Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for β-Alanine Production.

Xin-Jun Yu,Chang-Yi Huang,Zhao Wang,Zhe-Xian Xu,Xiao-Dan Xu,Miao-Jie Liang,Hong Chen,Hui-Xia Xu

doi:10.3390/molecules25061280

Xin-Jun Yu, Chang-Yi Huang + Show 6 more

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https://doi.org/10.3390/molecules25061280

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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 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.

Highlights

The β-alanine (3-aminopropionic acid) is the only β-type amino acid in nature and non-protein amino acid in vivo, which is not involved in protein synthesis, while it has various biological functions.the β-alanine is widely used in multiple industries, such as the medicine, food, and biochemical industry
The TcPanD gene from T. castaneum was randomly and site-directed mutated, and the mutants were cloned into pET-28a(+) and expressed in E. coli BL21(DE3)
These results suggested that the mutant TcPanD-R98H/K305S had higher pH stability than the 37 ◦ C

Summary

Introduction

The β-alanine (3-aminopropionic acid) is the only β-type amino acid in nature and non-protein amino acid in vivo, which is not involved in protein synthesis, while it has various biological functions. The β-alanine is widely used in multiple industries, such as the medicine, food, and biochemical industry. Β-alanine is used to synthesize a series of high-value products, such as pantothenic acid, calcium pantothenate, carnosine, disodium pamidronate, balsalazide, and so on [1,2,3]. Β-alanine serves as a food additive to improve the flavour of seasonings. Β-alanine is an important nutritional supplement for athletes to improve their body functions [4,5]. The chemical method is the traditional routine for β-alanine production [6,7,8]

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