假单胞菌多铜氧化酶CumA和CopA的漆酶活性以及锰离子氧化活性研究

发布时间：2018-01-12 10:15

  本文关键词：假单胞菌多铜氧化酶CumA和CopA的漆酶活性以及锰离子氧化活性研究　出处：《湖北大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 假单胞菌593 铜绿假单胞菌27853 细菌漆酶 酶动力学 多铜氧化酶 锰离子氧化

【摘要】：细菌漆酶有着真核漆酶所不具备的优势,如基因序列简单,没有真核生物漆酶基因的外显子——内含子复杂结构,酶蛋白中也没有糖基化修饰等。同时一些细菌漆酶具有广泛的底物特异性、良好的热稳定性以及偏碱的最适pH值范围等。这些都使得细菌漆酶可能是一种有利的生物催化剂,应用于真菌漆酶不适合的地方,弥补了传统漆酶的不足,扩大了漆酶的应用范围。本研究从Pesudomonas sp.593中分别克隆出多铜氧化酶cumA593和copA基因,并将cumA593和copA分别转入大肠杆菌BL21(DE3)pLysS,经IPTG诱导表达后,目的蛋白通过钴离子琼脂糖凝胶柱纯化,并对纯化好的两种蛋白分别做了漆酶活性的酶学研究。多铜氧化酶CumA593的最适反应pH对于底物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的最适反应温度对于底物DMP是55℃,SGZ是60℃,ABTS是60℃。CumA593的pH稳定性研究发现,酶蛋白在在偏弱碱性条件下比较稳定,在pH 9.5保存24小时后,其相对酶活仍保留60%左右的活力,而相反的,在pH 3.0保存12小时后,基本检测不到酶活力。CumA593的热稳定性并不是太好,当温度在60℃保存2小时,CumA593相对酶活仅仅剩余10%左右。二价金属离子对CumA593漆酶活力的影响发现Cu~(2+)能极大的促进CumA593酶活力。相反的,Fe~(2+)对CumA593酶活具有明显的抑制作用。对于底物DMP,CumA593的Km为4.38×10-4mol/L,Vmax等于1.187×10-6 mol·L-1·min-1,kcat为 0.056 s-1;对于底物 SGZ,Km为 1.714×10-5 mol/L,Vmax等于 0.705×10-6 mol·L-1·min-1,kcat 为 0.03 s-1;对于底物 ABTS,Km 为 1.06×10-4 mol/L,Vmax 等于 2.807×10-6 mol·L-1·min-1,kcat为 0.393 s-1。多铜氧化酶CopA和CumA593在氨基酸序列上具有21.75%的相同性,二者在蛋白结构和漆酶活性的酶学性质上都不相同。多铜氧化酶CopA的最适反应pH对于底物DMP是pH 7.5,SGZ是pH 7.5,ABTS是pH 3.5。CopA的最适反应温度对于底物DMP是50℃,SGZ是42℃,ABTS是50℃。CopA的pH稳定性研究发现,酶蛋白在中性条件下(pH 7.0)比较稳定。CopA的热稳定并不是太好,当温度在60℃保存0.5小时,CopA相对酶活仅仅剩余40%左右,而继续保存到3小时时,酶活力就基本检测不到了。二价金属离子对CopA漆酶活力的影响发现Cu~(2+)能极大的促进CopA酶活力,不加铜离子或加其它二价金属离子CopA显示出很微弱的漆酶活性。对于底物DMP,CopA的Km为1.41×10-4mol/L,Vmax等于4.54×10-6mol·L-1·min-1,kcat为2.2s-1;;对于底物8GZ,Km为0.25×10-4mol/L,Vmax等于0.7×10-6mol·L-1·min-1,kcat为0.87 3-1;对于底物 ABTS,Km为2.81×10-4mol/L Vmax等于 3.02×10-6mol·L-1·min-1,为 1.8 s-1。同时为了揭示多铜氧化酶CumA本身是否具有锰离子氧化能力,本研究分别从来源于具有锰离子氧化能力的Pesudomonas sp.593和非锰离子氧化能力的Pseudomonas aeruginosa 27853克隆了两种多铜氧化酶基因cumA593和cumA27853,并在大肠杆菌BL21(DE3)pLyS中进行了表达,相关蛋白进行了纯化。在体外证实了不管来源的宿主菌是否具有锰离子氧化能力,两种多铜氧化酶本身都能够催化氧化二价锰离子,并对二者相关酶动力学做了研究。CumA27853和CumA593虽然在氨基酸序列上仅有81%的相同性,但对于锰离子氧化的最适反应pH都是7.5,也都在30～37℃,酶活力相对较大,加入Cu~(2+)也都能极大的促进酶活力。CumA27853的Km为27.27μmol/L,Vmax等于0.124μmol·L-1·min-1,kcat 为 0.016 s-1;而 CumA593 的 Km 为 42.75 μmol/L,Vax 等于 0.153μmol·L-1·min-1,kcat 为 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.

【学位授予单位】：湖北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q936;R378.991

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