Abstract
Purpose of work
Is to report an oligoalginate lyase with high enzymatic activity and high-level expression.
Using site-finding PCR and degenerate PCR, a gene (designated oalS17) encoding a new oligoalginate lyase was cloned from Shewanella sp. Kz7 and expressed in Escherichia coli. The gene consisted of 2,292 bp with deduced amino acid size of 763 including a putative signal peptide of 44 amino acid residues belonging to polysaccharide lyase (PL) family 17. The recombinant protein was most active at 50 °C and pH 6.2 in 50 mM phosphate buffer. It degraded alginate more efficiently than polyM and polyG block into a monomeric sugar acid, with a specific activity of 32 U mg−1 toward alginate, 24 U mg−1 toward polyM and 5 U mg−1 toward polyG. With the high-level expression and high enzymatic activity, the recombinant oligoalginate lyase OalS17 could be a potential enzyme for further research on alginate saccharification and biofuels production.
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Acknowledgments
This study was supported by the Special Fund for Marine Scientific Research in the Public Interest (201105027-3), National High-tech R&D Program of China (2011AA09070304), National Natural Science Foundation of China (41376144), and Key Technologies Research and Development Program of China (2013BAB01B02).
Supporting information
Supplementary Table 1. Primers used for cloning of the gene oalS17.
Supplementary Fig. 1 The metabolic process of alginate. Alginate lyase depolymerizes alginate into oligosaccharides by cleaving the glycosidic bonds through a β-elimination reaction. Then, the oligosaccharides are degraded into monomeric unit by oligoalginate lyase, which spontaneously convert into 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Subsequently, DEH reductase (DehR) reduces DEH into 2-keto-3-deoxygluconate (KDG), a common metabolite that is fed into the Entner–Doudoroff (ED) pathway to produce ethanol. Thick and dotted arrows indicate the cleavage sites for oligoalginate lyases and alginate lyases, respectively. This scheme was based on the modification of the figure made by Miyake et al. (2003).
Supplementary Fig. 2 Conserved domains in the protein sequences of oligoalginate lyase OalS17 and other oligoalginate lyases. Protein sequences were analyzed using Conserved Domain Search Service of NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/). GenBank accession numbers of exotype oligoalginate lyases atu3025 from Agrobacterium tumefaciens strain C58, A1-IV from Sphingomonas sp. A1, OAL from Stenotrophomonas maltophilia KJ-2, AlgL from Sphingomonas sp. MJ3, Alg17C from Saccharophagus degradans 2-40 and AlyA5 from Zobellia galactanivorans DsiJT are AAK90358, BAB03319, AGM38186, AEM45874, ABD82539 and CAZ98266, respectively.
Supplementary Fig. 3 Protein sequence alignment of oligoalginate lyase OalS17, alginate lyase AlyII from Pseudomonas sp. OS-ALG-9, oligoalginate lyase AlgL from Sphingomonas sp. MJ3, oligoalginate lyase Alg17C from Saccharophagus degradans 2-40 and putative alginate lyase Sfri-3104 from Shewanella frigidimarina NCIMB 400 using ClustalX. Identical amino acid residues are boxed in dark shade, and amino acid residues above 70 % consensus are boxed in pale shade. The filled triangle and filled circle indicate the proposed catalytic sites, and substrate interacting sites for Alg17C, respectively.
Supplementary Fig. 4. ESI−MS analysis of the major end products from the reaction with alginate by oligoalginate lyase OalS17.
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Wang, L., Li, S., Yu, W. et al. Cloning, overexpression and characterization of a new oligoalginate lyase from a marine bacterium, Shewanella sp.. Biotechnol Lett 37, 665–671 (2015). https://doi.org/10.1007/s10529-014-1706-z
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DOI: https://doi.org/10.1007/s10529-014-1706-z