基于λ噬菌体裂解基因表达的PHB分离新工艺

NEW TECHNIQUE FOR RECOVERY OF PHB FROM RECOMBINANT Escherichia coli BASED ON EXPRESSION OF LYTIC GENES OF PHAGE λ WITH S AMBER MUTATION

将S基因琥珀突变的λ噬菌体裂解基因 (S-RRz)引入产聚 β 羟基丁酸酯 (PHB)的重组大肠杆菌VG1(pTU14 )中以实现细胞的可控裂解破壁 .采用EDTA/Tris (pH值 8 0 )缓冲液处理结果表明 ,S-RRz在VG1(pTU14 )中能够成功表达 ,且EDTA对细胞裂解的决定性作用是由于它模拟了S基因产物的功能 .当细胞内PHB含量为 85 %～ 90 %时 ,大量积累的PHB颗粒可以改变细胞膜的通透性 ,实现重组细胞的内控自裂解 .对PHB与细胞进行直接分离的后处理工艺研究表明 ,在S-RRz成功表达的基础上 ,采用升温处理模拟S基因产物的功能诱导细胞自裂解 ,PHB产品纯度可以达到 95 %以上 .

The lytic genes of phage λ with S amber mutation(S- RRz) were introduced into the recombinant E. coli VG1 (pTU14) producing poly-β-hydroxybutyrate (PHB) to attain controllable lysis of cells. The results of EDTA/Tris (pH8.0) buffer treatment showed that S- RRz were successfully expressed in VG1 (pTU14), and cell lysis was realized due to the action of EDTA on cytoplasm membrane. Here the function of EDTA was similar to that of S gene product. When PHB content was 85%-90%, membrane permeability would be increased by the abundantly accumulated in-cell PHB granules, and then the autolysis of recombinant cells occurred. After studies on different projects for direct separation of PHB from fermentation broth, a new technique, in which temperature treatment was introduced to simulate the function of S gene product, was presented, and the autolysis of cells was then easily realized based on the successful expression of S- RRz. By this simple technique, the final purity of PHB product could be up to 95%.