A genetic recombinant hEGF producer E.coli JM101 was immobilized in polyurethane foam matrix and a three-phase fluidized-bed bioreactor was used to produce human epidermal growth factor (hEGF).The results indicated th...A genetic recombinant hEGF producer E.coli JM101 was immobilized in polyurethane foam matrix and a three-phase fluidized-bed bioreactor was used to produce human epidermal growth factor (hEGF).The results indicated that polyurethane foam is an adequate immobilization material. With the addition of polyurethane foam matrix, the free cell density, plasmid stability and hEGF productivity were higher than those without the foam matrix, respectively. The two-stage culture of the immobilized cells was further carried out, in which, the first stage was performed in a three-phase fluidized-bed bioreactor for cell growth and the second stage was performed in bubbling bed for product expression. At a dilution rate of 0.2 h -1 , after continuous cultivation for more than 60 h, the free cell density and product expression were maintained relatively stable. The hEGF productivity was still higher than 140 mg·L -1 and the fraction of plasmid-carrying was more than 27%. The hEGF productivity in two-stage process was higher than the batch process.展开更多
3'-磷酸腺苷-5'-磷酰硫酸(PAPS)是生物肝素酶法制备途径的硫磺基供体,价格高且易分解。芳基硫磺基转移酶(ASTIV,EC 2.8.2.1)可以转化对硝基硫酸苯酯(PNPS)和3'-磷酸腺苷-5'-磷酸(PAP)生成PAPS。利用大肠杆菌系统高效可...3'-磷酸腺苷-5'-磷酰硫酸(PAPS)是生物肝素酶法制备途径的硫磺基供体,价格高且易分解。芳基硫磺基转移酶(ASTIV,EC 2.8.2.1)可以转化对硝基硫酸苯酯(PNPS)和3'-磷酸腺苷-5'-磷酸(PAP)生成PAPS。利用大肠杆菌系统高效可溶性表达ASTIV。对鼠源ASTIV基因序列进行密码子优化并全合成;转入大肠杆菌BL21(DE3)中诱导表达;对表达条件进行优化,提高可溶表达量;Ni2+亲和层析纯化目标蛋白,重组ASTIV产量达到80 mg/L,纯度高达95.3%,比酶活为42.5 m U/mg;用碱性磷酸酶(CIAP)转化ASTIV构型,比酶活进一步提高到85.0 m U/mg。该研究为ASTIV重组表达,PAPS酶法制备以及生物肝素合成奠定了坚实的基础。展开更多
文摘A genetic recombinant hEGF producer E.coli JM101 was immobilized in polyurethane foam matrix and a three-phase fluidized-bed bioreactor was used to produce human epidermal growth factor (hEGF).The results indicated that polyurethane foam is an adequate immobilization material. With the addition of polyurethane foam matrix, the free cell density, plasmid stability and hEGF productivity were higher than those without the foam matrix, respectively. The two-stage culture of the immobilized cells was further carried out, in which, the first stage was performed in a three-phase fluidized-bed bioreactor for cell growth and the second stage was performed in bubbling bed for product expression. At a dilution rate of 0.2 h -1 , after continuous cultivation for more than 60 h, the free cell density and product expression were maintained relatively stable. The hEGF productivity was still higher than 140 mg·L -1 and the fraction of plasmid-carrying was more than 27%. The hEGF productivity in two-stage process was higher than the batch process.
文摘3'-磷酸腺苷-5'-磷酰硫酸(PAPS)是生物肝素酶法制备途径的硫磺基供体,价格高且易分解。芳基硫磺基转移酶(ASTIV,EC 2.8.2.1)可以转化对硝基硫酸苯酯(PNPS)和3'-磷酸腺苷-5'-磷酸(PAP)生成PAPS。利用大肠杆菌系统高效可溶性表达ASTIV。对鼠源ASTIV基因序列进行密码子优化并全合成;转入大肠杆菌BL21(DE3)中诱导表达;对表达条件进行优化,提高可溶表达量;Ni2+亲和层析纯化目标蛋白,重组ASTIV产量达到80 mg/L,纯度高达95.3%,比酶活为42.5 m U/mg;用碱性磷酸酶(CIAP)转化ASTIV构型,比酶活进一步提高到85.0 m U/mg。该研究为ASTIV重组表达,PAPS酶法制备以及生物肝素合成奠定了坚实的基础。
