Objective To explore an economic, efficient, easy metallothionein production process, with biological fermentation engineering as a means, and to select the optimal production extraction conditions as well as verify the extracted metallothionein nature by orthogonal experimental method. Methods Submerged fermentation Kyushu as genetically engineered Cordyceps fungi, ZnSO4 stimulation was used to induce the synthesis of metallothionein. The metallothionein was isolated after cell disruption, crude extraction and chromatography steps. Preliminary study was made on the optimum conditions for zinc sulfate inducing cordyceps secreting metallothionein,and determination was made of the relative molecular mass, sulfhydryl content, and the content of zinc metal of the resulting metallothionein after purification. Results With zinc atom concentration of 15 g· L-1 metallothionein achieves maximum yield in the fermentation process,up to 10. 86 mg ·g-1 mycelium( wet weight ). The molecular weight of the zinc metallothionein is about 7 kD,each molecule metallothionein containing 21 cysteine and seven zinc atoms. Conclusion Through protein column chromatogram and ultraviolet scan pattern analysis the extraction of protein proves to accord with the characteristics of metallothionein.%目的 以生物发酵工程为手段,探寻一种经济,高效,简易的金属硫蛋白生产工艺,并用正交试验法优选出最适的生产提取条件及验证所提取的金属硫蛋白性质.方法 以经过基因工程改良的九州虫草真菌为菌种,采用液态深层发酵增殖菌丝体并用ZnSO4刺激诱导其合成金属硫蛋白,后经细胞破碎,粗提取和层析等步骤,得到锌金属硫蛋白,同时初步研究硫酸锌诱导虫草金属硫蛋白的最佳条件,提纯后所得金属硫蛋白的相对分子质量,巯基含量及金属锌的含量.结果 较传统动物肝脏提取方法,九州虫草菌株发酵法可快速高效的制得金属硫蛋白,发酵过程中培养基中锌原子浓度达15 g·L-1时金属硫蛋白达到最高产率,可达10.86mg·g-1菌丝体(湿重).该种锌金属硫蛋白分子量约为7 kD,每分子金属硫蛋白含有由21个半胱氨酸和7个锌原子.结论 通过蛋白柱层析图谱,紫外光扫描图谱分析,证明所提取蛋白符合金属硫蛋白的相应特征.
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