發(fā)布時(shí)間：2018-01-12 10:15

  本文關(guān)鍵詞：假單胞菌多銅氧化酶CumA和CopA的漆酶活性以及錳離子氧化活性研究　出處：《湖北大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 假單胞菌593 銅綠假單胞菌27853 細(xì)菌漆酶 酶動(dòng)力學(xué) 多銅氧化酶 錳離子氧化

【摘要】：細(xì)菌漆酶有著真核漆酶所不具備的優(yōu)勢(shì),如基因序列簡(jiǎn)單,沒有真核生物漆酶基因的外顯子——內(nèi)含子復(fù)雜結(jié)構(gòu),酶蛋白中也沒有糖基化修飾等。同時(shí)一些細(xì)菌漆酶具有廣泛的底物特異性、良好的熱穩(wěn)定性以及偏堿的最適pH值范圍等。這些都使得細(xì)菌漆酶可能是一種有利的生物催化劑,應(yīng)用于真菌漆酶不適合的地方,彌補(bǔ)了傳統(tǒng)漆酶的不足,擴(kuò)大了漆酶的應(yīng)用范圍。本研究從Pesudomonas sp.593中分別克隆出多銅氧化酶cumA593和copA基因,并將cumA593和copA分別轉(zhuǎn)入大腸桿菌BL21(DE3)pLysS,經(jīng)IPTG誘導(dǎo)表達(dá)后,目的蛋白通過鈷離子瓊脂糖凝膠柱純化,并對(duì)純化好的兩種蛋白分別做了漆酶活性的酶學(xué)研究。多銅氧化酶CumA593的最適反應(yīng)pH對(duì)于底物DMP(2,6-dimethoxyphenoI)是pH 5.0,SGZ(syringaldazine)是 pH 7.5,ABTS(2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid))是pH 5.0。CumA593的最適反應(yīng)溫度對(duì)于底物DMP是55℃,SGZ是60℃,ABTS是60℃。CumA593的pH穩(wěn)定性研究發(fā)現(xiàn),酶蛋白在在偏弱堿性條件下比較穩(wěn)定,在pH 9.5保存24小時(shí)后,其相對(duì)酶活仍保留60%左右的活力,而相反的,在pH 3.0保存12小時(shí)后,基本檢測(cè)不到酶活力。CumA593的熱穩(wěn)定性并不是太好,當(dāng)溫度在60℃保存2小時(shí),CumA593相對(duì)酶活僅僅剩余10%左右。二價(jià)金屬離子對(duì)CumA593漆酶活力的影響發(fā)現(xiàn)Cu~(2+)能極大的促進(jìn)CumA593酶活力。相反的,Fe~(2+)對(duì)CumA593酶活具有明顯的抑制作用。對(duì)于底物DMP,CumA593的Km為4.38×10-4mol/L,Vmax等于1.187×10-6 mol·L-1·min-1,kcat為 0.056 s-1;對(duì)于底物 SGZ,Km為 1.714×10-5 mol/L,Vmax等于 0.705×10-6 mol·L-1·min-1,kcat 為 0.03 s-1;對(duì)于底物 ABTS,Km 為 1.06×10-4 mol/L,Vmax 等于 2.807×10-6 mol·L-1·min-1,kcat為 0.393 s-1。多銅氧化酶CopA和CumA593在氨基酸序列上具有21.75%的相同性,二者在蛋白結(jié)構(gòu)和漆酶活性的酶學(xué)性質(zhì)上都不相同。多銅氧化酶CopA的最適反應(yīng)pH對(duì)于底物DMP是pH 7.5,SGZ是pH 7.5,ABTS是pH 3.5。CopA的最適反應(yīng)溫度對(duì)于底物DMP是50℃,SGZ是42℃,ABTS是50℃。CopA的pH穩(wěn)定性研究發(fā)現(xiàn),酶蛋白在中性條件下(pH 7.0)比較穩(wěn)定。CopA的熱穩(wěn)定并不是太好,當(dāng)溫度在60℃保存0.5小時(shí),CopA相對(duì)酶活僅僅剩余40%左右,而繼續(xù)保存到3小時(shí)時(shí),酶活力就基本檢測(cè)不到了。二價(jià)金屬離子對(duì)CopA漆酶活力的影響發(fā)現(xiàn)Cu~(2+)能極大的促進(jìn)CopA酶活力,不加銅離子或加其它二價(jià)金屬離子CopA顯示出很微弱的漆酶活性。對(duì)于底物DMP,CopA的Km為1.41×10-4mol/L,Vmax等于4.54×10-6mol·L-1·min-1,kcat為2.2s-1;;對(duì)于底物8GZ,Km為0.25×10-4mol/L,Vmax等于0.7×10-6mol·L-1·min-1,kcat為0.87 3-1;對(duì)于底物 ABTS,Km為2.81×10-4mol/L Vmax等于 3.02×10-6mol·L-1·min-1,為 1.8 s-1。同時(shí)為了揭示多銅氧化酶CumA本身是否具有錳離子氧化能力,本研究分別從來源于具有錳離子氧化能力的Pesudomonas sp.593和非錳離子氧化能力的Pseudomonas aeruginosa 27853克隆了兩種多銅氧化酶基因cumA593和cumA27853,并在大腸桿菌BL21(DE3)pLyS中進(jìn)行了表達(dá),相關(guān)蛋白進(jìn)行了純化。在體外證實(shí)了不管來源的宿主菌是否具有錳離子氧化能力,兩種多銅氧化酶本身都能夠催化氧化二價(jià)錳離子,并對(duì)二者相關(guān)酶動(dòng)力學(xué)做了研究。CumA27853和CumA593雖然在氨基酸序列上僅有81%的相同性,但對(duì)于錳離子氧化的最適反應(yīng)pH都是7.5,也都在30～37℃,酶活力相對(duì)較大,加入Cu~(2+)也都能極大的促進(jìn)酶活力。CumA27853的Km為27.27μmol/L,Vmax等于0.124μmol·L-1·min-1,kcat 為 0.016 s-1;而 CumA593 的 Km 為 42.75 μmol/L,Vax 等于 0.153μmol·L-1·min-1,kcat 為 0.035 s-1。
[Abstract]:A bacterial laccase eukaryotic laccase does not have the advantages such as simple gene sequence, no eukaryotic laccase gene exon - intron complex structure, there is no glycosylation modification of enzyme protein. At the same time some bacterial laccase has broad substrate specificity, good thermal stability and the optimum bias alkali pH value range. These are made of bacterial laccase may be a biological catalyst used in fungal laccase beneficial, not suitable for areas that make up the lack of traditional laccase, to expand the scope of application of laccase. This study from Pesudomonas sp.593 were cloned multicopper oxidase cumA593 and copA genes, and cumA593 and copA were transformed into Escherichia coli BL21 (DE3) pLysS, induced by IPTG, the target protein by cobalt agarose gel column purification, and two kinds of purified protein were good the enzyme laccase activity. The optimum pH for substrate DMP multicopper oxidase CumA593 (2,6-dimethoxyphenoI) pH 