鱼腥藻苯丙氨酸脱氨酶的基因克隆、表达及最适反应pH改造

【目的】表达鱼腥藻苯丙氨酸脱氨酶(AvPAL)，并经分子改造降低其最适反应pH。【方法】PCR克隆AvPAL编码基因，并在大肠杆菌中表达，用Ni2+亲和层析柱和凝胶柱纯化重组蛋白。利用GETAREA软件筛选与催化残基距离较近的暴露于酶分子表面的氨基酸位点，将其突变为带电性质不同的氨基酸，并对突变体进行酶学性质研究。【结果】在大肠杆菌中成功表达了AvPAL，纯化后得到电泳纯的重组酶。突变体E75Q和E75R的最适反应pH从8.5分别偏移到7.5和7.0。E75Q在pH 7.5时的比酶活较原酶提高了25%，在pH 6.5–9.5之间酶的稳定性良好，其最适反应温度为50 °C，在此温度下保温1 h酶活无显著变化。在最适反应条件下，E75Q的kcat/Km值较原酶提高了26.6%。【结论】改变AvPAL酶分子中起路易斯碱作用的关键氨基酸残基(质子受体)附近与之有相互作用的氨基酸的带电性质，降低了AvPAL的最适反应pH，提升了其在医疗领域的应用前景。

Gene cloning and expression of phenylalanine ammonia lyase from Anabaena variabilis and modification of the optimal reaction pH

Objective] Phenylalanine ammonia lyase from Anabaena variabilis (AvPAL) was expressed and molecular modified to decrease its optimum reaction pH. Methods] The full length AvPAL gene was cloned and expressed in E. coli BL21(DE3). The recombinant protein was purified through Ni2+ affinity chromatography and gel filtration chromatography. The mutation sites were decided by GETAREA software to choose the amino acid sites both closed to the catalytic group and exposed to the surface of the enzyme. After changing the electric properties of the target amino acid residues, enzymatic properties of the mutants were studied. Results] The recombinant AvPAL was successfully expressed in E. coli BL21(DE3). SDS-PAGE analysis showed that the recombinant enzyme of electrophoresis pure was obtained. The optimal reaction pH of E75Q and E75R mutants were shifted to 7.5 and 7.0, respectively, in contrast to pH 8.5 of the wild type (WT). The E75Q had a 25% higher specific activity than AvPAL WT at pH 7.5, and there was a good stability between pH 6.5 and pH 9.5. The optimal reaction temperature of E75Q was 50 °C. At the same time, the E75Q had a good stability under 50 °C with no obvious reduction in enzymatic activity after incubation of 1 h. Under the optimal reaction conditions, the kcat/Km was increased by 26.6% compared to the AvPAL WT. Conclusion] The optimal reaction pH of AvPAL was decreased by altering the charge of amino acid residues which were in a hypothetical position to interact with the general base catalyst. The resulting mutant would improve the application prospect of AvPAL in therapeutic.