疏绵状嗜热丝孢菌的糖基化热稳定锰超氧化物歧化酶及真菌锰超氧化物歧化酶的系统学

摘要

疏绵状嗜热丝孢菌Thermomyces lanuginosus是一种广泛分布的嗜热真菌,产生的酶在高温条件下具有高活性和稳定性.从该嗜热真菌中克隆了一种锰超氧化物歧化酶(manganese superoxide dismutase,MnSOD)基因Tlmnsod1(EU364837),cDNA全长791bp,开放阅读框654bp,编码217个氨基酸,没有信号肽和前导肽序列.Tlmnsod1基因在酵母中高效表达,经纯化获得了电泳均一分子量约为27kDa的重组蛋白TIMnSOD1.表达的TIMnSOD1酶是糖基化蛋白,具高的热稳定性,最适反应pH和温度分别为8.0和55℃.通过对真菌的40种锰超氧化物歧化酶的细胞定位和系统学分析可知:真菌的锰超氧化物歧化酶可以分为胞内MnSOD、线粒体MnSOD和胞外MnSOD;锰超氧化物歧化酶可以作为分子标记来研究真菌的进化关系;3种嗜热真菌MnSOD分别与非嗜热真菌MnSOD聚类在3个不同的组中.本研究为MnSOD的糖基化和作为分子标记用于真菌种的鉴定提供了证据.%A manganese superoxide dismutase gene (Tlmnsod1) from Thermomyces lanuginosus was cloned and expressed in Pichia pastoris.The full-length cDNA of the MnSOD was 791bp and contained a 654bp open reading frame encoding 217 amino acid residues.No signal peptide or leader peptide was present in the putative amino acid sequence of TIMnSOD1.The enzyme belongs to the Mn/Fe-binding family.The highly conserved motif of the MnSOD family,DXWEHAYY,was found in TIMnSOD1.The gene was transformed into Pichia pastoris.The recombinant TIMnSOD1 was shown to be a glycosylated MnSOD.The enzyme exhibited optimal activity at 55℃ and pH 8.0 and had high thermostability.Analysis of subcellular localization and phylogeny of the TIMnSOD1 and 39 other fungal MnSODs from Ascomycota predicted three kinds of MnSODs (mitochondrial,cytoplasmic,and extracellular MnSODs).Each of three MnSODs from thermophilic fungi was clustered together with non-thermophilic mesophilic fungi in different groups in the phylogeny tree.The results of this study provide evidences that MnSOD may be glycosylated and used as a molecular marker for identification of closely related fungal species.