食品科学

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油橄榄苯丙氨酸解氨酶基因的克隆及其在毕赤酵母中的表达

陈文拴1,黄乾明1,*,陈华萍1,杨泽身2,王安逸2,苏光灿2   

  1. 1.四川农业大学理学院,四川 雅安 625014;2.凉山州中泽新技术开发有限责任公司,四川 西昌 615000
  • 出版日期:2015-04-15 发布日期:2015-05-05

Cloning of Phenylalanine Ammonia Lyase Gene from Olea europaea and Its Expression in Pichia pastoris

CHEN Wenshuan1, HUANG Qianming1,*, CHEN Huaping1, YANG Zeshen2, WANG Anyi2, SU Guangcan2   

  1. 1. College of Science, Sichuan Agricultural University, Ya’an 625014, China;
    2. Liang Shan Zhong Ze New Technology Development Co. Ltd., Xichang 615000, China
  • Online:2015-04-15 Published:2015-05-05

摘要:

采用同源克隆、反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)、融合引物嵌套PCR(fusion primer and nested integrated PCR,FPNI-PCR)与3’-cDNA末端快速扩增(rapid amplificationof cDNA end,RACE)技术相结合,从油橄榄(Olea europaea)中克隆得到苯丙氨酸解氨酶(phenylalanineammonia lyase,PAL)基因全长,命名为OePAL。序列分析表明,OePAL的DNA全长2 970 bp(GenBank登录号KJ511867),含一个内含子(393~1 220 bp);全长cDNA有2 142 bp(GenBank登录号KJ511868),开放阅读框编码713 个氨基酸,与其他植物有较高的同源性。利用该基因构建重组质粒pPICZα A-OePAL,且在毕赤酵母X33中进行诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectrophoresis,SDS-PAGE)检测显示该重组酶的分子质量在77.5 kD左右,纯化后的酶比活力为196.3 U/mg。酶学性质研究表明:该重组酶的最适反应条件为40 ℃,pH 8.8;在供试范围内Cu2+与Na+可以提高该酶活性;以L-苯丙氨酸为底物,在最适条件下该酶的Km为4.89×10-4 mol/L。结果表明成功地克隆到OePAL,构建了表达载体,并进行了功能验证。

关键词: 苯丙氨酸解氨酶, 基因克隆, 表达载体, 毕赤酵母X33

Abstract:

The phenylalanine ammonia lyase gene (PAL) was cloned from Olea europaea by homology cloning, RT-PCR,
FPNI-PCR (fusion primer and nested integrated PCR) and 3’-RACE, and named as OePAL. Sequencing showed that the
full-length DNA of OePAL was 2 970 bp (GenBank, KJ511867) with an intron (393–1 220 bp). Meanwhile, the full-length
cDNA of OePAL was 2 142 bp (GenBank, KJ511868). The open reading frame (ORF) of OePAL encoded 713 amino acid
residues, and sequence analysis suggested that OePAL had high homology with other botanic PALs. The full-length exon
of OePAL expression vector pPICZα A-OePAL was constructed and expressed in Pichia pastoris strain X33. SDS-PAGE
analysis showed that the molecular weight of recombinant enzyme was approximately 77.5 kD, and the specific activity
of purified enzyme was 196.3 U/mg. The enzymatic properties of recombinant 6×His-OePAL indicated that the optimum
reaction conditions were 40 ℃ and pH 8.8. Ions such as Cu2+ and Na+ could enhance the enzyme activity within the tested
range. The enzyme had a Km of 4.89 × 10-4 mol/L for L-phenylalanine at the optimum reaction conditions. Results indicated
that OePAL was amplified and sub-cloned into the vector of pPICZα A, and its function was validated successfully.

Key words: phenylalanine ammonia lyase (PAL), gene cloning, expression vector, Pichia pastoris strain X33

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