|本期目录/Table of Contents|

[1]冯娟,覃炎锋,李荷.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[J].应用与环境生物学报,2014,20(06):1076-1081.[doi:10.3724/SP.J.1145.2014.06004]
 FENG Juan,QIN Yanfeng,LI He.Expression optimization and dye degradation of laccase gene lac1338 in Escherichia Coli[J].Chinese Journal of Applied & Environmental Biology,2014,20(06):1076-1081.[doi:10.3724/SP.J.1145.2014.06004]
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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()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
20卷
期数:
2014年06期
页码:
1076-1081
栏目:
研究论文
出版日期:
2014-12-31

文章信息/Info

Title:
Expression optimization and dye degradation of laccase gene lac1338 in Escherichia Coli
作者:
冯娟 覃炎锋 李荷
Department of Biochemistry &Molecular Biology, Guangdong Pharmaceutical University, Guangzhou 510006, China
Author(s):
FENG Juan QIN Yanfeng LI He
Department of Biochemistry &Molecular Biology, Guangdong Pharmaceutical University, Guangzhou 510006, China
关键词:
laccase Escherichia coli optimization of expression conditions dye degradation
Keywords:
accase Escherichia coli optimization of expression conditions dye degradation
分类号:
Q78 : X172
DOI:
10.3724/SP.J.1145.2014.06004
摘要:
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)/pET32a(+)-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(SDS-PAGE)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)/pET32a(+)-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. 图13参24
Abstract:
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 cells induced under the optimized conditions (incubation at 30 °C, 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.

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备注/Memo

备注/Memo:
国家自然科学基金项目(30970107)、广东省科技厅项目(2012B010300021)和广东省教育厅项目(2013KJCX0107)资助 Supported by the National Natural Science Foundation of China (30970107), the Project of Guangdong Provincial Department of Science and Technology (2012B010300021) and the Project of Guangdong Department of Education (2013KJCX0107)
更新日期/Last Update: 2015-01-05