Lisboa Helen Cristina Fávero, Biasetto Carolina Rabal, de Medeiros João Batista, Âraújo Angela Regina, Silva Dulce Helena Siqueira, Teles Helder Lopes, Trevisan Henrique Celso
Instituto de Química, Universidade Estadual Paulista "Julio de Mesquita Filho", Araraquara, SP, Brazil. ; Instituto de Ciências Exatas e Naturais, Universidade Federal de Mato Grosso, Rondonópolis, MT, Brazil.
Instituto de Química, Universidade Estadual Paulista "Julio de Mesquita Filho", Araraquara, SP, Brazil.
Braz J Microbiol. 2014 Jan 10;44(3):923-6. doi: 10.1590/s1517-83822013005000067. eCollection 2013.
A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS). The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project "Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest". The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1--carboxyl-esterases) for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue), changing the color of the reaction medium (from blue to yellow), that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea as a potential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms.
为开发高通量筛选（HTS）技术，优化了一种灵敏高效的比色法，用于检测内生真菌产生的酯酶。这些真菌先前是从巴西圣保罗州环境保护区内的塞拉多和大西洋森林的植物物种中分离得到的，这是“与塞拉多和大西洋森林植物物种相关的内生真菌的化学和生物学勘探”项目的一部分。使用丁酸乙酯、乙酸乙酯和丙酸甲酯作为标准酯，它们被内生真菌的细胞外酶（EC. 3.1.1.1 - 羧基酯酶）水解生成羧酸。因此，pH值的降低会增加质子化指示剂（溴百里酚蓝）的浓度，从而改变反应介质的颜色（从蓝色变为黄色），这可以通过在616 nm处的分光光度法进行观察和测量。采用酸碱指示剂的方法对13种微生物进行了检测，将紫黑附球菌作为短链酯生物转化的潜在酯酶来源。结果还证明，该方法高效、快速、廉价，试剂消耗低且易于操作，可用于筛选大量微生物中的羧酸酯水解酶。
