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

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

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’-c DNA末端快速扩增(rapid amplification of c DNA 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(phenylalanine ammonia lyase,PAL)基因全长,命名为Oe PAL。序列分析表明,Oe PAL的DNA全长2 970 bp(Gen Bank登录号KJ511867),含一个内含子(393~1 220 bp);全长c DNA有2 142 bp(Gen Bank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p PICZαA-Oe PAL,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 k D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℃,p H 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Oe PAL,构建了表达载体,并进行了功能验证。

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 Oe PAL. Sequencing showed that the full-length DNA of Oe PAL was 2 970 bp(Gen Bank, KJ511867) with an intron(393–1 220 bp). Meanwhile, the full-length c DNA of Oe PAL was 2 142 bp(Gen Bank, KJ511868). The open reading frame(ORF) of Oe PAL encoded 713 amino acid residues, and sequence analysis suggested that Oe PAL had high homology with other botanic PALs. The full-length exon of Oe PAL expression vector p PICZα A-Oe PAL was constructed and expressed in Pichia pastoris strain X33. SDS-PAGE analysis showed that the molecular weight of recombinant enzyme was approximately 77.5 k D, and the specific activity of purified enzyme was 196.3 U/mg. The enzymatic properties of recombinant 6×His-Oe PAL indicated that the optimum reaction conditions were 40 ℃ and p H 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 Oe PAL was amplified and sub-cloned into the vector of p PICZα A, and its function was validated successfully.