1. Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis.
- Author
-
Ji W, Sun W, Feng J, Song T, Zhang D, Ouyang P, Gu Z, and Xie J
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins genetics, Biocatalysis, Cloning, Molecular, Corynebacterium glutamicum classification, Corynebacterium glutamicum enzymology, Escherichia coli enzymology, Escherichia coli genetics, Gene Expression, Hexosamines metabolism, Hydro-Lyases chemistry, Hydro-Lyases classification, Hydro-Lyases genetics, Hydro-Lyases metabolism, Kinetics, Molecular Sequence Data, Oxo-Acid-Lyases chemistry, Oxo-Acid-Lyases genetics, Phylogeny, Pyruvic Acid metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Substrate Specificity, Bacterial Proteins metabolism, Corynebacterium glutamicum chemistry, N-Acetylneuraminic Acid biosynthesis, Oxo-Acid-Lyases metabolism
- Abstract
N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the high Neu5Ac cleavage activity in most isozyme forms, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a "GRAS" (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest kcat/Km value for Neu5Ac and highest kcat/Km values for ManNAc and pyruvate, which makes CgNal favor industrial Neu5Ac synthesis process in a non-equilibrium condition. The recombinant CgNal reached the highest expression level (480 mg/L culture), and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition.
- Published
- 2015
- Full Text
- View/download PDF