Design and Engineer L-Amino Acid Deaminase from Proteus vulgaris As a Robust Biocatalyst for One-Step Biosynthesis of a-Keto Acids

Design and engineer L-amino acid deaminase from Proteus vulgaris as a robust biocatalyst for one-step biosynthesis of a-keto acids

Long Liu

School of Biotechnology,Jiangnan University, Lihu road 1800, Wuxi, China

Tel.: +86-510-85918312, Fax:+86-510-85918309,E-mail: longliu@jiangnan.edu.cn

Abstract

L-Amino acid oxidases (LAAOs), which catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids and ammonia, are flavin adenine dinucleotideâ??containing homodimeric proteins [1, 2]. L-Amino acid oxidases are widely distributed in diverse organisms and have a range of properties. In this work, we systematially engineered the LAAO from Proteus vulgaris as a robust biocatalyst for the efficient one-step biosynthesis of a-keto acids, including α-ketoglutaric acid, α-keto-γ-methylthiobutyric acid, and phenylpyruvic acid, which are currently produced by multi-step chemical synthesis with heavy environmental pollution. First, the directed evolution of LAAO was done by error-prone polymerase chain reaction (PCR), and then the site-saturation mutagensis was conducted at the mutation sites of the positive mutants yielded by error-prone PCR. The biotransformaton conditions including biocatalyst concentration, the substrates concentration, temperature, and pH and so on. The maximum titers of α-ketoglutaric acid, α-keto-γ-methylthiobutyric acid, and phenylpyruvic acid are 12.3 g/L, 63.6 g/L, and 11.8 g/L, respectively, under the specific optimal conditions. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of a-keto acids has an advantage in terms of less environmental pollution and thus has great potential for industrial production of a-keto acids.

[1]Liu L et al., Appl Microbiol Biotechnol. doi: 10.1007/s00253-013-5444-2. 2014.

[2]Hossain GS et al., J Biotechnol. 169, 112-120, 2014.

Key-words: L-amino acid deaminase, Proteus vulgaris, a-keto acids, protein engineering