导读:本文包含了乙醇酸氧化酶同工酶论文开题报告文献综述、选题提纲参考文献及外文文献翻译，主要关键词:氧化酶,乙醇,菠菜,同工,特性,电泳,论文。
乙醇酸氧化酶同工酶论文文献综述
罗莎,黄璐瑶,殷勤,张钰莹,赵燕[1]（2019）在《菠菜乙醇酸氧化酶的酶学特性及同工酶电泳分析》一文中研究指出为了解析菠菜乙醇酸氧化酶(Sp GLO)的酶学特性及同工酶谱。首先提取菠菜的RNA并反转录成c DNA,通过NCBI数据库中提供的菠菜GLO基因mRNA序列信息,设计特异性引物,扩增目标序列并连接到p MD19-T载体上进行测序鉴定;随后再次设计引物并在上游引物加入His标签,克隆Sp GLO基因构建到p YES2载体上,将该载体转入酿酒酵母中进行表达并通过His-tag亲和柱纯化,然后在不同诱导时间点取样测定Sp GLO酶活。结果显示,转化后的酿酒酵母菌株在诱导发酵20 h后能得到最高的GLO活性。以乙醇酸为底物测定Sp GLO在不同p H、不同温度条件下的催化活性,其最适p H值为8. 0,最适温度为39℃。然后分别以乙醇酸、乙醛酸、甘油酸为反应底物,系统分析了Sp GLO的酶学特性,数据显示,Sp GLO对乙醇酸的亲和力最高,其Km为0. 41 mmol/L,Vm为45. 92μmol/(min·mg)。以乙醇酸、乙醛酸为底物,使用草酸抑制其催化活性,其Ki分别为4. 61,2. 09 mmol/L,表明以乙醛酸为底物时Sp GLO的催化活性更易被草酸抑制。同时将纯化后的Sp GLO通过Caps-氨水电泳体系进行同工酶电泳,经过染色后出现2条同工酶带,表明菠菜叶片中可能存在2种GLO同工酶。为将来深入研究植物GLO同工酶之间的生化特性差异并分析其不同生理功能奠定良好的基础。(本文来源于《华北农学报》期刊2019年01期）
尹汉萍,徐杰,曾秋莲,王再花,叶庆生[2]（2004）在《菜心中高等电点高活性的乙醇酸氧化酶同工酶的纯化和特性》一文中研究指出Glycolate oxidase (GO) isozyme with high specific activity (75.0～279.0 U/mg) is purified quickly on DEAE- Cellulose column from Brassica parachinensis Bailey. Its pI is greater than 10.0 assayed by acetate cellulose membrane electrophoresis for 1 hour. In view of about ten kinds of pI varied from 4.5 to 10.0 are observed when the same GO isozyme is assayed in IEF for 14 hours, it is obvious that its pI decreases in IEF. Its pI also decreases when this GO isozyme is assayed in PAGE for 14 hours. Based on the results in SDS-PAGE, CGE-SDS, and IEF, it is most likely that this GO isozyme comprises two noncovalently associated 66 kD basic subunit and 40 kD acidic subunit, the phenomenon of pI change is related to subunit dissociation. The basic/acidic amino acid residues ratios in GO isozyme and its 40 kD acidic subunit are detected to be 0.66 and 0.54, respectively, a value much lower than that (0.96) in 40 kD peptide encoded by GO cDNA reported previously, indicating neither M r nor charge characteristic of this 40 kD peptide is similar to that of GO isozyme subunits, two subunits of GO isozyme may be the modified products of the same GO gene after post-translation.(本文来源于《中国生物化学与分子生物学报》期刊2004年05期）
