【摘要】 大肠杆菌自身代谢特征具有发酵生产双乙酰的天然优势。利用PCR技术,以广泛用于双乙酰生产的Lactococcus lactis基因组DNA为模板,克隆得到α-乙酰乳酸合成酶基因α-als,将其构建在表达载体pET-30 a上。与能够高效表达3种大肠杆菌来源HSP 70家族分子伴侣的pKJE 7质粒共同转化E.coli BL21(DE3)。利用目的蛋白质分子伴侣共表达的方法,首次获得了能够高效表达具有酶活力的α-乙酰乳酸合成酶的重组大肠杆菌。在静置培养条件下,能够在该菌株的培养基中检测到双乙酰的生成。融合蛋白酶在温度为30-40℃和pH在6-7之间时具有较高的酶活,以丙酮酸为底物,该酶最适pH为6.8,最适温度为39℃。更多还原

【Abstract】 Escherichia coli has many advantages in produce diacetyl.Since Lactococus lactis is commonly used to produce diacetyle,its genome was used as a template to amplify the gene encoding α-Acetolactate synthase.The PCR fragment was inserted in to pET-30a vector,and the recombinant expression plasmid and the plasmid pKJE7 was transformed into Escherichia coli BL21(DE3).Co-expression of these two plasmids perform high efficiency to produce active α-Acetolactate synthase,and the bacteria were detected to produce diacetyl.Using pyruvate as substrate,the recombinant enzyme had an optimum temperature of 35-40℃ and pH of 6.0-7.0.更多还原