L-天冬氨酸-β-脱羧酶(L-aspartate-β-decarboxylase, Asd)催化L-天冬氨酸脱羧生成L-丙氨酸,该研究对Acinetobacter radioresistens来源的Asd酶进行酶学性质解析,为工业生产L-丙氨酸提供参考。构建表达质粒pET28a-ArAsd,转化大肠杆菌E. coli BL21 (DE3)实现ArAsd基因的异源表达。利用亲和层析纯化获得携带His标签的纯酶后进行酶学性质研究,并考察重组菌底物、产物耐受的能力。结果表明,重组酶比酶活力为753 U/mg,其最适催化温度为55 ℃,最适反应pH为4.5,在40~45 ℃、pH 6.0~7.0条件下较稳定,45 ℃处理3 h酶活力剩余70%左右,pH 7.0处理12 h酶活力剩余90%左右。产物L-丙氨酸浓度超过500 mmol/L时重组菌细胞酶活力有明显降低,底物L-天冬氨酸对重组菌细胞催化活性有促进作用。该研究首次在E.coli中异源表达Acinetobacter radioresistens来源Asd酶,其比酶活力高于目前已有报道的Asd酶,具有一定的工业应用潜力。
L-aspartate-β-decarboxylase (Asd) catalyzes the decarboxylation of L-aspartic acid to produce L-alanine. The enzymatic properties of the Asd enzyme derived from Acinetobacter radioresistens were analyzed to provide a reference for industrial production of L-alanine. The expression plasmid pET28a-ArAsd was constructed and transformed into E. coli BL21 (DE3) to achieve heterologous expression of the ArAsd gene. After purification by affinity chromatography to obtain a pure enzyme carrying the His-tag, the enzymatic properties were studied. And the tolerances of the recombinant bacteria to substrates and products were investigated. The results showed that the specific enzyme activity of the recombinant enzyme was 753 U/mg, and its optimum catalytic temperature and reaction pH were 55 ℃ and 4.5, respectively. It was stable under the condition of 40-45 ℃ and pH 6.0-7.0. The enzyme maintained 70% activity after treated at 45 ℃ for 3 h, and maintained 90% activity after treated at pH 7.0 for 12 h. When the concentration of the product L-alanine exceeded 500 mmol/L, the activity of the recombinant cells was significantly reduced, and the substrate L-aspartic acid could promote the catalytic activity of the recombinant cells. The Asd derived from Acinetobacter radioresistens was expressed in E. coli for the first time. The activity of ArAsd is the highest to date, and it has great potential for the industrial production of L-alanine.
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