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.

中文翻译：

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使用替代底物对脂肪酶活性进行高通量筛选的分光光度法和荧光测定法的优化

研究了反应条件对使用替代底物（对硝基苯基棕榈酸酯和 4-甲基伞形酮油酸酯）的分光光度法和荧光测定法的影响，以优化它们以对来自农产品的脂肪酶活性进行高通量筛选。从四个模型脂酶粘稠色，荧光假单胞菌，野猪胰腺和小麦胚芽（小麦) 被允许在不同的底物浓度 (1-5 mM)、操作 pH (5.0-8.0) 和操作温度 (25-55°C) 下水解替代底物。结果表明，分光光度法和荧光法在标准反应条件（pH 7.0 和 30°C）下均能很好地找到典型的脂肪酶，但应考虑 pH 条件来检测脂肪酶的催化活性，这适用于更多的脂肪酶。酸性或碱性 pH 值环境。为了验证优化条件，对17种国内农产品进行了脂肪酶活性的高通量筛选。杏鲍菇菌盖在分光光度法 (633.42 μ U/mg) 和荧光法 (101.77 μ U/mg) 中均表现出最高活性 U/mg) 测定。这项研究的结果为在微孔板上使用替代底物对脂肪酶进行高通量筛选提供了实用信息。