可降解酚之Candida albicans TL3及其catechol 1,2-dioxygenase之單離與特性探討

Abstract

本研究已從土壤裡單離出一株可利用酚做為唯一碳源的酵母菌－Candida albicans TL3；相較於其它可降解酚之微生物而言，這菌株不僅對酚具有較高的耐受性而且分解酚的速率也相對較快，它對酚的降解能力目前是第一次被發現的。基於酵素活性、層析及質譜等分析，我們推論此菌株是經由ortho-fission途徑分解酚。涉及此途徑的相關酵素－phenol hydroxylase和catechol 1,2-dioxydanase兩者皆是可誘導性酵素，當C. albicans TL3被培養在培養液分別含酚濃度是22 mM和10 mM時，可達到最大活性。另外，此菌株除了能降解甲醛樹脂工廠廢液中的酚之外，亦可對甲醛加以降解。 藉由硫酸銨沉澱、Sephadex G-75凝膠過濾和HiTrap Q Sepharose管柱層析，可從C. albicans TL3中分離出一高純度的catechol 1,2-dioxydanase（1,2-CTD）；此酵素是由相同的兩個單體所組成的，每一個單體的分子量是32,000 Da且含一個鐵離子。此真菌的1,2-CTD的pI值、最適溫度和最適酸鹼值分別為5.3~5.7、25℃和pH 8.0。由受質特異性之研究顯示，此酵素應屬於type Ι catechol 1,2-dioxydanase。這是首次有關來自真核細胞之catechol 1,2-dioxygenase的研究報導。在二維電泳膠片上，可看到此純化的1,2-CTD具有五個分子量相近但等電點稍微不同的蛋白質點；這五種異構型態的1,2-CTD可能是不同程度的轉譯後修飾作用所造成的。利用Edman降解和MALDI-TOF/TOF對經胰蛋白酶水解後的此1,2-CTD裂解之胜肽片段進行序列分析，所得的序列結果可比對到一與其具高度相同性來自於Candida albicans SC5314的假想蛋白質－CaO19_12036 (GenBank accession no. XM 717691)；我們建議此一假想蛋白質應該是一1,2-CTD。

A yeast strain isolated from soil was able to utilize phenol as the sole carbon source and was further identified as Candida albicans TL3. This microbe possesses higher tolerance on phenol (24 mM) as well as stronger activity on the rate of phenol degradation than other microorganisms at 30℃. The capability of this strain on phenol degradation is first reported herein. Based on the enzymatic, chromatographic and mass spectrometric analyses, we concluded that C. albicans TL3 follows the ortho-fission pathway on phenol degradation. The optimal activity of phenol hydroxylase and catechol 1,2-dioxygenase were found when this strain grew in culture media containing 22 mM and 10 mM phenol, respectively. In addition to phenol, C. albicans TL3 also exhibited catalytic power on degrading formaldehyde in wastewater directly obtained from phenolic resin-producing factory. The catechol 1,2-dioxygenase (1,2-CTD) induced from Candida albicans TL3 was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and characterized to be a homodimer, with a molecular weight of 32,000 Da for each subunit. The investigation of this eukaryotic 1,2-CTD revealed that the iron content for each subunit, pI value, optimal temperature, and optimal pH are 1 iron/subunit, 5.3~5.7, 25℃ and pH 8.0, respectively. Substrate analysis showed that the purified enzyme belongs to the type I catechol 1, 2-dioxygenase. The study on this eukaryotic 1,2-CTD was reported for the first time. On 2-D gel analysis of the purified 1,2-CTD, five spots with approximately similar molecular weight but with different pIs were found. These spots were further analyzed by MALDI-TOF mass spectrometry. Results suggested that these spots (isotypes) were derived from the same 1,2-CTD. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF analysis provide information of amino acid sequences for BLAST search, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314 (GenBank accession no. XM 717691). We, thus, suggested that the hypothetical protein should be 1,2-CTD.