溫度對於Pseudomonas putida鄰苯二酚加氧酵素之活性與結構的效應

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許原彰(Yuan-Chang Hsu) 查詢紙本館藏

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論文名稱

溫度對於Pseudomonas putida鄰苯二酚加氧酵素之活性與結構的效應

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 鄰苯二酚加氧酵素
★ 熱穩定性
★ 小角度中子散射

關鍵字(英)

★ Pseudomonas putida
★ catechol 2
★ 3-dioxygenase
★ SANS

論文目次

中文摘要……………………………………………………I
英文摘要……………………………………………………IV
目錄…………………………………………………………VI
圖目錄………………………………………………………VII
表目錄………………………………………………………VI
壹、緒論……………………………………………………1
一、芳香族化合物及微生物分解…………………………1
二、鄰苯二酚加氧酵素之特性……………………………2
三、鄰苯二酚加氧酵素之結構……………………………4
四、鄰苯二酚加氧酵素之熱穩定性………………………6
五、小角度中子散射於酵素結構之應用…………………7
六、研究背景與目的………………………………………9
貳、材料與方法……………………………………………13
一、微生物培養……………………………………………13
二、鄰苯二酚加氧酵素活性測定…………………………15
三、蛋白質測定……………………………………………16
四、鄰苯二酚加氧酵素之純化……………………………17
五、分子量測定……………………………………………20
六、鄰苯二酚加氧酵素熱穩定性測定……………………21
七、小角度中子散射………………………………………22
八、酵素穩定因子測試……………………………………22
九、N端胺基酸定序…………………….…………………23
十、化學藥品………………………………………………23
十一、儀器設備……………………………………………24
參、結果……………………………………………………26
一、C23Os N端胺基酸序列………………….……………26
二、C23O活性分析…………………………………………26
三、C23Os之純化………………………………….………28
四、溫度對C23Os活性之效應………………….…………31
五、溫度對C23Os結構之效應……….……………………33
六、C23O穩定因子測試……………………………………34
肆、討論……………………………………………………35
伍、參考文獻………………………………………………41
陸、圖………………………………………………………47
柒、表………………………………………………………68

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2000-7-18

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