Date Published: June 11, 2019
Publisher: Public Library of Science
Author(s): Man Kumari Gurung, Bjørn Altermark, Ronny Helland, Arne O. Smalås, Inger Lin U. Ræder, Fernando Rodrigues-Lima.
N-acetylneuraminate lyases (NALs) are enzymes that catalyze the reversible cleavage and synthesis of sialic acids. They are therefore commonly used for the production of these high-value sugars. This study presents the recombinant production, together with biochemical and structural data, of the NAL from the psychrophilic bacterium Aliivibrio salmonicida LFI1238 (AsNAL). Our characterization shows that AsNAL possesses high activity and stability at alkaline pH. We confirm that these properties allow for the use in a one-pot reaction at alkaline pH for the synthesis of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) from the inexpensive precursor N-acetylglucosamine. We also show that the enzyme has a cold active nature with an optimum temperature for Neu5Ac synthesis at 20°C. The equilibrium constant for the reaction was calculated at different temperatures, and the formation of Neu5Ac acid is favored at low temperatures, making the cold active enzyme a well-suited candidate for use in such exothermic reactions. The specific activity is high compared to the homologue from Escherichia coli at three tested temperatures, and the enzyme shows a higher catalytic efficiency and turnover number for cleavage at 37°C. Mutational studies reveal that amino acid residue Asn 168 is important for the high kcat. The crystal structure of AsNAL was solved to 1.65 Å resolution and reveals a compact, tetrameric protein similar to other NAL structures. The data presented provides a framework to guide further optimization of its application in sialic acid production and opens the possibility for further design of the enzyme.
Sialic acids are sugars found on the surface of both prokaryotic- and eukaryotic cells and belong to the family of nine carbon α-keto acidic monosaccharides. N-acetylneuraminic acid (Neu5Ac, also often called sialic acid) is the most studied sugar within this family [1–3]. Extensive research has been done after its discovery in 1936 [4, 5] due to its interesting and important biological roles [6–10]. The applications of sialic acid and its derivatives are increasing. They have a wide range of potential medical applications, such as anti-viral and anti-microbial agents [11–14]. Furthermore, Neu5Ac has potential as a glyconutrient and its importance for fetal brain development has made it an attractive component for infant formulas . As a marker, increased concentrations of free serum sialic acid is an indicator of several diseases .
This study has described the recombinant production, biochemical characterization and structural determination of the N-acetylneuraminate lyase from A. salmonicida. The protein is a tetramer with high purity and yield after purification and with a tetrameric structure similar to other NALs. Based upon sequence and structural data we constructed a mutant that was important for the high kcat observed for Neu5Ac cleavage. We identified interesting enzymatic features of the enzyme, such as high activity and stability at alkaline pH, high activity at low temperature and a higher specific activity compared to the commercially available homologue from E. coli. We proved that the enzyme can be used at alkaline pH for synthesis of Neu5Ac from the inexpensive precursor N-acetylglucosamine. These enzymatic properties make the enzyme a promising biocatalyst, and the data presented provides a framework to guide further exploration of the enzyme. To evaluate the economic viability of its use, we suggest a further optimalization of the application of the enzyme in the synthesis of sialic acid using industrially relevant parameters, such as for example higher substrate concentrations and industrially relevant buffers.