重组毕赤酵母高密度发酵表达水蛭素过程中产物的生成和降解

Production and Degradation of Hirudin During Expression in Pichia pastoris by High Density Fermentation

目的 研究重组毕赤酵母高密度发酵表达水蛭素过程中产物的生成和降解。方法 通过质谱和氨基酸序列分析确定水蛭素的 4种活性异构体 ,再用HPLC监测发酵过程中水蛭素的降解情况。结果 发酵过程中产生了 4种具有较高比活的水蛭素异构体 ,它们分别是目的产物水蛭素Hir6 5及Hir6 5的C 末端降解 1～ 3个氨基酸的产物。发酵时细胞干重达到 16 2g/L ,水蛭素的总活性一直在增加 ,最高为 2 .4 5× 10 4ATU/ml,相当于总产量1.8g/L。但Hir6 5在 4种水蛭素异构体总量中的比例一直在下降。Hir6 5的产量先升高再降低 ,最高为 2 80mg/L。结论 毕赤酵母发酵过程中水蛭素存在降解 ,Hir6 5降解产生了Hir6 2和Hir6 3。

Objective To analyze the production and degradation of hirudin during expression in Pichia pastoris by high density fermentation.Methods Identify the 4 kinds of isomers of hirudin by Time of Flight Mass Spectrum and amino acid sequencing,and monitor the degradation of hirudin during fermentation by RP HPLC.Results Four kinds of isomers with specific activities,i.e.goal product Hir65,and the products in which the 1,2 and 3 amino acids at the C terminal were degraded,were produced during fermentation.The cell dry weight reached 162g/L,and the total activity of hirudin increased during fermentation to a peak value of 2.45×10 4 ATU/ml,which was equivalent to a total yield of 1.8g/L.However,the proportion of Hir65 in the 4 kinds of isomers decreased all the time.The yield of Hir65 increased to a peak value of 280mg/L then decreased.Conclusion Hir65 may be degraded to Hir62 and Hir63 during fermentation of Pichia pastoris .