腾冲嗜热厌氧杆菌6-磷酸-β-葡萄糖苷酶的表达与结晶及其功能鉴定

被引:7
作者
尹捷
刘一苇
李洁
马延和
向华
严景华
高福
机构
[1] 中国科学院微生物研究所
关键词
6-磷酸-β-葡萄糖苷酶(PbgL); 糖基水解酶家族4(GH4); 蛋白结晶; 耐热性;
D O I
10.13865/j.cnki.cjbmb.2008.10.006
中图分类号
Q936 [微生物生物化学];
学科分类号
071010 ;
摘要
6-磷酸-β-葡萄糖苷酶(PbgL,EC3.2.1.86)催化由磷酸烯醇式丙酮酸-糖磷酸转移酶系统(PEP-PTS)转运入胞内的某些磷酸化二糖水解.一段来自腾冲嗜热厌氧杆菌(Thermoanaerobacter tengcongensis)编码PbgL(436个氨基酸残基)的开放阅读框(ORF TTE0337)被成功克隆,并在大肠杆菌BL21(Escherichia coli BL21)中有活性地表达.序列分析显示,该6-磷酸-β-葡萄糖苷酶属于糖基水解酶家族4(GH4),与枯草杆菌(Bacillus subtilis)的LicH,海栖热袍菌(Thermotoga Maritima)的BglT的一致性分别为62%和40%.纯化后的重组PbgL(rPbgL)经SDS-PAGE分析,为1条分子量约50kD的蛋白条带,与推测的分子量大小一致.该酶需要Mn2+、NAD+和还原剂为辅因子,能专一性地水解pNPβG6P,并在85℃达到最高酶活性.Western免疫印迹实验,利用针对rPbgL的多克隆抗体血清,能从腾冲嗜热厌氧杆菌胞内检测到PbgL的表达.此外,rPbgL的蛋白晶体已被筛选获得,并收集到2.4分辨率的衍射数据.采用分子置换法的结构解析目前仍在进行中.
引用
收藏
页码:916 / 924
页数:9
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