| 红冬孢酵母苯丙氨酸解氨酶基因pal在大肠杆菌中的克隆与表达 |
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| 引用本文： | 侯文婷,张梁,顾正华,丁重阳,石贵阳.红冬孢酵母苯丙氨酸解氨酶基因pal在大肠杆菌中的克隆与表达[J].生物加工过程,2012,10(5):61-66. |
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| 作者姓名： | 侯文婷 张梁 顾正华 丁重阳 石贵阳 |
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| 作者单位： | 江南大学工业生物技术教育部重点实验室粮食发酵工艺与技术国家工程实验室,无锡,214122 |
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| 基金项目： | 国家高技术研究发展计划(863计划)资助项目(2011AA100905);2008年度江苏省高校“青蓝工程”科技创新团队;江苏高校优势学科建设工程资助项目 |
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| 摘 要： | 以红冬孢酵母总RNA为模板反转录获得其苯丙氨酸解氨酶基因pal,测序与已公布蛋白序列进行比对,相似度为99％.并以含有T7强启动子的pET - 28a(+)为载体构建重组质粒pET - 28a(+)- pal,通过异丙基硫代-β-D-半乳糖苷(IPTG)诱导实现苯丙氨酸解氨酶(PAL)在大肠杆菌中的表达.对诱导条件进行初步优化后,重组茵PAL比酶活可达到42.99 U/g.转化实验中肉桂酸的转化率为48.52％,L-苯丙氨酸生成量为1.73 g/L.结果表明,红冬孢酵母pal基因通过表达载体pET - 28a(+)在E.coli中获得了高效表达.
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| 关 键 词： | 苯丙氨酸解氨酶(PAL) 反转录 重组表达 |
Cloning and expression of phenylalanine ammonia-lyase gene of Rhodosporidium toruloides in Escherichia coli |
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| HOU Wenting
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ZHANG Liang
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GU Zhenghua
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DING Chongyang
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SHI Guiyang.Cloning and expression of phenylalanine ammonia-lyase gene of Rhodosporidium toruloides in Escherichia coli[J].Chinese Journal of Bioprocess Engineering,2012,10(5):61-66. |
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| Authors: | HOU Wenting ZHANG Liang GU Zhenghua DING Chongyang SHI Guiyang |
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| Institution: | (National Engineering Laboratory for Cereal Fermentation Technology,Key Laboratory of Industrial Biotechnology of the Ministry of Education,Jiangnan University,Wuxi 214122,China) |
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| Abstract: | The cDNA fragment of pal gene was amplified from Rhodosporidium toruloides by RT-PCR.Nucleotide sequencing identified the gene of 99% similarity with reported pal gene.Using pET-28a(+) with T7 promoter as a vector,the recombinant plasmid pET-28a(+)-pal was constructed and then expressed in E.coli BL21(DE3).After optimization,the specific activity of PAL reached 42.99 U/g.The conversion rate of trans-cinnamic acid was 48.52% and the concentration of L-phenylalanine was 1.73 g/L.It was shown that the gene pal from R.toruloides was highly expressed. |
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