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

Abstract:

[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.

Key words:Anabaena variabilis, Phenylalanine ammonia lyase, Enzymatic properties, Optimal reaction pH