Chou C P, Yu C C, Lin W J, Kuo B Y, Wang W C
Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan, ROC.
Biotechnol Bioeng. 1999 Oct 20;65(2):219-26.
A novel and simple method of using penicillin for screening of mutant strains with a high penicillin acylase (PAC) activity was developed. Random mutagenesis was conducted using a PAC-producing strain resistant to 6-aminopenicillanic acid (6-APA) as the parent strain and mutants were screened with penicillin at a high concentration. Results suggest that mutants with a high minimum inhibitory concentration for penicillin (MIC(penG)) usually overproduce PAC. Both volumetric and specific PAC activities of a mutant, MD7, were significantly higher than those of the parent strain, HBPAC101 harboring pCLL2902. The mutation(s) resulting in the enhanced expression was mapped on the host chromosome rather than the plasmid. In addition, the mutant strain of MDDeltaP7, derived by elimination of the harbored plasmid in MD7, was demonstrated to be efficient in production of PAC by using the expression plasmids for which expression of the pac gene is limited by translation. An extremely high specific PAC activity of more than 350 U/L/OD(600) was reached upon cultivation of MDDeltaP7 harboring pTrcKnPAC2902 in a bioreactor. As such, the strategy is effective in terms of constructing PAC overproducers and improving the process yield for production of PAC.
开发了一种新颖且简单的利用青霉素筛选高青霉素酰化酶（PAC）活性突变株的方法。以对6-氨基青霉烷酸（6-APA）耐药的产PAC菌株作为亲本菌株进行随机诱变，并用高浓度青霉素筛选突变体。结果表明，对青霉素具有高最小抑菌浓度（MIC(penG)）的突变体通常会过量产生PAC。突变体MD7的体积活性和比活性均显著高于携带pCLL2902的亲本菌株HBPAC101。导致表达增强的突变定位在宿主染色体而非质粒上。此外，通过消除MD7中携带的质粒得到的突变菌株MDDeltaP7，被证明使用pac基因表达受翻译限制的表达质粒时，在PAC生产中效率很高。在生物反应器中培养携带pTrcKnPAC2902的MDDeltaP7时，达到了超过350 U/L/OD(600)的极高比活性。因此，该策略在构建PAC过量生产者和提高PAC生产的工艺产量方面是有效的。
