铜离子浓度是影响CotA漆酶在大肠杆菌中异源表达的重要因素。然而,对大肠杆菌表达细菌漆酶重组蛋白的发酵条件探索和优化研究较少。该文通过在培养过程中控制铜离子添加量,探究铜离子浓度对大肠杆菌重组菌及突变菌漆酶表达量的影响。重组CotA漆酶表达菌株在铜离子浓度为1 mmol/L时破碎液漆酶活性较高,达到约2 207 U/mL;突变细菌漆酶I421A表达菌株在铜离子浓度为1.5 mmol/L时破碎液漆酶活性较高,达到631 U/mL;突变细菌漆酶T466A表达菌株在铜离子浓度为1.5 mmol/L时破碎液漆酶活性较高,达到852 U/mL;突变细菌漆酶K464A表达菌株在铜离子浓度为1 mmol/L时破碎液漆酶活性较高,达到295 U/mL。菌株的表达量受铜离子浓度影响变化幅度较大,通过对铜离子添加条件的探究,对改善细菌漆酶表达量较低的问题有重要参考价值。
Abstract
Copper ion concentration is a critical factor influencing the heterologous expression of CotA laccase in Escherichia coli.However, limited studies have focused on optimizing the fermentation conditions for the expression of recombinant bacterial laccase proteins in E.coli.The study investigated the effect of copper ion concentration on the expression levels of recombinant and mutant laccase in E.coli by controlling the amount of copper ion added during cultivation.The recombinant CotA laccase-expressing strain showed the highest laccase activity in cell lysate at a copper ion concentration of 1 mmol/L, reaching approximately 2 207 U/mL.The mutant laccase I421A-expressing strain exhibited maximum laccase activity at 1.5 mmol/L copper ion, reaching 631 U/mL.Similarly, the mutant laccase T466A-expressing strain reached the highest activity at 1.5 mmol/L copper ion, with 852 U/mL, and the K464A mutant strain showed the highest activity at 1 mmol/L copper ion, reaching 295 U/mL.The expression levels of these strains varied significantly with changes in copper ion concentration.The study provides valuable insights into improving the low expression levels of bacterial laccases by optimizing copper ion supplementation.
关键词
细菌漆酶 /
异源表达 /
定点突变 /
蛋白表达量 /
铜离子
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Key words
bacterial laccase /
heterologous expression /
site-directed mutagenesis /
protein expression level /
copper ion
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脚注
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基金
安徽科技学院引进人才项目(SKYJ202006);安徽省高校自然科学研究重点项目(2022AH051635);安徽科技学院大学生创新课题项目(S202210879289);安徽科技学院横向合作项目(880578)
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