【作者】 郭芳先；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 微生物学， 2007， 硕士

【摘要】 超氧化物歧化酶(Superoxide Dismutase,简称SOD)(EC.1.15.1.1)系一类金属酶,广泛存在于生物体中。它能够催化超氧阴离子(O2.-)发生歧化反应,从而清除超氧阴离子。在生物体防御氧化损伤的过程中,发挥着重要作用。根据酶分子中所含金属辅基的不同,超氧化物歧化酶主要可分为CuZn-SOD(存在于真核生物中),Mn-SOD(存在于原核生物和真核生物中),Fe-SOD(存在于原核生物和高等植物的叶绿体中)等类型。Mn-SOD和Fe-SOD的氨基酸组成相似,可能起源于同一祖先。嗜热毛壳菌(Chaetomium thermophilum)是一种广泛分布的,生长上限温度较高的嗜热真菌,从该菌中已分离了多种嗜热酶,但未见超氧化物歧化酶的报道。本研究中C. thermophilum在含干酪素的合成培养基中生长产生超氧化物歧化酶,通过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等步骤获得了凝胶电泳条带均一的超氧化物歧化酶。分别用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)的方法和Sephacryl S-100凝胶过滤层析的方法,测定出本研究所获得的超氧化物歧化酶的分子量分别为23.5kDa和94.4kDa,证明在本研究中所得到的超氧化物歧化酶是一种由4个相同的亚基所构成的蛋白质。本研究所得到的超氧化物歧化酶的反应最适pH值为7.5,酶的反应最适温度为60℃。在pH值为7.5的条件下,该酶在50℃和60℃时保温1h是基本稳定的,在70℃时保温1h后,仍保留有60%的酶活性;在80℃时,酶的半衰期约为25min。C. thermophilum超氧化物歧化酶(Mn-SOD) N-端10个氨基酸序列为KATLPDLKYD,与Aspergillus fumigatus和Paracoccidioides brasiliensis超氧化物歧化酶(Mn-SOD)的N-端氨基酸序列有较高的同源性。根据超氧化物歧化酶的氨基酸保守序列设计兼并引物,克隆了C. thermophilum热稳定超氧化物歧化酶CuZn-SOD和Mn-SOD的基因,随后提取C. thermophilum的基因组DNA,克隆了CuZn-SOD DNA序列,该序列包含三个内含子区域。这三个基因在GenBank中的注册号分别为DQ493760、EF569987、DQ683184。将超氧化物歧化酶的基因cz1与酵母表达载体pPIC9K用EcoRⅠ和NotⅠ双酶切后体外连接构建了酵母分泌型表达载体pPIC9K/cz1,转化巴斯德毕赤酵母获得了转酵母工程菌GS-CZ-10,酶蛋白表达量为0.56mg/mL,同时对重组超氧化物歧化酶的性质进行了研究。更多还原

【Abstract】 The superoxide dismutase (SOD, EC1.15.1.1) are metallo-enzymes that catalyze the dismutation of superoxide(O2.-)to hydrogen peroxide(H2O2) and molecular oxygen(O2). They have been found in nearly all organisms examined to date and play a critical role in the defense against oxidative stress. There are three general classes of SODs in organisms, which differ in their metal cofactor: copper zinc-containing SOD (CuZn-SOD), manganese- containing SOD (Mn-SOD) and iron-containing SOD (Fe-SOD).Chaetomium thermophilum is a widely distributed soil-inhibiting fungus of considerable interest producer of thermostable enzymes, including cellulase, endocellulase, xylanase and laccase which have been purified and well studied. However, the superoxide dismutase from the genus C. thermophilum has not yet been studied. During growth in a medium containing Casein, C. thermophilum also produced superoxide dismutase. The enzyme was purified to homogeneity from the culture supernatant of the strain by fractional ammonium sulphate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography.The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60oC. It was thermostable at 50 and 60oC and retained 60% activity after 60 min at 70oC. The half-life of the SOD at 80oC was approximately 25 min.The first 10 amino acids from the N-terminal of the Mn-SOD were KATLPDLKYD. The sequence showed a certain similarity with some other fungal manganese superoxide dismutase such as those from Aspergillus fumigatus and Paracoccidioides brasiliensis.Degenerate primers based on the conserved domain of other reported superoxide dismutase, and the cDNA fragments encoding the copper zinc superoxide dismutase and manganese superoxide dismutase were obtained through RT-PCR. The RACE-PCR was used to generate full-length cDNA clones. Then partial DNA encoding the copper zinc superoxide dismutase was cloned and it contained three introns. They have been registered in GenBank with accession number DQ493760, EF569987 and DQ683184.The cz1 gene and expression vector pPIC9K were digested with EcoR I and Not I, then ligationed in vitro to construct the expression plasmid pPIC9K/cz1. The pPIC9K/cz1 was transformed to Pichia GS115 competent cell after linearized with restriction enzyme Sal I. The recombinant Pichia GS-CZ-10 was got and the expressed superoxide dismutase was purified and characterized. The expression level was up to 0.56 mg/mL.更多还原