摘要
目的建立简便、快速纯化D-氨基酸氧化酶(DAAO)的新方法,以利用高纯度的DAAO转化头孢菌素C制备戊二酰-7-氨基头孢霉烷酸(GL-7ACA)。方法通过基因工程手段,构建N端和C端各镶嵌6个组氨酸的DAAO重组质粒,电转化Pichia pastoris GS115电感受态细胞,利用PCR方法筛选阳性转化子,再经甲醇诱导筛选出高表达菌。结果高表达重组菌(PHD12)摇瓶发酵单位达到2 000 IU/L,发酵罐内达到22 500 IU/L。并利用Ni螯合型树脂对表达的DAAO经一步分离纯化,以60%的总收率获得纯化产物。纯化产物保持良好的转化CPC-Na活性。结论利用基因工程表达组氨酸标记蛋白,可简化基因工程的下游工序。
Purpose To develop a new method to purify D-amino acid oxidase (DAAO) efficiently and fast. Methods Six additional histidine amino acid residues were tagged at N and C-termini of DAAO. His-tagged DAAO gene was cloned into pPIC3.5k of Pichia pastoris. Then it was purified by nickel-chelate chromatography. Results His-tagged protein was successfully expressed in Pichia pastoris GSll5 with 22 500 IU per liter and purified quickly to total yield of 60% by one step. Conclusions His-tagged enzyme still keeps the capability of bioconversion cephalosporin C to 7-glutary cephalosporanic. The purification was more fast and efficiently.
出处
《复旦学报(医学版)》
CAS
CSCD
北大核心
2006年第5期622-625,共4页
Fudan University Journal of Medical Sciences