人工合成苯丙氨酸脱氨酶基因在乳酸乳球菌中的食品级表达

Food-grade expression of artificial phenylalanine-ammonia-lyase gene in Lactococcus lactis

目的将人工合成具有乳酸菌偏好密码子的苯丙氨酸脱氨酶基因(PALart),在乳酸乳球菌中克隆并进行食品级表达,以期进一步用于治疗苯丙酮尿症的研究。方法以欧芹苯丙氨酸脱氨酶氨基酸序列为准,人工合成以乳酸菌偏爱密码子编码相同氨基酸序列的基因PALart,将该基因克隆到食品级乳酸乳球菌高效表达系统pNZ8149/NZ3900中而获得基因工程菌,用HPLC法测定诱导表达出的苯丙氨酸脱氨酶活性。结果获得了能表达苯丙氨酸脱氨酶活性的阳性克隆pNZ8149-PALart/NZ3900。该酶的表达量与诱导物乳链菌肽的浓度有关。反应液pH值能明显影响工程菌转化苯丙氨酸为肉桂酸的转化率;以10mmol/L的苯丙氨酸作为底物,在pH8.0的磷酸盐缓冲液中,于37℃反应18h,其转化率可达64%。结论获得具有活性PALart食品级表达的乳酸乳球菌株,为进一步将其制成口服制剂用于治疗经典型苯丙酮尿症模型动物的实验研究打下基础。

Objective To done and express designed Lactococcus-lactis-codon-preferred phenylalanine-ammonia-lyase （PAL） gene in food-grade L. lactis expression system, and to find whether active PAL in L.lactis can be produced to degrade phenylalanine. Methods PAL gene is synthesized, which originates from PAL eDNA of Petroselinum crispum. The artificial PAL gene was cloned into food-grade expression system pNZS149/NZ3900. After pNZS149-PAL^art/NZ3900 was induced by nisin, the activity of PAL expressed in L. lactis was analyzed. Re. suits Artificial PAL gene was cloned and expressed in L.lactis food-grade expression system. The PAL could convert phenylalanine to trans-cinnamic acid. The conversion rate was varied in different pH of reaction buffer, In phosphate buffer （pH8.0） of 10 mg/ml wet cells of the engineering L. lactis and 10 mmol/L phenylalanine were incubated at 37℃ for 18 h, and the conversion rate reached 64%. Conclusion Active PAL^art was successfully expressed in L.lactis food-grade expression system, and it could be used in the Phenylketonuria treatment research.