苯丙氨酸脱氨酶cDNA在大肠杆菌中的克隆与表达及酶法合成L-苯丙氨酸

CLONING AND EXPRESSION OF PHENYLALANINE AMMONIA LYASE (PAL) cDNA IN E.coli AND SYNTHESIS OF L-PHENYLALANINE ENZYMATICALLY

利用基因重组技术 ,在大肠杆菌中克隆并表达苯丙氨酸脱氨酶 (PAL) (EC4 .3 .1 .5) ,并应用此酶转化肉桂酸生成L 苯丙氨酸。方法是将欧芹苯丙氨酸脱氨酶cDNA亚克隆到组成型表达载体pMG3 6e启动子P3 2下游 ,以菌落PCR法鉴定插入片段的大小和方向都正确的克隆 ,进而以HPLC检测肉桂酸浓度的方法鉴别重组质粒有催化肉桂酸生成L 苯丙氨酸的酶活力。结果获得能表达PAL酶活性的阳性克隆 ,在pH1 0 ,含 1 .0 %肉桂酸、8.0mol/L氨的转化液中 ,3 0℃反应 2 0h ,肉桂酸重量转化率可达 60 %。该基因工程菌有希望用于工业化生产L 苯丙氨酸。

Phenylalanine ammonia lyase (PAL), or phenylalanine deaminase (PheD), cDNA from parsley ( Petroselinum crispum ) was subcloned into downstream of P32 promoter of a constitutive expression vector pMG36e. The obtained plasmid pMG36ePAL was then transformed into E. coli DH5α. The length and direction of the fragment was checked by colony PCR methods. Expression of PAL was identified by HPLC technique. In converting solution containing 1.0% cinnamic acid and \{8.0\} mol/L of ammonium ion (pH 10), the recombinant strains showed their PAL activity converting cinnamic acid into L phenylalanine. The yield of L phenylalanine reached up to 60% of the substrate at 37℃ after 20h's incubation. The engineering bacteria may provide an industrial way to produce L phenylalanine.\;