Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.
Molecules
; 25(6)2020 Mar 12.
Article
em En
| MEDLINE
| ID: mdl-32178239
ABSTRACT
In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-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% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ 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 l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.
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Base de dados:
MEDLINE
Assunto principal:
Fosfato de Piridoxal
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Engenharia de Proteínas
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Beta-Alanina
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Glutamato Descarboxilase
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article