| 摘要: | 本實驗室從台灣受石油污染地區篩選出一株可分解、酚和甲酚等芳香族類化合物之分解菌 Pseudomonas putida SH1 。在之前研究中，我們已了解本株菌在異化（catabolize ）、酚和鄰-甲酚時分別生誘發成四種生化性質不同之苯環切割酵素：皆具有鄰苯二酚加氧酵素（catechol 2,3-dioxygenase, C23O ）活性；分別命名為C23Onap(SH1)、C23OpheI(SH1)、C23OpheII(SH1)及 C23Oo-cre(SH1)。並分析P. putida SH1 菌株中不同的鄰苯二酚加氧酵素之生化特性，同時以小角度中子散射（small-angle neutron scattering, 簡稱SANS ）技術，在5-80 ℃範圍內研究酵素在水溶液中的結構與高溫對結構的效應。實驗結果裡都顯示在催化活性與小角度中子散射實驗中已知的分解菌分離出之（P. putida NCIB9816-4 ）C23Onap 比C23Onap(SH1)有較高的熱穩定性。 A soil bacterium, Pseudomonas putida SH1, was isolated from petroleum-contaminated area in Taiwan. In our previously study, this bacterial strain catabolizes naphthalene, phenol, o-, m-, or 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 C23Os were purified and characterized namely C23Onap(SH1), C23OpheI(SH1), C23OpheII(SH1) and C23Oo-cre(SH1). The thermal effect on C23Onap(SH1) was observed by enzyme stability as well as conformational change. The optimally catalytic temperature of C23Onap(SH1) was at 50?XC in phosphate buffer at pH 7.5. 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 at 50?XC. The structural conformation the enzymes in solution was analyzed by using small-angle neutron scattering (SANS) technique. SANS measurements revealed distinct changes on the size of C23Onap(SH1) between 57 and 80?XC, where the size can not be restored even the temperature was then reduced. At 80?XC, the size of the enzyme (230? ) becomes more than twice of its native one (95?). Accordingly, the enzyme starts to denature at 57?XC and the structure was destroyed as temperature reaches 80?XC. The C23Onap(NCIB9816-4), was also studied under the same experimental conditions with the transition temp of conformation change at 62?XC. This enzyme shows slightly higher heat stability in both catalytic activity and conformation by SANS studies. 研究期間：9001 ~ 9012 |
