Photobacterium sp.QH氨肽酶的基因克隆及其序列、酶学性质分析  

Gene cloning, sequencing and enzymological property of aminopeptidase secreted by Photobacterium sp. QH

作　　者：武翠玲[1] 万兵[1] 李钰娜[1] 钟妞妞 周静 裴晋红[1] 

机构地区：[1]长治医学院生物化学教研室,山西长治046000

出　　处：《中国生物制品学杂志》2017年第10期1017-1021,1027,共6页Chinese Journal of Biologicals

基　　金：长治医学院科研启动基金(QDZ201504;QDZ201517);2015年山西省高等学校大学生创新创业训练项目(2015299)

摘　　要：目的克隆盐湖菌株Photobacterium sp.QH(Ps sp.QH)氨肽酶的基因,并对其进行序列及酶学性质分析。方法从青海盐湖采集水样,筛选Ps sp.QH菌株,进行16S rRNA分子生物学测定。通过硫酸铵沉淀、阴离子交换层析、凝胶过滤层析法从筛选菌株的发酵液中纯化氨肽酶,同时对其进行质谱分析,基因克隆及酶学性质分析。结果获得的菌株Ps sp.QH与Photobacterium halotolerans为同一属。该菌株分泌的氨肽酶属于金属螯合氨肽酶M28家族,全基因序列长1 527 bp,共编码508个氨基酸,预测其全酶分子相对分子质量为55 900,氨基酸序列与Photobacterium halotolerans氨肽酶(KKC99795.1)同源性达98%。该氨肽酶在50℃时酶活最高,40~50℃及pH 8.5时稳定性较好;Co^(2+)对其有激活作用,Mg^(2+)、Mn^(2+)、Zn^(2+)、Ca^(2+)、Fe^(2+)及Cu^(2+)对其有不同程度的抑制作用;该酶在1~3 mol/L的Na Cl溶液中仍保持较高活性。结论 Ps sp.QH分泌的氨肽酶对高盐有一定的耐受性,具有潜在的工业应用价值。Objective To clone the aminopeptidase gene of Photobacterium sp. QH（Ps sp. QH）and analyze its sequence and enzymological property. Methods Water samples were taken from Qinghai Salt Lake, from which Ps sp. QH was screened and analyzed for the scquence of 16 S r RNA. Aminopeptidase was purified from the fermentation liquid of Ps sp. QH by ammonium sulfate precipitation, DEAE Sepharose FF and gel filtration chromatography for mass spectro-metry, gene cloning and analysis of enzymological property. Results The obtained Ps sp. QH belonged to the genus of Photobacterium halotolerans, by which the secreted aminopeptidase belonged to M28 family, at a full-length of 1 527 bp,and encoded 508 amino acids. It was predicted that the relative molecular mass of aminopeptidase was 55 900, while the homology of amino acid sequence was 98% to that of Photobacterium halotolerans（KKC99795. 1）. The aminopeptidase showed the highest enzyme activity at 50 ℃ and high stability at 40 - 50 ℃ and p H 8. 5. The enzyme activity was promoted by cobalt ion while inhibited at different degrees by magnesium, manganese, zinc, calcium, iron and copper ions. Aminopeptidase showed high activity in 1 - 3 mol/L sodium chloride solution. Conclusion The aminopeptidase secreted by Photobacterium sp. QH showed high halo-tolerance, which was of potential value in application to industry.

关 键 词：Photobacterium sp.QH 氨肽酶 基因克隆 酶学性质 质谱分析 

分 类 号：Q566.3[生物学—生物化学]