• Mycobiology
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• v.34 no.2
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• pp.108-110
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• 2006

To understand the ability of producing cellulolytic enzyme activity in the sapstaining fungi, four species of Ophiostoma and two species of Leptographium were investigated in the culture media containing each of cellulose substrates such as CM-cellulose, Avicel and D-cellobiose and each of chromogenic dyes such as Congo-Red, Phenol Red, Remazol Brilliant Blue and Tryphan Blue. When the fungi were grown for $5{\sim}7$ days at $25^{\circ}C$, the formation of clear zone by chromogenic reaction around the margin of the fungal colony was demonstrated in all the culture media Congo-Red containing CM-cellulose. There was difference in the formation of clear zone among the dyes. Only Ophiostoma setosum and Leptographium spp. showed cellulolytic activity to the three substrates. Overall, the results of this study show that ophiostomatoid sapstaining fungi can produce cellulolytic enzymes.

• YAKHAK HOEJI
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• v.43 no.5
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• pp.606-613
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• 1999

To survey the possibility of replacing the pyrogen test with Limulus Amebocyte Lysate(LAL) test and to find out a standard methods suitable to our blood products made in Korea, 100 samples of 20% human serum albumin were tested by commercial LAL test kits and results of those were compared with rabbit pyrogen test. The LAL test is used both dinetic-chromogenically and kinetic-turbidimetrically. Both methods equally showed broad detection range (5.0~0.005 EU/ml), excellent sensitivity ($\geq$ 0.005 EU/ml) and predominant recovery rate within valid dilution range, but kinetic-turbidimetric method seemed to be more reproducible than kinetic-chromogenic method(kinetic-chromogenic method : S.D. = 15.88, kinetic-turbidimetric method : S.D. = 8.12). After heating the sample at 75$^{\circ}C$ for 15 min, the results showed a little elevated recovery rate with both methods. After performing the test on 100 albumin samples with both kits, the results were analysed using the USP standard (1.33 EU/ml). 7% of samples in kinetic-chromogenic methods and 1% of samples in kinetic-turbidimetric method exceeded the limit of endotoxin levels regulated for blood products in USA. Because this phenomenon was not observed in both methods at the same time and both methods have high sensitivity ($\geq$0.005 EU/ml), these results seemed to depend on nonspecific reaction. Considering its sensitivity and reproducibility, we could assure that LAL test is proper to detecting pyrogenic with good sensitivity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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• pp.132-136
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• 2004

The LAL (Limulus amebocyte lysate) test for the detection and quantification of endotoxin is based on the gelation reaction between endotoxin and LAL from a blood extract of Limulus polyphemus. The test is labor intensive, requiring dedicated personnel, a relatively long reaction time (approximately 1 h), relatively large volumes of samples and reagents and the detection of the end-point is rather subjective. To solve these problems, a miniaturized LOC (lab-on-a-chip) prototype, 62mm (L) ${\times}$ 18 mm (W), was fabricated using PDMS (polydimethylsiloxane) bonded to glass. Using this prototype, in which 2mm (W) ${\times}$ 44.3mm (L) ${\times}$ 100 $\mu\textrm{m}$ (D) microfluidic channel was constructed, turbidometric and chromogenic assay detection methods were compared, and the chromogenic method was found the most suitable for a small volume assay. In this assay, the kinetic-point method was more accurate than the end-point method. The PDMS chip thickness was found to be minimized to around 2 mm to allow sufficient light transmittance, which necessitated the use of a glass slide bonding for chip rigidity. Due to this miniaturization, the test time was reduced from 1 h to less than 10 min, and the sample volume could be reduced from 100 to ca. 4.4 ${\mu}$L. In summation, this study suggested that the LOC using the LAL test principle could be an alternative as a semi-automated and reliable method for the detection of endotoxin.

• KSBB Journal
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• v.18 no.5
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• pp.429-433
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• 2003

LAL (Limulus amebocyte lysate) test to detect and quantity endotoxin is based on gellation reaction between endotoxin and LAL from a blood extract of Limulus polyphemus. The test is labor intensive requiring dedicated personnel, takes relatively long reaction time (approximately 1 hr), requires relatively large volume of samples and reagents, and its end-point detection method is rather subjective. To solve these problems, we attempted to develop a miniaturized LOC (lab-on-a-chip) prototype using PDMS and glass. Using the 62 mm (length) ${\times}$ 18 mm (width) prototype in which 2 mm (width) ${\times}$ 44.34 mm (length) ${\times}$ 100 $\mu\textrm{m}$ (depth) microfluidic channel was provided, we compared the various detection methods of gellation, turbidometric, and chromogenic assays to find the chromogenic method to be the most suitable for small volume assay. In this assay, kinetic point method was more accurate than end point method. We also found the PDMS chip thickness should be minimized to around 2 mm to allow sufficient light transmittance, which necessitated a glass slide bonding for chip rigidity. Through the miniaturization, the test time was reduced from 1 hr to less than 10 minutes, and the sample volume could be reduced from 100 ${\mu}\ell$ to 4.4 ${\mu}\ell$. In sum, this study revealed that the mini LOC could be an alternative for a semi-automated and reliable method for LAL test.

• Mycobiology
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• v.39 no.2
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• pp.129-132
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• 2011

To determine the optimal media conditions for the detection of the extracellular cellulase activity in Ganoderma neo-japonicum, we varied three media conditions: dye reagent, pH, and temperature. We evaluated the use of four dyes, Congo red, phenol red, remazol brilliant blue, and trypan blue. To observe the effect of pH on the chromogenic reaction, we tested media ranging from 4.5 to 8.0. To research the effect of temperature on the clear zone and the fungus growing zone, we tested temperatures ranging from 15 to $35^{\circ}C$. On the whole, the best protocol called for Ganoderma neo-japonicum transfer onto media containing Congo red with a pH of 7.0, followed by incubation at $25^{\circ}C$ for 5 days. Our results will be useful to researchers who study extracellular enzyme activity in Ganoderma neo-japonicum.