尹汉萍,徐杰,曾秋莲,王再花,叶庆生[3]（2004）在《菠菜中高活性高等电点的乙醇酸氧化酶同工酶纯化方法的改进》一文中研究指出通过缩短DEAE-celluloses柱长度,提高其洗脱液pH,可快速地从菠菜绿叶中纯化得到乙醇酸氧化酶同工酶,证实其具高活性113.0 U/mg·min和高等电点(pI)10.6.纯化后产率为0.14％,纯化倍数为66.5.经SDS-PAGE和毛细管电泳(CE-SDS)均只有40kD单带,说明已纯化至电泳纯.(本文来源于《华南师范大学学报(自然科学版)》期刊2004年03期）
尹汉萍[4]（2004）在《高等电点高活性的乙醇酸氧化酶同工酶的纯化及特性》一文中研究指出前人认为乙醇酸氧化酶(EC 1.1.3.15，简称GO)是由一个40kD亚基和黄素单核苷酸(FMN)组成的寡聚酶，但该观点无法解释GO具有从7.1到10.0的近10种等电点(pI)。本实验组徐杰首次提出菠菜GO是一个同工酶，可能含40kD酸性和66kD碱性两种亚基，由于亚基间容易解离，导致等电点的变化。GO同工酶在纯化过程中很不稳定，其全酶分子量(Mr)、活性和pI均会下降，这可能是造成目前报道的GO的Mr和pI等基本参数相差很大的原因。所有纯化后的GO的pI均≤9.6，而粗蛋白中pI＞9.6的GO始终未被纯化出来，这可能与GO同工酶的稳定性差有关。因此，有必要找到一种更简便快速的纯化方法，以便获得pI＞9.6的更接近生理状态的GO同工酶。 本文通过一种改进后的方法，即通过缩短DEAE-cellulose-52柱的长度，以及提高洗脱液的pH，可在9h内从菠菜、菜心和豆角绿叶中纯化GO同工酶。证实该GO同工酶具高活性(90～197．U／mg．min)和高pI(pI＞10.0)。SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)表明，该GO同工酶均有40kD主带。初步证实不同植物中的GO同工酶的亚基M_r是相同的。 多次纯化菜心中的GO同工酶。醋酸薄膜电泳表明其pI＞10.0，而等电聚焦电泳(IEF-PAGE)则表明，该GO同工酶可产生从4.5～10.0的近10种pI。在SDS-PAGE电泳中，GO同工酶始终只存在40kD主带，表明GO同工酶pI的变化与40kD亚基降解无关。 菜心GO同工酶的SDS-PAGE和SDS-毛细管电泳(CE-SDS)显示，该酶除了含40kD主带外，还有很浅的66kD带，和之前我们提出的菠菜GO同工酶含40kD酸性亚基和66kD碱性亚基相似；SOS-PAGE和CE-SDS电泳中，无论加入β-巯基乙醇与否，GO同工酶都只有40kD主带和66kD浅带，表明菜心GO同工酶中40kD酸性亚基和66kD碱性亚基不是以共价二硫键相连；用制备性SDS-PAGE法获得菜心GO同工酶的40kD亚基，并和菜心GO同工酶一起测定其氨基酸组成，该GO同工酶及40kD亚基的碱／酸性氨基酸的比例分别为0.66和0.54，表明40kD亚基可能是个酸性蛋白，而66kD带则是个碱性蛋白。 SDS-PAGE、CE-SDS和氨基酸测定表明，和菠菜GO同工酶类似，菜心GO同工酶也可能含4OkD酸性亚基和66kD碱性亚基。鉴于在醋酸薄膜电泳中菜心G0同工酶的PI>10.0，以及在IEF能聚焦在PI4.5的结果，表明菜心G0同工酶66kD碱性亚基的pl>10.0，而菜心G0同工酶40kD酸性亚基的PI<4 .5。在电场中，非共价相连的G0同工酶两种亚基很容易分离，导致G0同工酶在工EF中出现近10种pl。(本文来源于《华南师范大学》期刊2004-06-01）
徐杰,吴燕燕[5]（2001）在《菠菜乙醇酸氧化酶同工酶的亚基组成》一文中研究指出Protein components of which p I <8.3 and molecular weights 35±2 kD and 25±2 kD may be degradation products of GO isozyme 40±2 kD subunit.But protein band of which p I >8 3 and molecular weight 67±2 kD from the proteins containing GOⅢ and GOⅡ was stable.There was a cross immunoreaction between specific GOⅠ antibody and only 40±2 kD protein band from crude protein of spinach green leaves assayed by SDS PAGE Western blot.Specific antigen from spinach crude protein immunoadsorbed by specific GOⅠ antibody showed four bands with the molecular weights 67±2 kD,40±2 kD,38±2 kD,and 25±2 kD assayed by SDS PAGE, Western blot using the antibody against the protein containing GOⅡ.So 67±2 