Two hundred thirty-two nonfilamentous bacterial strains, including saprophytes, plant pathogens, and opportunistic plant and human pathogens, were screened for the ability to produce cutinases (cutin-degrading esterases). Initially, esterase activity of culture filtrates of strains grown in nutrient broth-yeast extract medium supplemented with 0.4% apple or tomato cutin was determined by a spectrophotometric assay utilizing the model substrate p-nitrophenyl butyrate. The culture filtrates of the 10 Pseudomonas aeruginosa strains tested exhibited the highest esterase activity, with values of >500 nmol/min/ml. Of these 10 strains, 3 (K799, 1499A, and DAR41352) demonstrated significant induction (10-fold or above) of esterase activity by addition of cutin to nutrient broth-yeast extract medium. The ability of culture filtrates of the three strains to cause release of apple cutin monomers was confirmed by a novel high-performance liquid chromatography technique. Monomer identification was confirmed by gas chromatography-mass spectroscopy analyses. Addition of the nonionic detergent n-octylglucoside stimulated cutinase activity of culture filtrates from strains K799 and DAR41352, but not that of filtrates from strain 1499A. Time course studies in nutrient broth-yeast extract medium supplemented with apple cutin indicated maximal levels of cutinase in the culture fluids after cultures entered stationary phase. Incubation temperatures below the optimal temperature for growth (37°C) led to maximal production of cutinase.
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机译:筛选了包括腐生植物,植物病原体以及机会性植物和人类病原体在内的232个非丝状细菌菌株产生角质酶(角质素降解酯酶)的能力。最初,使用模型底物对硝基苯基丁酸酯通过分光光度法测定在补充有0.4%苹果或番茄角质的营养肉汤酵母提取培养基中生长的菌株的培养滤液的酯酶活性。测试的10个铜绿假单胞菌菌株的培养物滤液表现出最高的酯酶活性,其值> 500 nmol / min / ml。在这10个菌株中,有3个(K799、1499A和DAR41352)通过在营养肉汤酵母提取培养基中添加角质显示出明显的酯酶活性诱导作用(10倍或以上)。通过新型高效液相色谱技术证实了这三种菌株的培养滤液引起苹果角质单体释放的能力。单体鉴定通过气相色谱-质谱分析确认。加入非离子去污剂正辛基葡糖苷刺激了菌株K799和DAR41352的培养滤液的角质酶活性,但不刺激菌株1499A的滤液的角质酶活性。在补充了苹果角质的营养肉汤酵母提取培养基中进行的时程研究表明,培养液进入稳定期后,培养液中的角质酶含量最高。孵化温度低于最佳生长温度(37°C)导致角质酶产量最大化。
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