摘要
对用乳糖替代异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组产朊假丝酵母尿酸酶基因在E.coliJM109(DE3)中表达进行了研究,拟建立一种高效低成本的生产重组尿酸酶的工艺路线。通过摇瓶试验对诱导所采用的乳糖浓度,诱导时机和诱导持续时间进行了优化,并考察在乳糖诱导下的目的产物表达动力学,随后在5L发酵罐上进行扩大化培养以验证摇瓶优化的结果,进一步将乳糖作为诱导剂应用于高密度发酵过程。实验结果表明乳糖诱导的最佳浓度为5g/L,最佳诱导时机是对数生长期中后期,诱导持续时间为9～10h;按照优化的条件在摇瓶和5L发酵罐上进行分批培养,重组尿酸酶最大表达量可达菌体总蛋白的26%左右,可溶性蛋白的36%左右,略高于IPTG的诱导效果;高密度发酵过程菌体终密度达到OD600值40以上,尿酸酶表达量占菌体总蛋白25%左右。
Using lactose as a succedaneous inducer of IPTG to induce the expression of Candida utilis uricase gene cloned in E. coli JM109（DE3） was deeply investigated for establishing a high-performance but low-cost method of recombinant uricase production. The adopted lactose concentration for induction, the time point of induction, the duration of induction and the dynamics of uricase expression were optimized and studied in detail by shake flask experiment. The results of optimization were verified by enlarged fermentation in 5 L fermentor and then lactose was used as the inducer in the high density fermentation. As showed by the experiments, the best inducing concentration of lactose was 5 g/L, the midanaphase of logarithmic phase was the best time for induction and the duration of induction were 9 -10h; according to the optimum conditions, compared with IP-FG, both in the shake flask and 5 L fermentor with batch culture, the better inducing effect could be obtained： the expression level of uricase was about 26% of the total bacterial protein and about 36% of the soluble protein, the final cell density （OD600） was over 40 in the 5 L fermentor and uricase expression level was about 25% with high density fermentation.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第7期74-79,共6页
China Biotechnology
基金
国家科技支撑计划资助项目(2006BAF07B01)
关键词
尿酸酶
乳糖
诱导表达
优化
高密度发酵
Uricase Lactose Induced expression Optimization High density fermentation