kD protein could be immunoadsorbed by specific GOⅠ antibody.By native PAGE and SDS PAGE,Western blot using the antibody against the protein containing GOⅡ,rice GOⅡ and a rice 67 kD protein of which p I >8 3 was confirmed to be induced by glycolate substrate.On the results above,a new theory was put forwards that spinach GOⅡ consisted of 67±2 kD and 40±2 kD subunits.(本文来源于《中国生物化学与分子生物学报》期刊2001年05期）
徐杰,吴燕燕[6]（2001）在《菠菜叶片中乙醇酸氧化酶3种同工酶的生化特性》一文中研究指出By DEAE cellulose and Sepharose 6B chromatography, the proteins containing glycolate oxidase isozymes GOⅡ and GOⅢ were extracted from spinach green leaves. The protein containing GOⅡ showed two bands of 67±2 kD and 40±2 kD in SDS PAGE whose specific activity of glycolate oxidase was 33.4 U·mg -1 ·min -1 .It migrated towards cathode in Native PAGE in pH 8.3 buffer system. pI of GOⅡwas about 9.4 detected by IEF. The protein containing GOⅢ showed three bands of 67±2 kD, 40±2 kD and 38±2 kD in SDS PAGE whose specific activity of glycolate oxidase 14.4 U·mg -1 ·min -1 and could not migrate anywhere in the same Native PAGE. pI of GOⅢ was about 8.3 detected by IEF. The 40±2 kD might be the subunits of GOⅡ and GOⅢ. Antibodies of the protein containing GOⅡ and GOⅢ were prepared respectively. GOⅡ was very unstable and could change into GOⅢ artifact; GOⅢ was also unstable and could change into GOⅠartifact whose Mr ≈470 kD and pI ≈7.4 . This GOⅠ(specific activity: 9.8 U·mg -1 ·min -1 ), showing one 40±2 kD band in SDS PAGE, could be purified on another Sepharose 6B chromatography. The specific activity of GOⅡ decreased rapidly to about half of its original value and then was relatively stable when stored in 50% glycerol at -20℃. The results above explained why GOⅡ was extracted difficultly, and GOⅢ were easily confused with GOⅠ and GOⅡ.(本文来源于《中国生物化学与分子生物学报》期刊2001年04期）
徐杰,吴燕燕[7]（2001）在《菠菜乙醇酸氧化酶同工酶GO Ⅰ的纯化和特性》一文中研究指出从菠菜绿叶中获得ＳＤＳ－ＰＡＧＥ为４０ ０００±２０００Ｍｒ单带的乙醇酸氧化酶同工酶ＧＯⅠ。其ＧＯ比活为 ８．４ Ｕ ｍｉｎ－１ｍｇ－１ ｐｒｏｔｅｉｎ，经 ＮＤ－ＰＡＧＥ后显示 Ｍｒ为 ４７０ ０００。用 ＩＥＦ测得其等电位为 ７．４。用氧电极法证实该ＧＯⅠ能同时催化乙醇酸和乙醛酸的氧化。制备ＧＯⅠ的抗体并对菠菜绿叶粗蛋白作免疫双扩散，有免疫沉淀线；经蛋白Ａ－Ｓｅｐｈａｒｏｓｅ ＣＬ－４Ｂ柱吸附法和抗原亲和吸附法，得到单特异性ＧＯⅠ抗体。用该ＧＯⅠ抗体对菠菜绿叶粗蛋白作ＳＤＳ－ＰＡＧＥ Ｗｅｓｔｅｒｎ ｂｌｏｔ后只有４００００±２０００－条染色带；作 ４％－ ２０％ＮＤ－ＰＡＧＥ Ｗｅｓｔｅｒｎ ｂｌｏｔ后有多条染色带， Ｍｒ分别大约为 ４７０ ０００、 ２８０ ０００和８０ ０００，表明菠菜绿叶中的 ＧＯＩ有多个聚合态。(本文来源于《热带亚热带植物学报》期刊2001年01期）
