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

Cloning,Expression,Crystallization and Characterization of a Novel 6-Phospho-β-glucosidase from Thermoanaerobacter tengcongensis

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.

6-Phospho-β-glucosidase （PbgL, EC 3.2.1.86） catalyzes the hydrolysis of some kinds of phosphodisaccharides imported by phosphoenolpyruvate-dependent phosphotransferase system （PEP-PTS）. An open reading frame （ORF TTE0337） from thermophilic bacterium Thermoanaerobacter tengcongensis, encoding a novel 6-phospho-β-glucosidase （436 amino acid residues） was cloned and actively expressed in E. coli system. Sequence analysis indicates that it belongs to the glycoside hydrolase family 4 （GH4）, with 62% identity to LicH from Bacillus subtilis and 40 % identity to BglT from Thermotoga Maritima. The recombinant form of PbgL （rPbgL） was purified as a single band with molecular weight of about 50 kD on SDS-PAGE, in agreement with the molecular weight deduced from the amino acid sequences. The recombinant enzyme requires Mn^2＋ , NAD^＋ and reducing agents for catalysis, and exhibits substrate specificity for pNPβG6P with optimal catalytic activity at 85 ℃. Western immunoblots using highly specific polyclonal antibody against rPbgL revealed the expression of the enzyme in the modified MB medium grown cells of T. tengcongeusis. Crystals of rPbgL were obtained and the diffraction data were collected to 2.4 A resolution. Structure determination by the molecular replacement method is in progress.