5, SGZ (syringaldazine) pH 7.5, ABTS (2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) pH 5.0.CumA593) is the optimal reaction temperature is 55 DEG C for substrate DMP, SGZ is 60, ABTS is the research on the stability of pH 60 C the discovery of.CumA593 enzyme protein is relatively stable in weak alkaline condition in pH 9.5 after 24 hours of preservation, the relative enzyme activity retained about 60% of the activity, on the contrary, in pH 3 after 12 hours of preservation, and can not be detected in the thermal stability of.CumA593 activity is not very good, when the temperature in the 60 stored at 2 hours, relative activity of CumA593 only remaining about 10%. Two effects of divalent metal ions on the CumA593 laccase activity found that Cu~ (2+) can greatly promote the activity of CumA593. On the contrary, Fe~ (2+) has obvious inhibition on CumA593 activity. For substrate DMP, CumA593 Km 4.38 * 10-4mol/L, Vmax * 10-6 mol is equal to 1.187 L-1 min-1, kcat 0.056 S-1; for the substrate SGZ, Km 1.714 * 10-5 mol/L Vmax * 10-6 mol is equal to 0.705 L-1 min-1, kcat 0.03 S-1; for the substrate ABTS, Km 1.06 * 10-4 mol/L Vmax * 10-6 mol is equal to 2.807 L-1 min-1, kcat for the same 0.393 s-1. copper oxidase CopA and CumA593 with 21.75% in amino acid sequence, the two are not the same in protein structure and enzymatic properties of laccase activity. The multicopper oxidase CopA the optimum reaction pH is 7.5 for pH at the end of DMP, SGZ, pH 7.5, ABTS pH 3.5.CopA is the optimal reaction temperature is 50 DEG C for substrate DMP, SGZ is 42, ABTS is the research on the stability of pH 50 C.CopA, the enzyme protein in neutral condition (pH 7) and the thermal stability of.CopA is stable when the temperature is not too good, in 60 degrees to save 0.5 Hours, relative activity of CopA only remaining around 40%, and continue to save up to 3 hours, the enzyme activity basically not detected. Two effects of divalent metal ions on the CopA laccase activity found that Cu~ (2+) can greatly promote CopA activity, without copper ions or other divalent metal ions CopA two showed very weak laccase activity. For substrate DMP, CopA Km 1.41 * 10-4mol/L, Vmax is equal to 4.54 * 10-6mol L-1 min-1, kcat 2.2s-1; 8GZ Km; for the substrate, 0.25 * 10-4mol/L, Vmax * 10-6mol is equal to 0.7 L-1 min-1, kcat 0.87 3-1; for the substrate ABTS. Km 2.81 * 10-4mol/L Vmax is equal to 3.02 x 10-6mol - L-1 - min-1, 1.8 s-1. at the same time, in order to reveal the multicopper oxidase CumA is manganese oxidizing ability, this study respectively from with Manganese Oxidizing Ability of Pesudomonas sp.593 and non Manganese Oxidizing Ability of Pseudomonas AE Ruginosa 27853 was cloned two multicopper oxidase gene cumA593 and cumA27853 in Escherichia coli BL21 (DE3) pLyS in the expression of related protein was purified. In vitro confirmed whether host bacteria is regardless of the source of Manganese Oxidizing Ability of two multicopper oxidase itself can be catalyzed oxidation of two manganese ions on the two, and related enzyme kinetics studied.CumA27853 and CumA593 are the same only 81% in amino acid sequence, but for the optimum reaction pH of manganese ion oxidation is 7.5, also in 30 to 37 DEG C, the enzyme activity is relatively large, adding Cu~ (2+) can greatly promote the enzyme the activity of.CumA27853 Km 27.27 mol/L Vmax 0.124 mol or L-1 min-1, kcat S-1 and CumA593 0.016; Km 42.75 mol/L, Vax is equal to 0.153 mol - L-1 - min-1, kcat 0.035 s-1.

【學(xué)位授予單位】：湖北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q936;R378.991

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