一株SOD生产菌的分离、鉴定和产酶条件优化

Identification and Fermentation Condition Optimization of a SOD-producing Strain Isolated from Soil

为得到高产超氧化物歧化酶(SOD)的菌株,通过稀释平板法从土壤中进行SOD生产菌株分离和筛选,利用非变性聚丙烯酰胺凝胶电泳和氯化硝基四氮唑蓝(NBT)染色法鉴定SOD,采用抑制剂实验分析SOD类型,利用生理生化性质分析和16S rDNA亲缘关系分析进行种属鉴定,并进一步研究碳源、氮源、碳氮比、接种量和装液量对发酵产酶的影响。结果表明:SOD活性染色显示一条条带,说明只产一种SOD同工酶。该SOD对氯仿-乙醇敏感,对H2O2不敏感,为Mn-SOD。理化性质和16S rDNA序列分析初步鉴定该菌为肠杆菌科沙雷氏菌属。发酵条件优化结果表明产酶最佳条件为:最佳碳源为玉米粉,最佳氮源为蛋白胨,碳氮比4:1,接种量8%,250mL三角瓶装液量70mL。

In order to obtain SOD-producing strains,diluted plate method was used to isolate strains from soil.Non-denaturing gel electrophoresis and NBT staining were applied to identify SOD.The types of SOD were analyzed by inhibition test.Homological analysis was conducted according to physiological and biochemical characteristics as well as 16S rDNA sequence analysis.Furthermore,the effects of carbon source,nitrogen source,carbon/nitrogen ratio,inoculation amount,and medium volume in a 250 mL flask on the yield of SOD were investigated.The results showed that among 10 strain isolates,Lshm-3 was found to have higher ability to produce SOD.The SOD produced by the strain Lshm-3 was sensitive to chloroform-ethanol and insensitive to H2O2 and therefore could be identified as Mn-SOD.NBT staining revealed that this enzyme was a SOD isozyme.The strain Lshm-3 was identified as a member of the genus SerratiaBizio.Its fermentation conditions were optimized to be using maizena as the carbon source,using peptone as the nitrogen source,carbon/nitrogen ratio of 4：1,inoculation amount of 8%,and medium volume in a 250 mL flask of 70 mL.