漆酶基因lac1338在大肠杆菌中的表达条件优化及其染料降解作用

Expression optimization and dye degradation of laccase gene lac1338 in Escherichia Coli

在摇瓶水平上,采用单因素试验方法,对已构建好的表达漆酶蛋白HIS-Lac1338的工程菌BL21(DE3)/p ET32a(+)-lac1338的诱导表达条件进行优化,以期提高蛋白产量,降低生产成本.研究了培养温度、诱导时间、异丙基硫代半乳糖苷(IPTG)浓度、铜离子(Cu2+)浓度等不同条件对HIS-Lac1338表达量和活性的影响.通过聚丙烯酰氨凝胶电泳(SDSPAGE)分析确定最佳表达条件,用酶标仪测其酶活性.结果显示,温度为30℃,初始菌体浓度D600 nm为1.6,加终浓度为0.2 mmol/L的IPTG及终浓度0.5 mmol/L的Cu2+,培养16 h,融合蛋白表达量提高了约2.5倍,为450 mg/L,是已报道的最高表达量;以2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)为底物测纯酶的比酶活为22.8 U/mg.染料降解作用显示其对刚果红和靛红有95%以上的降解能力.研究表明,工程菌BL21(DE3)/p ET32a(+)-lac1338产漆酶量高且大部分为可溶性蛋白,可以快速生产,具有较好的应用价值.

In order to improve the expression level of lac1338 gene in Escherichia coli, four factors for producing HIS- Lac1338 by recombinant strain BL21（DE3）/pET32a（＋）-lac1338 were investigated, including the culture temperature, inducing time, and final concentrations of inductor Isopropyl beta-D-thiogalactopyranoside （IPTG） and Cu2＋. The optimized factors were determined by single factor analysis experiments. The optimum expression conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with the activities detected by enzyme-labeled instrument. The experiments showed that BL21（DE3）/pET32a（＋）-lac1338 ceils induced under the optimized conditions （incubation at 30 ℃, at an initial bacteria density of D600 nm 1.6, induction with 0.2 mmol/L IPTG and 0.5 mmol/L Cu2＋ for 16 h） resulted in the accumulation of large amounts of soluble laccase. The culture provided 450 mg/L of laccase, 250% higher than that under the original fermentation condition. Its activity was determined by one-step purification through His-Binding-Resin affinity chromatography to be up to 22.8 U/mL of specific activity to ABTS under the optimal conditions. It could degrade more than 95% of Congo red and Indigo carmine. Overall, the optimized process to express HIS-Lac1338 in E. Coli can produce large amount of soluble and active recombinant protein, therefore it is valuable in application.