U/mL of neuraminidase. According to the amount of neuraminidase in human serum, the PNAS could monitor the enzyme activity with a good linearity ($R^2$ > 0.99). In addition, the initial performance of the PNAS has been maintained up to 70 days in the $4^{\circ}C$. Finally, we demonstrated whether the Michaelis-Menten kinetics is applied to the PNAS, which can show the reliability of the enzyme reactions. The kinetic studies indicated that the PNAS provides the good condition for enzyme reactions ($K_m=8.327{\times}10^{-3}M$), but they were performed on paper chip nonetheless. The paper-based neuraminidase assay sensor may be useful in a wide range of rapid and safe detection of influenza virus.

• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1269-1274
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• 2014

Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

• Journal of Environmental Health Sciences
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• v.15 no.2
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• pp.41-49
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• 1989

The assay on microbial dehydrogenase activity by using TTC was investigated. The experimental results are summarized as follows: 1. The TF production was affected by dissolved oxygen. Dissolved oxygen in samples could be effectively removed by adding $Na_{2}SO_{3}$ as reductant prior to incubation. 2. The toxic effect on TF formation was observed at up to 0.1% of final TTC concentration when VSS concentration was 2.7g/l. As the VSS concentration increased the effect decreased. 3. It was clearly revealed that the TF generation rate decreased with increasing VSS concentration. The dilution of sludge could be applicable when VSS level is below 3.5g/l. 4. The TF production of endogenous phase (De) could be used as a measure for the viable fraction of sludge over various sludge ages. The additional TF production (Ds) due to extracellular substrate responded to De, not to VSS concentration.

• Biomedical Science Letters
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• v.12 no.3
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• pp.225-231
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• 2006

Fibrinolytic enzyme was purified from the fruiting bodies of Lepista nuda, using DEAE-Cellulose chromatography, Phenyl Sepharose chromatography, and Mono-S column chromatography. The substance has a molecular weight of 30006.62 Da as measured by MALD-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. The activity of the enzyme was inhibited by PMSF, indicating that the enzyme is a serine protease. No inhibition was found with E-64, pepstatin, and EDTA. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $30^{\circ}C$. The enzyme hydrolyzes both Aa and y chains of human fibrinogen but did not show any reactivity for $B{\beta}$ chain of human fibrinogen.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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• pp.67-71
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• 1996

The plasmid pK8 was constructed to verify the existence of an adenylate domain in peptide synthetase by using pGC12. 1.2 kb fragment, coding tyrocidine synthetase 1 (123 kDa) was deleted, and 79.6 kDa one was expressed in Escherichia coli XL1-blue. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over expressed synthetase. ATP-[$^{32}P$]PPi exchange reaction was measured for the enzyme assay.

• BMB Reports
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• v.31 no.2
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• pp.170-176
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• 1998

Phosphatidylethanolamine N-methyltransferase (PEMT) is known to be a membrane-associated protein. However, cytosolic PEMT was detected when sufficient amounts of exogenous phospholipids were added in the incubation media. The methylation of phospholipids was measured by the incorporation of the $[^3H]-methyl$ group from S-adenosylmethionine and the methylated phospholipids were analyzed by thinlayer chromatography. The essence of the assay condition for the cytosolic enzyme was the inclusion of 200 ${\mu}g$ of each substrate, phosphatidylethanolamine (PE), phosphatidyl N-monomethylethanolamine (PME) and phosphatidyl N,N-dimethylethanolamine (PDE), in the reaction mixture of 100 ${\mu}l$. The subcellular fractionation of brain PEMT activities revealed that approximately 38.1 % for PME, 39.5% for PDE, and 22.4% for PC formation was present in the cytosolic fraction. The general properties of cytosolic PEMT were characterized and compared with those of neuronal nuclei PEMT.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.695-700
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• 2007

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans (GenBank access code AF222787) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein Aga1p. The surface display of CFTase was confirmed by immunofluorescence microscopy and enzymatic assay. The optimized reaction conditions of surface-displayed CFTase were as follows; pH, 8.0; temperature, $50^{\circ}C$; enzyme amount, 30 milliunit; substrate concentration, 5%; inulin source, Jerusalem artichoke. As a result of the reaction with inulin, cycloinulohexaose was produced as a major product along with cycloinuloheptaose and cycloinulooctaose as minor products.

• Food Science and Biotechnology
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• v.14 no.6
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• pp.743-746
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• 2005

A dipstick-type immunochemical biosensor for the detection of the organophosphorus insecticide fenthion was developed using a screen-printed electrode system as an amperometric transducer with polyclonal antibodies against fenthion as a bioreceptor. The assay of the biosensor involved competition between the pesticide in the sample and pesticide-glucose oxidase conjugate for binding to the antibody immobilized on the membrane. This was followed by measurement of the activity of the bound enzyme by the supply of the enzyme substrate (glucose) and amperometric determination of the enzyme reaction product ($H_2O_2$). The activity of the bound enzyme was inversely proportional to the concentration of pesticide. The optimized sensor system showed a linear response against the logarithm of the pesticide concentration ranging from $10^{-2}$ to $10^3\;{\mu}g/L$.