title: Semi-Rational Engineering of Leucine Dehydrogenase for L-2-Aminobutyric Acid Production.
abstract: L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD+regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The "best" variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency (kcat/KM) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L-ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.
authors.affiliation: Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
authors.forename: Jian-Miao
authors.initials: JM
authors.lastname: Xu
authors.affiliation: Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
authors.forename: Feng
authors.initials: F
authors.lastname: Cheng
authors.affiliation: Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
authors.forename: Fang-Tian
authors.initials: FT
authors.lastname: Fu
authors.affiliation: Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
authors.forename: Hai-Feng
authors.initials: HF
authors.lastname: Hu
authors.affiliation: Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. zhengyg@zjut.edu.cn.|Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D019943:Amino Acid Substitution; D000613:Aminobutyrates / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D050803:Leucine Dehydrogenase / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D020125:Mutation, Missense
chemical_list: D000613:Aminobutyrates; D001426:Bacterial Proteins; C012223:alpha-aminobutyric acid; D050803:Leucine Dehydrogenase
keywords: High catalytic efficiency; L-2-aminobutyric acid; Leucine dehydrogenase; Semirational protein engineering