| 摘要: | 本實驗室從台灣本土受石油污染地區篩選出一株可分解?（naphthalene）、酚（phenol）、和甲酚（cresols）等芳香類化合物之分解菌Pseudomonas putida SH1。在之前研究中，我們已了解本株菌在異化?、酚和鄰-甲酚（o-cresol）分別生成四種鄰苯二酚加氧酵素（catechol 2,3-dioxygenase, C23O）將苯環切割形成直鏈再進入下游中央代謝系統，故可以利用此類苯環化合物為唯一碳源與能源而生長；先前已純化之四種鄰苯二酚加氧酵素分別命名為C23Onap(SH1)、C23OpheI(SH1)、C23OpheII(SH1)及C23Oo-cre(SH1)，由其他細菌異化苯環化合物之基因組成皆為形成operon型式且單獨誘發各自之C23O基因表現，推測P. putida SH1在異化不同碳源時是經由不同的operon和代謝途徑來轉化中間產物鄰苯二酚（catechol），因而產生多套的鄰苯二酚加氧酵素。本實驗針對此菌株在異化甲酚之三種異構物時是否使用不同的鄰苯二酚加氧酵素，則分別以?、鄰-甲酚、間-甲酚（m-cresol）和對-甲酚（p-cresol）作為P. putida SH1的單一生長碳源來誘導菌體產生鄰苯二酚加氧酵素，再以鄰苯二分及其衍生物（3-methylcatechol、4- methylcatechol和4-chlorocatechol）四種基質進行此酵素之活性分析，發現P. putida SH1以三種甲酚異構物為單一生長碳源所誘導產生的酵素對上述基質之催化特異性相似，因此推測P. putida SH1在異化三種甲酚異構物時可能是產生同一種鄰苯二酚加氧酵素。 在了解SH1菌株中不同的鄰苯二酚加氧酵素生化特性後，我們以C23Onap(SH1)和已知?的分解菌P. putida NCIB9816-4所純化出之C23Onap(NCIB9816-4)研究溫度對酵素活性的影響；同時根據小角度中子散射（small angle neutron scattering, SANS）實驗，在不同溫度下研究酵素在水溶液中的結構，如分子大小與四級結構等。從生化實驗顯示C23Onap(SH1)是由4個相同的次單元(subunit)所組成，最適催化溫度為50℃；在50℃時，C23Onap(SH1)活性半衰期不及1小時而C23Onap(NCIB9816-4)半衰期則為4小時，在相同溫度但無氧條件下，C23Onap(SH1)之活性半衰期增為3小時；此二酵素之活性分別在55℃與60℃急速下降（1小時後為15%和9%）。從SANS實驗所得結果顯示，在5~55℃時，二者分子大小皆約為60 Å，但C23Onap(SH1)在57℃、C23Onap(NCIB9816-4)在62℃時分子大小開始變大，當溫度升至80℃間時二者分子大小分別增為130 Å與120 Å，且即使回溫至5℃後酵素分子大小也不會回復，顯示酵素結構是不可逆性地被破壞；溫度對此二酵素的效應在催化活性與小角度中子散射實驗結果裡都顯示C23Onap(NCIB9816-4)比C23Onap(SH1)高出5℃的熱穩定性。 此計畫未來研究方向將尋找影響酵素熱穩定性與結構中每個subunit相互連接處之胺基酸，並以蛋白質工程進行酵素之改良。 A soil bacterium, Pseudomonas putida SH1, was isolated from a petroleum-contaminated area in Taiwan. In our previously study, the bacterium catabolizes naphthalene, phenol, o-, m-, and p-cresol as sole source of carbon and energy to grow. Extradiol dioxygenases, catechol 2,3-dioxygenases (C23Os), involved in the cleavage of aromatic ring were induced when the bacterium was grew in minimal salts basal medium containing individual aromatic compound. Four of them were purified and characterized namely C23Os as C23Onap(SH1), C23OpheI(SH1), C23OpheII(SH1) and C23Oo-cre(SH1). The specific activities of C23Os induced by 3 isomers of cresol in crude cell extracts from P. putida SH1 was determined towards to catechol and and catabolic derivatives, 3-methylcatechol, 4- methylcatechol and 4-chrolocatechol. It showed the same substrate specificity, suggesting this strain converts o-, m-, and p-cresol to catechol probably by the same C23O. The thermal effect on C23O was then observed by the stability of activity as well as conformation change at various temperatures. The optimally catalytic temperature of C23Onap(SH1) was at 50℃ in 20 mM phosphate buffer, pH 7.5 but the half-life of C23Onap(SH1) at this temperature was 30 min under aerobic condition. The half-life of another C23O, C23Onap(NCIB9816-4) isolated from a known naphthalene-degrading bacterium, P. putida NCIB9816-4, was 4 hour under the same condition. It indicated that C23Onap(NCIB9816-4) was more stable than C23Onap(SH1). The structural conformation of the enzymes in solution was analyzed by using small-angle neutron scattering (SANS) technique. SANS measurements revealed distinct changes on the size of the C23Onap(SH1) between 57 and 80°C, where the size can not be restored even the temperature was then reduced. At 80°C, the size of the enzyme becomes more than twice of its native one. Accordingly, the enzyme starts to denature at 57°C and the structure was destroyed as temperature reaches 80°C. Another enzyme, C23Onap(NCIB9816-4), was also studied under the same experimental conditions. This enzyme shows slightly higher heat stability, about 5°C, in both catalytic activity and conformation. In the meantime, the factors influencing thermal stability, including Fe2+, acetone, ethanol, and starch, on C23O were further investigated by using C23O purified from p. putida mt-2. |
