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研究生:康吉良
研究生(外文):Kang, Chi-Liang
論文名稱:Micrococcussp.NTU-7C苯甲酸1,2-雙加氧脢之研究
論文名稱(外文):Studies on Benzoate 1,2-dioxygenase from Microccocus sp. NTU-7C
指導教授:劉文雄劉文雄引用關係
指導教授(外文):Wen-Hsiung Liu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學系
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:110
中文關鍵詞:苯甲酸雙加氧脢
外文關鍵詞:benzoatedioxygenase
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中 文 摘 要
本研究以苯甲酸、鄰苯二酚、 對-基苯甲酸 、磺酸基柳酸為碳源培養
Micrococcus sp. NTU-7C可同時誘導生產菌體內NADH -DCIP 還原及苯
甲酸1,2-雙加氧。當合成培養基中苯甲酸的濃度為5 g / L、酵母萃出
物濃度為10 g / L 、二價鐵離子濃度為1.5 g / L 時苯甲酸1,2-雙加氧
之生產最有利。菌體經破碎、 鏈黴素處理、硫酸銨鹽析、酸處理、膠
體過濾管柱層析法 ( Sepharose CL-6B ) 以及陰離子交換樹脂 ( DEAE-
Sepharose CL-6B ) 等純化步驟後,苯甲酸1,2-雙加氧中的NADH-DCIP
還原及氧化比活性分別可提高20倍及17倍,回收率為16 % 及19 %。
NADH-DCIP還原的最適溫度為 42* C,最適 pH為6.8,在 pH 7較為穩定
,本酵素能利用cytochrome c、DCIP、ferricyanide、 nitroblue
tetrazolium 作為電子接受者。對NADH之Km值為9.89μM。硫氫基抑制劑
pCMB ( p-chloromercuribenzoic acid )、iodoacetate、5,5*-
dithiobis可抑制部份活性。不受金屬嵌合劑 KCN、EDTA之影響。 氧化
的最適溫度為 42 * C,最適 pH為8.3,在pH 7較為穩定。本酵素能有效
利用鹵化苯酸類及基苯酸類化合物。對苯甲酸之Km值為32.5μM,Cu2+
、 Mg2+、及 Mn2+ 等金屬離子可增加活性。硫氫基抑制劑iodoacetate
及5,5*-dithiobis可完全抑制酵素活性,電子接受者DCIP亦為酵素之抑制
因子。苯甲酸1,2-雙加氧需NADH作為輔因子且NAD無法取代NADH,而添
加FAD、FMN、riboflavin、Mg2+或 Fe2+ 皆可有效提升酵素活性。 以
硫氫基抑制劑碘乙酸( iodoacetate )測定酵素活性與467nm吸光值之關係
, 得知467 nm吸光值增加與酵素活性減少成正比,顯示黃素為維持酵素活
性之重要因子。此外酵素在可見光下417nm處有吸收波長,而當酵素液 (
pH 2.7 ) 與FAD、 FMN ( pH 2.7 ) 以螢光光度計偵測450 nm激發波長時
,發現522-525 nm有最大發射波長,故本酵素應為黃素蛋白(
flavoprotein )。 本菌株在培養過程中培養液經調酸後會產生紅色變
化,此紅色化合物在分光光度計下於485 nm有吸收高峰﹔以完全培養基培
養22小時所得之紅色最深,而紅色化合物之生成和菌體生長成平行關係。
培養基成份中以苯甲酸與酵母萃出物對紅色化合物的生成影響最大,兩者
必須同時存在才會有紅色化合物之生成。
ABSTRACT
NADH-DCIP reductase and oxygenase component in benzoate
1,2-dioxygenase system of Micrococcus sp. NTU-7C could be
induced in a synthetic medium by the addition of sodium benzoate
, catechol , p-hydroxybenzoate or sulfosalicylic acid as carbon
sources. The benzoate 1,2-dioxygenase activity was improved
efficiently when the concentration of sodium benzoate , yeast
extract and ferrous sulfate contained in the synthetic medium
were 5 g / L , 10 g / L , 1.5 g / L , respectively. The
oxygenase component and NADH-DCIP reductase com- ponent in
benzoate 1,2-dioxygenase system were purified sequencially by
lysozyme treatment, sonication disruption, ammonium sulfate
fractionation, acid treatment, Sepharose CL-6B and DEAE-
Sepharose CL-6B . The specificity activities of purified
oxygenase and reductase increased to 20 and 17 fold , and the
recoveries were about 16 % and 19 % , respectively.NADH-DCIP
reductase showed the maximun activity at pH 6.8 and 42℃,
respectively. It was stable at pH 7. The enzyme showed the
activities toward a variety of electron acceptor , such as DCIP,
ferricyanide, nitroblue tetrazolium and cytochrome c. The
apparent Km values of reductase for NADH was 9.89μM . The
reductase activity was partially inhibited by sulfhydryl
inhibitor pCMB , iodoacetate , 5,5*-dithiobis . The metal
chelating agents EDTA﹑KCN did not influence the reductase
activity.The optimal pH and optimal temperature of the oxygenase
were 8.3 and 42℃ , respectively . The oxygenase was stable at
pH 7. Benzoate analogs such as halobenzoates and hydroxy-
benzoates could be assimilated efficiently . The apparent Km
values of oxygenase for benzoate was 32.5μM. The oxygenase
activity could be promoted by the addition of Cu2+, Mg2+, Mn2+ .
The oxygenase activity was inhibited by sulfhydryl inhibitors
iodoacetate , 5,5*-dithiobis and electron acceptor DCIP.
Benzoate 1,2-dioxygenase required NADH for its activity . NADH
could not be replaced by NAD. Exogenous FAD , FMN , riboflavin ,
Mg2+ or Fe2+ were favorable for the enzyme activity . The
relationship between benzoate 1,2-dioxygenase activity and
absorption at 467nm was determined by sulfhydryl inhibitor,
iodoacetate. It was found that the increase of absorption at
467nm was inparallel with the decrease of benzoate
1,2-dioxygenase activity. It was suggested that flavin is an
important cofactor for the enzyme activity. The enzyme showed
absorption peaks at 417nm. Under acidic condition ( pH 2.7-3.0 )
the fluorescence spectrum of enzyme at emmision wavelength of
522nm was similar to that of FAD and FMN. It indicated that
benzoate 1,2-dioxygenase belongs to a flavoprotein .After the pH
was adjusted to acidic level , the culturesupernatant became red
color. The red color solution showed an absorption peaks at
485nm. The maximum production of red compound could be obtained
at 22h-cultivation in the complete medium. The production of red
compound was in parallel with cells growth. The production of
red compound was greatly influenced by the addition of benzoate
and yeast extract. Both of benzoate and yeast extract were
necessary for the production of red compound.
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