Protein Engineering of a Pyridoxal-5′-Phosphate-Dependent <span style="font-variant: small-caps">l</span>-Aspartate-α-Decarboxylase from <i>Tribolium castaneum</i> for β-Alanine Production
Xin-Jun Yu,
Chang-Yi Huang,
Xiao-Dan Xu,
Hong Chen,
Miao-Jie Liang,
Zhe-Xian Xu,
Hui-Xia Xu,
Zhao Wang
Affiliations
Xin-Jun Yu
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
Chang-Yi Huang
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
Xiao-Dan Xu
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
Hong Chen
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
Miao-Jie Liang
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
Zhe-Xian Xu
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
Hui-Xia Xu
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
Zhao Wang
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, China
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 β-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 β-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.