短芽孢杆菌a-乙酰乳酸脱羧酶基因在大肠杆菌中的克隆和表达

CLONING AND EXPRESSION OF α-ACETOLACTATE DECARBOXYLASE FROM BACILLUS BREV1S IN ESCHERICHIA COLI

用PCR方法扩增短芽孢杆菌α—乙酰乳酸脱羧酶基因,引入原核表达载体pBV220中,得到重组质粒pALDl。pALDl中的ALDC基因在大肠杆菌中高效表达,每毫升发酵液产酶80单位以下,比原始菌株提高200余倍。SDS-PAGE蛋白质分析表明,大肠杆菌DH5α(pALDl)表达的ALDC占细胞总蛋白量的40％以上。研究了重组质粒稳定性,大肠杆菌DH5α(PALDl)和HB101(pALDl)分别在无选择压力下30℃连续培养50代以上,41℃诱导不同时间,DH5α(pALDl)在热诱导5h后,未发现质粒不稳定性,HB101(pALDl)在热诱导3h后,质粒基本稳定,但热诱导5h后,丢失质粒的细胞约占70％。

The a-acetolactate decarboxylase (ALDC) gene was amplified from Bacillus brevis by using PCR technique. The amplified 0.85kb DNA fragment was inserted into an expression vector, pBV220, to give the recombinant plasmid pALDl. ALDC gene in pALDl was overexpressed by E. coli DH5α (pALDl) and HB101(pALD1), the production of ALDC reached over 80 units / ml of the culture, 200 times more than the parental strain. The expressed ALCD protein in DHSα(pALDl) accounted for more than 40% of the total protein in the recombinent E. coli cell. The stability of the plasmid pALDl in E.coli was determined. E. coli DHSα(pALDl) and HB101(pALDl) were cultivated in LB broth at 30℃ for more than 50 generations without antibiotic selection, then induced at 41℃ for up to 5 hours. The plasmid in DHSα(pALDl) was proved to be stable, but about 70% of the plasmid in HB101 (pALD1) was lost after 5 hours of heat induction.