• Mycobiology
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• 제34권2호
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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.

• 약학회지
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• 제43권5호
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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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• 제9권2호
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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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• 제18권5호
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• pp.429-433
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• 2003

LAL 측정용 chip을 제작하기 위해서 우선 시료의 부피 감소에 대한 비탁법과 비색법을 비교하였다. 비색법은 낮은 부피에서 높은 감도를 보여 주었으며 시료의 부피와 무관하게 같은 endotoxin의 농도에서는 같은 흡광도를 보인다는 결론을 얻었다. Endotoxin의 농도에 따른 표준곡선을 end point법과 kinetic point법을 비교한 결과 대한약전의 기준에 적합한 kinetic point법이 적합하였다. 이러한 기초 실험결과를 통해 PDMS LOC를 제작하여 LAL 시험을 수행하였다. LOC를 이용하여 더 짧은 시간과 더 작은 시료로 시험이 가능하도록 하였다. 특히 PDMS LOC는 복잡한 channel을 쉽게 만들 수 있을 뿐 아니라 mold를 이용하여 상용화를 위한 대량 생산이 가능하다. 따라서 PDMS를 이용한 LOC의 제작과 실험을 통해 기존의 수작업의 LAL 시험을 LOC를 이용한 다중시료 측정과 자동화의 가능성을 제시하였다.

• Mycobiology
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• 제39권2호
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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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• 제37권4호
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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.

• 한국균학회지
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• 제39권2호
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• pp.99-104
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• 2011

표고버섯의 육종은 두 개의 다른 모균주에 의한 교배와 버섯생산에 좋은 형질을 지닌 교잡균주의 선발이 요구된다. 본 연구에서는 서로 다른 두 계통의 표고 모균주에서 유래한 단핵균주와 이들 단핵균주간의 교배로부터 만들어진 교잡균주에 대하여 발색반응 배지를 이용하여 세포외 분해효소의 분비능력 정도를 비교하여 생화학적 특성이 우수한 균주를 선발할 가능성에 대하여 조사하였다. 모균주로부터 유래한 단핵균주들 간에 ${\beta}$-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, protease의 분비능력에 차이가 있음을 확인하였다. 교잡균주에 있어서도 단핵균주의 조합에 따라 효소 분비능력이 달라지는 것을 볼 수 있었다. 이에 따라 발색반응배지를 이용한 세포외효소 평가법이 표고의 육종과정 중에 생성되는 교잡균주들의 평가에 활용 할 수 있을 것으로 사료된다.

• Bulletin of the Korean Chemical Society
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• 제18권1호
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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.

• 대한화학회지
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• 제19권4호
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• pp.246-251
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• 1975

Dideptidyl carboxypeptidases와 angiotenisn-coverting enzyme의 새로운 기질불질로서 thiol ester 인 S-Hippuryl thioglycolyl glycine을 합성하였으며, 이 기질에 의한 간편하고도 예민한 효소 활성도의 정량방법을 제시하였다. 이 경우 효소반응 생성물인 thioglycolyl glycine은 반응계중에 첨가한 5,5-dithio-bis-(2-nitrobenzoic acid), DTNB와 쉽게 반응하여 410nm에서 강한 흡광스펙트럼을 갖는 5-thio-2-nitrobenzoic acid(${\varepsilon}M=1.36{\times}10^4$)을 형성함으로서 효소의 새로운 미량정량 방법으로 이용 가치가 크다고 본다.

• Membrane and Water Treatment
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• 제14권4호
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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.