產鹼假單孢菌A25分解鄰苯二甲酸二酯(DEHP)之研究及其胞外酯解酵素分析

Abstract

鄰苯二甲酸酯 (Phthalates，PAEs) 為鄰苯二甲酸 (Phthalic acid) 酯化後生成的一類化合物，又統稱為鄰苯二甲基酯類，一般揮發性低，且穩定性高，為無色的黏稠油狀液體，對於水的溶解度極低。而PAEs經常被當作塑化劑 (plasticizers) 使用，塑化劑為塑膠添加物以改良塑膠的特性，能提供塑膠彈性、透明度、耐用性及增加使用壽命等功能，但塑化劑及塑膠製品之間並非共價鍵結合，因此若塑膠經陽光曝曬、高溫加熱或接觸有機溶液，便有機會使塑膠中的塑化劑釋放到環境中，除此之外塑膠老化也會加快塑化劑析出的進程。 在台灣行政院保護署的調查當中，發現台灣河川底泥及魚體所含最大宗的PAE即為DEHP (附錄二)。而在2011年時台灣發生塑化劑食品安全事件，不肖業者將廉價的DEHP當作食品添加劑代替合法的起雲劑添加到食品當中，經研究證明DEHP的毒性要比三聚氰胺毒上3.5至20倍，DEHP被認為是潛在的致癌劑、環境賀爾蒙及代謝干擾物，勢必會嚴重影響民眾的健康，因此解決環境中汙染物DEHP成為一項重要的課題。 本實驗發現產鹼假單孢菌A25 (Pseudomonas alcaligenes A25) 具有降解DEHP的能力，且分解效率佳，以高效液相色譜法分析DEHP的降解曲線，在低濃度 (100 ppm) 的DEHP條件下約24小時即可降解76%的DEHP，在高濃度 (600 ppm) 的DEHP條件下約48小時可以降解87%的DEHP，而經192小時可以降解98%的DEHP。又經實驗測試得知Pseudomonas alcaligenes A25經DEHP的誘導可以產生胞外酵素，該胞外酵素經過三丁酸甘油酯瓊脂培養基及酵素活性膠體染色的測驗中確認具有酯解酵素活性，在酵素活性測試中得知最佳反應溫度為40℃、酸鹼值為8.0，同時測試不同介面活性劑對於此酵素的活性影響。Pseudomonas alcaligenes A25為一菌種可以有效率的降解DEHP，且產生之降解酵素為胞外蛋白質易於大量收集，將來或許可應用於處理環境中的塑化劑汙染物DEHP。

Phthalates, or phthalate esters, are esters of phthalic acid and are with characteristics of general low volatility and high stability, as a colorless viscous oily liquid, very low solubility in water. Phthalates are mainly used as plasticizers, substances added to plastics to increase their flexibility, transparency, durability and longevity. There is no covalent bond between the phthalates and plastics. So if the plastic by exposure to sunlight, high temperature heating or organic solvents, it has the opportunity to make the plasticizers released into the environment. Moreover, plastic aging will accelerate this process. The studies by EPA Taiwan found that DEHP is most abundant phthalate in Taiwan river sediment and fish body (Appendix. 2). In 2011 Taiwan plasticizer food safety incidents occur, unscrupulous industry use cheap DEHP as a food additive instead of a legal Cloudy agent added to food products. The study shows that the DEHP toxicity is more than melamine 3.5 to 20 times. DEHP is considered a potential carcinogen, environmental hormone and metabolic disruptors and is bound to seriously affect the health of the people. Thus, to solve environmental pollutants, DEHP becomes an important issue. Our study found that Pseudomonas alcaligenes A25 have the ability to degrade DEHP with good degradation efficiency. We analyze the degradation rate of DEHP by High performance liquid chromatography. At low concentrations (100 ppm) of DEHP about 24 hours to degrade 76% of DEHP, and at high concentrations (600 ppm) for about 48 hours to degrade 87% of DEHP. The strain A25 could degrade 98% DEHP within 192 hours. We found that strain A25 produce extracellular enzyme by DEHP induction. The extracellular enzyme after tributyrin agar medium and esterase activity staining tests confirmed having esterase activity. The optimal DEHP degradation conditions were 40℃ and pH 8.0. The addition of different nutrient sources and surfactants will affect degradation of DEHP. P. alcaligenes A25 as a newly discovered specie can efficiently degrade DEHP and produces extracellular esterase for DEHP degradation. It has the potential to degrade DEHP, perhaps can be applied to treatment of environmental pollutants DEHP.