SDS-Chitin-PAGE电泳法分析大葱叶几丁质酶特性
详细信息Characterization of chitinase isoenzymes in Allium Fistulosum L leaves by Sodium dodecyl sulfate-Chitin-Polyacrylamide gel electrophoresis
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摘要: 使用Sodium dodecyl sulfate-几丁质-聚丙烯胺酰胺凝胶电泳（SDS-Chitin-PAGE）方法分析了大葱葱叶几丁质酶的特性。在大葱叶组织中共检测到三种几丁质酶同工酶,分别命名为CH-A、CH-B、CH-C。分析结果表明,这三种几丁质酶具有SDS抗性,β-巯基乙醇对其活性也无显著影响。在SDS-Chitin-PAGE前,样品缓冲溶液与样品蛋白粗提液孵化处理的最适温度为24℃、时间为10～20min。凝胶电泳酶活分析表明,大葱葱叶中CH-A、CH-B的耐热温度可达60℃,CH-C的耐热温度达到70℃。葱叶中CH-A的最适pH为5.2～5.6,CH-B的最适pH为4.0～4.4,CH-C的最适pH为6.6～8.0。
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关键词:
- 几丁质酶大葱; /
- 酶学特性; /
- 聚丙烯酰胺凝胶原位显示电泳;
Abstract: Allium Fistulosum L leaves was representatively selected to test their chitinase characteristics by the optimized SDS-Chitin-PAGE.The results showed that three chitinase isozymes, CH-A, CH-B and CH-C, were detected in Allium Fistulosum L leaves.Allium Fistulosum L had the very special characteristic of SDS resistance.β-mercaptoethanol had no significant effect on the chitinase activity.The optimal incubating temperature and time of the crude enzymes with the sample buffer were 24℃, 10～ 20min before the electrophoresis.The activity of CH-A and CH-B could be observed up to 60℃, the activity of CH-C could be observed up to 70℃.The optimal pH ranges for CH-A, CH-B and CH-C were pH 5.2～ 5.6, pH 4.0～ 4.4, and pH 6.6～ 8.0, respectively. -
[1] 马钊, 李景明, 李丽梅, 等.洋葱多糖提取工艺的研究[J].食品工业科技, 2005, 26 (5) :98-99. [2] 陈崇顺, 徐凤彩, 李明启.21科41种 (变种) 植物叶片几丁质酶系的研究[J].植物资源与环境, 1993, 2 (4) :28-33. [3] Trudel J, Asselin A.Detection of chitinase activity after polyacrylamide gel electrophoresis[J].Anal Biochem, 1989, 178:362-366.
[4] Bravo JM, Campo S, Murillo I, et al.Fungus and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein4 (PR-4) family of maize[J].Plant Mol.Biol, 2003, 52:745-759.
[5] Cordero MJ, Ravento's D, Segundo BS.Differential expression and induction of chitinases and beta-1, 3-glucanases in response to fungal infection during germination of maize seeds[J].Mol Plant-Microbe Interact, 1994, 7:23-31.
[6] 费凡, 闫巧娟, 江正强, 等.膜荚黄芪种子中2种几丁质酶的分离纯化及活性测定[J].西北植物学报, 2009, 29 (12) :2521-2526. [7] 曹建康, 姜微波, 赵玉梅.果蔬采后生理生化实验指导[M].北京:中国轻工业出版社, 2007. [8] Andres M, Gisbert E, Diaz M, et al.Ontogenetic changes in digestive enzymatic capacities of the spider crab, Majabrachydactyla (Decapoda:Majidae) [J].Journal of Experimental Marine Biology and Ecology, 2010, 389:75-84.
[9] Goto Y, Calciano LJ, Fink AL.Acid induced folding of proteins[J].Proceeding of National Academy of Sciences, 1990, 87 (2) :573-577.
[10] 孙静.番茄果实衰老相关蛋白酶的特性分析及鉴定[D].北京:中国农业大学, 2009. [11] Liu CL, Shen CR, Hsu FF, et al.Isolation and Identification of two novel SDS-resistant secreted chitinases from aeromonas schubertii[J].Biotechnol Prog, 2009, 25 (1) :124-131.
[12] Xia G, Jin C, Zhou J, et al.A novel chitinase having a unique mode of action from Aspergillus fumigatusYJ-407[J].Eur J Biochem, 2001, 268:4079-4085.
[13] Brurberg MB, Nes IF, Eijsink VG.Comparative studies of chitinases A and B from Serratia marcescens[J].Microbiology, 1996, 142 (7) :1581-1589.
[14] Lonhienne T, Mavromatis K, Vorgias CE, et al.Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp.strainTAD20:isolation and partial characterization of the enzymes[J].J Bacteriol, 2001, 183:1773-1779.
[15] Park SK, Kim CW, Kim H, et al.Cloning and high-level production of a chitinase from Chromobacterium sp.and the role of conserved or nonconserved residues on its catalytic activity[J].Appl Microbiol Biotechnol, 2007, 74:791-804.
[16] Kopparapu NK, Liu ZQ, Yan QJ, et al.A novel thermostable chitinase (PJC) from pomegranate (Punica granatum) juice[J].Food Chemistry, 2011, 127:1569-1575.
[17] 阎瑞香, 吴仲, 侯建华.重组水稻几丁质酶的发酵生产、纯化及酶学性质[J].农业生物技术学报, 2008, 16 (1) :148-153. [18] Wang SY, Hong BS, Fu H, et al.Isolation of a thermostable legume chitinase and study on the antifungal activity[J].ApplMicrobiol Biotechnol, 2009, 85:313-321.
[19] Onaga S, Chinen K, Ito S, et al.Highly thermostable chitinase from pineapple:Cloning, expression, and enzymatic properties[J].Process Biochemistry, 2011, 46:695-700.
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