Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity
The effects of reaction conditions on the spectrophotometric and fluorometric assays using alternative substrates (p-nitrophenyl palmitate and 4-methylumbelliferyl oleate) were investigated to optimize them for the high-throughput screening of lipase activity from agricultural products. Four model l...
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| Format: | Article |
| Language: | English |
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Wiley
2021-01-01
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| Series: | Journal of Chemistry |
| Online Access: | http://dx.doi.org/10.1155/2021/3688124 |
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| author | Jun-Young Park Jisu Ha Yoonseok Choi Pahn-Shick Chang Kyung-Min Park |
| author_facet | Jun-Young Park Jisu Ha Yoonseok Choi Pahn-Shick Chang Kyung-Min Park |
| author_sort | Jun-Young Park |
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| description | The effects of reaction conditions on the spectrophotometric and fluorometric assays using alternative substrates (p-nitrophenyl palmitate and 4-methylumbelliferyl oleate) were investigated to optimize them for the high-throughput screening of lipase activity from agricultural products. Four model lipases from Chromobacterium viscosum, Pseudomonas fluorescens, Sus scrofa pancreas, and wheat germ (Triticum aestivum) were allowed to hydrolyze the alternative substrates at different substrate concentrations (1–5 mM), operating pH (5.0–8.0), and operating temperatures (25–55°C). The results show that both the spectrophotometric and fluorometric assays worked well at the standard reaction conditions (pH 7.0 and 30°C) for finding a typical lipase, although pH conditions should be considered to detect the catalytic activity of lipases, which are applicable to more acidic or alkaline pH circumstances. To validate the optimized conditions, the high-throughput screening of lipase activity was conducted using 17 domestic agricultural products. A pileus of Pleurotus eryngii showed the highest activity in both the spectrophotometric (633.42 μU/mg) and fluorometric (101.77 μU/mg) assays. The results of this research provide practical information for the high-throughput screening of lipases using alternative substrates on microplates. |
| format | Article |
| id | doaj-art-2021fffe824d4c69a8fde3ccc0cb66df |
| institution | Kabale University |
| issn | 2090-9063 2090-9071 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Wiley |
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| series | Journal of Chemistry |
| spelling | doaj-art-2021fffe824d4c69a8fde3ccc0cb66df2025-02-03T01:24:45ZengWileyJournal of Chemistry2090-90632090-90712021-01-01202110.1155/2021/36881243688124Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase ActivityJun-Young Park0Jisu Ha1Yoonseok Choi2Pahn-Shick Chang3Kyung-Min Park4Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of KoreaDepartment of Food Science and Biotechnology, Wonkwang University, Iksan 54538, Republic of KoreaDepartment of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of KoreaDepartment of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of KoreaDepartment of Food Science and Biotechnology, Wonkwang University, Iksan 54538, Republic of KoreaThe effects of reaction conditions on the spectrophotometric and fluorometric assays using alternative substrates (p-nitrophenyl palmitate and 4-methylumbelliferyl oleate) were investigated to optimize them for the high-throughput screening of lipase activity from agricultural products. Four model lipases from Chromobacterium viscosum, Pseudomonas fluorescens, Sus scrofa pancreas, and wheat germ (Triticum aestivum) were allowed to hydrolyze the alternative substrates at different substrate concentrations (1–5 mM), operating pH (5.0–8.0), and operating temperatures (25–55°C). The results show that both the spectrophotometric and fluorometric assays worked well at the standard reaction conditions (pH 7.0 and 30°C) for finding a typical lipase, although pH conditions should be considered to detect the catalytic activity of lipases, which are applicable to more acidic or alkaline pH circumstances. To validate the optimized conditions, the high-throughput screening of lipase activity was conducted using 17 domestic agricultural products. A pileus of Pleurotus eryngii showed the highest activity in both the spectrophotometric (633.42 μU/mg) and fluorometric (101.77 μU/mg) assays. The results of this research provide practical information for the high-throughput screening of lipases using alternative substrates on microplates.http://dx.doi.org/10.1155/2021/3688124 |
| spellingShingle | Jun-Young Park Jisu Ha Yoonseok Choi Pahn-Shick Chang Kyung-Min Park Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity Journal of Chemistry |
| title | Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity |
| title_full | Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity |
| title_fullStr | Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity |
| title_full_unstemmed | Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity |
| title_short | Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity |
| title_sort | optimization of spectrophotometric and fluorometric assays using alternative substrates for the high throughput screening of lipase activity |
| url | http://dx.doi.org/10.1155/2021/3688124 |
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