来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni²⁺柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.

Gene Synthesis and Fusion Expression D-Aminoacylase Gene from Alcaligenes A-6†

The codons of D-ANase gene from Alcaligenes A-6 were substituted by the codons abundant in E. coli., then the D-ANase gene was synthesized by the two-step method based on PCR technology. Synthetic gene and pET-32a vector were digested with BglⅡ and XhoⅠ, ligated by T4 DNA ligase. The ligation mixture transformed into E.coli BL21(DE3) competent cell. Recombinant protein was detected by SDS-PAGE, Western-blot and activity assay. D-ANase can be expressed efficiently in E. coli and the expressed protein content can reach to 69.2% of the total bacterial protein content. In addition, the fermentation activity can achieve 96 U/mL. After the ultrasonic cell disruption, the recombinant protein was purified by Ni²⁺ affinity chromatography column. The specific activity of the purified recombinant enzyme was 1692.3 U/mg and the purity could be up to 95%. Furthermore a firm foundation was laid for the industrial use of the D-ANase.