摘要
【目的】实现黑曲霉来源的阿魏酸酯酶在毕赤酵母(Pichia pastoris GS115)中的组成型表达。【方法】以黑曲霉(Aspergillus niger)基因组为模板,经重叠延伸PCR扩增得到阿魏酸酯酶基因(AnfaeA),将其与载体pGAP9K相连,构建重组表达载体p GAP9KAnfae A,经SalI线性化后电转入毕赤酵母GS115中,得到重组菌株。高效液相色谱法测定发酵液中阿魏酸酯酶活力,并对重组菌进行了发酵优化。【结果】克隆得到783 bp的阿魏酸酯酶编码基因并实现了其在毕赤酵母中的组成型表达。重组菌发酵84 h后,上清液中酶活达5.72±0.10 U/m L。重组酶(reAnfaeA)经分离纯化后比酶活为59.75 U/mg,大小约为40 k D。发酵优化结果为:葡萄糖40.0 g/L,蛋白胨10.0 g/L,酵母膏30.0 g/L,CaCO_3 0.2 g/L,种龄28 h,接种量3%(体积比),装液量50 m L/250 m L。在此条件下发酵培养,酶活达15.60±0.23 U/m L。【结论】阿魏酸酯酶在毕赤酵母中的组成型表达,对研究毕赤酵母组成型表达系统和阿魏酸酯酶的发酵生产具有一定的借鉴意义。
[Objective] This study is aimed to constitutively express feruloyl esterase gene（Anfae A） from Aspergillus niger in Pichia pastoris GS115. [Methods] The Anfae A gene was amplified from A. niger by overlap extension PCR and cloned into the expression vector p GAP9 K.The recombinant expression vector（p GAP9 KAnfae A） was linearized by Sal I, then transformed into P. pastoris GS115. Feruloyl esterase activity of the recombinant strain was determined by high-performance liquid chromatography（HPLC） and we optimized the fermentation conditions. [Results] We cloned Anfae A gene（783 bp） and constitutively expressed Anfae A gene in P. pastoris GS115. Feruloyl esterase activity reached its peak（5.72±0.10 U/m L） after 84 h fermentation, its specific activity was 59.75 U/mg, and the molecular weight of re Anfae A was about 40 k D. The optimal fermentation conditions were as follows： glucose 40.0 g/L, tryptone 10.0 g/L, yeast extract 30.0 g/L, CaCO3 0.2 g/L, inoculum age 28 hours, inoculation level 3%（V/V）, medium volume 50 m L in 250 m L flask. Under these conditions, feruloyl esterase activity in fermentation supernatant was 15.60±0.23 U/m L. [Conclusion] We constitutively expressed feruloyl esterase in P. pastoris GS115. It was helpful to study the constitutive expression system in P. pastoris and improve the fermentation production of feruloyl esterase.
出处
《微生物学通报》
CAS
CSCD
北大核心
2017年第1期68-78,共11页
Microbiology China
基金
国家高技术研究发展计划项目(863计划)(No.2011AA100905)~~
关键词
黑曲霉
阿魏酸酯酶
GAP启动子
组成型表达
发酵优化
Aspergillus niger
Feruloyl esterase
GAP promoter
Constitutive expression
Fermentation optimization