• Mycobiology
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• v.37 no.4
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• pp.313-316
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• 2009

To determine the optimal medium conditions for the detection of the cellulolytic activity in Ganoderma lucidum, we varied three media conditions: dye reagent, pH, and temperature. First, we evaluated the use of four dyes, Congo Red, Phenol Red, Remazol Brilliant Blue, and Trypan Blue. To observe the effect of pH on the chromogenic reaction, we also made and tested various media spanning acidic and alkaline pHs, ranging from 4.5 to 8.0. Furthermore, in order to research the effect of temperature on the clear zone and the fungus growing zone, we tested temperatures ranging from 15 to $35{^{\circ}C}$. On the whole, the best protocol called for Ganoderma lucidum transfer onto media containing Congo red with pH adjusted to 7.0, followed by incubation at $25{^{\circ}C}$ for 5 days. Our results will be useful to researchers who aim to study extracellular enzyme activity in Ganoderma lucidum.

• The Korean Journal of Mycology
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• v.39 no.2
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• pp.99-104
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• 2011

Shiitake breeding requires the procedures of mating of two different parental strains and selection of hybrid strains that have good traits for the mushroom production. In this study, we tested the possibility of the use of chromogenic plate-based assay for extracellular enzyme production in order to assess and find good biochemical properties-possessed hybrid strains that were generated from genetic cross of the monokaryotic strains derived from two different parental strains of Lentinula edodes Sanjo-101ho and Sanjo-108ho. We observed that there was difference in the ability of producing ${\beta}$-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease among the monokaryotic strains. We could also comparatively assess that the ability of the seven extracellular enzymes production in the hybrid strains depended on the mating combination of the monokaryotic strains. Our results demonstrate that the assessment method for extracellular enzyme production using chromogenic plate assay could be usefully applied to the assessment of the hybrid strains derived from the breeding procedure of L. edodes.

• Bulletin of the Korean Chemical Society
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• v.18 no.1
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• pp.44-50
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• 1997

A method of dual-signal generation from two different enzymes was developed and utilized to simultaneously perform dual immunoassays in a single microwell. Two enzymes selected as tracers were horseradish peroxidase (HRP) and β-galactosidase (GAL). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) and chlorophenolred-β-galactopyranoside (CPRG) as chromogenic substrates for the respective enzyme were used. Although the two enzymes showed their maximum activities at distinct pH conditions (pH 5.1 for HRP and 7.5 for GAL), the enzyme reactions were able to be concurrently carried out at pH 5.75 in a dual-substrate solution without signal loss. This performance was achieved by increasing TMB concentration two-fold, introducing potassium salt as activator of GAL reaction, and extending total reaction time 50%. The signal generation method was then used for dual-enzyme immunoassays to detect antibodies with co-immobilized Hepatitis C virus antigens (core and NS5) and a Hepatitis B virus antigen (PreS(2)) in a microwell. Dose-response curves of the assays revealed cooperativity between different antigen-antibody complex formation, which suggested that dual immunoassays can only be used for qualitative screening tests unless the antigens immobilized were spatially separated.

• Journal of the Korean Chemical Society
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• v.19 no.4
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• pp.246-251
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• 1975

A spectrophotometric assay technique is descriead for the measurement of free SH-groups in the enzyme reaction mixture. The method utilizes a new substrate, S-hippuryl-thioglycolyl-glycine(S-Hip-thioglycol-Gly) which is the basis for a convenient assay of angiotensin-converting enzyme and other dipeptidyl carboxypeptidases. This substrate contains an appropriately located thioester linkage that is hydrolyzed by the converting enzyme and other dipeptidyl carboxypeptidases. One of the products, thioglycolyl glycine, is readily measured by reaction with Ellman's reagent, 5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB, to produce 5-thio-2-nitrobenzoic acid which has a strong absorption band at 410 nm. The method is sensitive (${\varepsilon}M = 1.36{\times}10^4$ at 412 nm) and can be applied as a continuous recording with DTNB present in the enzymatic reaction mixture.

• Membrane and Water Treatment
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• v.14 no.4
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• pp.181-189
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• 2023

The Covid-19 pandemic has increased demand for chlorine-based sanitizing solutions, most of which contain hypochlorous acid (HOCl) as an active agent. Free chlorine (HOCl) in these sanitizers is crucial for their efficacy. Disposable test strips are affordable and convenient tools for determining various qualitative and quantitative parameters. In this study, disposable opto-chemical test strips were developed by physically immobilizing 3,3',5,5'-tetramethylbenzidine (TMB) and o-dianisidine (o-D) reagents on chromatography and filter paper-based test strips for the visualization and detection of free chlorine in the form of HOCl. The reagents undergo a rapid color change upon reaction with chlorine through a redox reaction. The paper-based test strips showed rapid color change within a minute and a low sample volume requirement (1 ml). This portable, disposable paper-based test strip is a simple and cost-effective way to rapidly detect the presence of HOCl sanitizers for home and field applications. Both TMB and o-D successfully detected chlorine. Chromatography paper proved to be the more efficient option among the two papers used as substrates for the reagents (TMB and o-D). It exhibited high retention capacity and high performance in terms of color transformation when reacting with HOCl, even after two months of storage.