| Summary: | In the present study, a pyridoxal-5′-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from <i>Tribolium castaneum</i> (TcPanD) was selected for protein engineering to efficiently produce β-alanine. A mutant <i>Tc</i>PanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (<i>m/v</i>), 50 °C, and 7.0, respectively. The 1mM of Na<sup>+</sup>, Ni<sup>2+</sup>, Co<sup>2+</sup>, K<sup>+</sup>, and Ca<sup>2+</sup> stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni<sup>2+</sup> and Na<sup>+</sup> could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate <span style="font-variant: small-caps;">l</span>-aspartic acid was consumed and 170.5 g/L of β-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.
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