徐杰[8]（1998）在《菠菜中的乙醇酸氧化酶是一个同工酶》一文中研究指出乙醇酸氧化酶（ＥＣ．１．１．３．１５．ＧＯ）是光呼吸途径的关键酶，降低其活性可提高Ｃ３植物如水稻的产量，在目前中国乃至世界人口不断增加和可耕种土地日益减少的情况下，对ＧＯ的研究具有重要的理论意义和实际应用价值．在光呼吸途径被提出后的几十年间，人们对如...(本文来源于《中国生物化学与分子生物学报》期刊1998年06期）
乙醇酸氧化酶同工酶论文开题报告
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Glycolate oxidase (GO) isozyme with high specific activity (75.0～279.0 U/mg) is purified quickly on DEAE- Cellulose column from Brassica parachinensis Bailey. Its pI is greater than 10.0 assayed by acetate cellulose membrane electrophoresis for 1 hour. In view of about ten kinds of pI varied from 4.5 to 10.0 are observed when the same GO isozyme is assayed in IEF for 14 hours, it is obvious that its pI decreases in IEF. Its pI also decreases when this GO isozyme is assayed in PAGE for 14 hours. Based on the results in SDS-PAGE, CGE-SDS, and IEF, it is most likely that this GO isozyme comprises two noncovalently associated 66 kD basic subunit and 40 kD acidic subunit, the phenomenon of pI change is related to subunit dissociation. The basic/acidic amino acid residues ratios in GO isozyme and its 40 kD acidic subunit are detected to be 0.66 and 0.54, respectively, a value much lower than that (0.96) in 40 kD peptide encoded by GO cDNA reported previously, indicating neither M r nor charge characteristic of this 40 kD peptide is similar to that of GO isozyme subunits, two subunits of GO isozyme may be the modified products of the same GO gene after post-translation.
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乙醇酸氧化酶同工酶论文参考文献
[1].罗莎,黄璐瑶,殷勤,张钰莹,赵燕.菠菜乙醇酸氧化酶的酶学特性及同工酶电泳分析[J].华北农学报.2019
[2].尹汉萍,徐杰,曾秋莲,王再花,叶庆生.菜心中高等电点高活性的乙醇酸氧化酶同工酶的纯化和特性[J].中国生物化学与分子生物学报.2004
[3].尹汉萍,徐杰,曾秋莲,王再花,叶庆生.菠菜中高活性高等电点的乙醇酸氧化酶同工酶纯化方法的改进[J].华南师范大学学报(自然科学版).2004
[4].尹汉萍.高等电点高活性的乙醇酸氧化酶同工酶的纯化及特性[D].华南师范大学.2004
[5].徐杰,吴燕燕.菠菜乙醇酸氧化酶同工酶的亚基组成[J].中国生物化学与分子生物学报.2001
[6].徐杰,吴燕燕.菠菜叶片中乙醇酸氧化酶3种同工酶的生化特性[J].中国生物化学与分子生物学报.2001
[7].徐杰,吴燕燕.菠菜乙醇酸氧化酶同工酶GOⅠ的纯化和特性[J].热带亚热带植物学报.2001
[8].徐杰.菠菜中的乙醇酸氧化酶是一个同工酶[J].中国生物化学与分子生物学报.1998
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