Molecular cloning, overexpression and purification of a novel laminarinase from Mesoflavibacter zeaxanthinifaciens S86

Abstract

Laminarin is a linear polymer of β-1,3-D-glucose with some β-1,6-D-glucose branching. It is a major structural and storage polysaccharide of various brown seaweeds and microalgae. Laminarinase (endo-1,3(4)-D-glucanase) catalyse the hydrolysis of β-1,3-D-glucosidic linkages in glucan. Laminarinase coding genes have not been demonstrated in Mesoflavibacter speicies. Here, we first report a novel laminarinase gene from the Meso&#64258 avibacter zeaxanthinifaciens S86. Meso&#64258 avibacter zeaxanthinifaciens S86 was isolated from seawater of Chuuk State in Micronesia. The genome of M. zeaxanthinifaciens S86 was sequenced by Genome Sequencer-FLX (GS-FLX), a next generation sequencing (NGS) technology. A unique nucleotide sequence that showed homology to known laminarinase was identified by the BLAST algorithm. It was cloned and designated as MzLam. MzLam was 2106 bp which is encoding 702 amino acid residues and includes a glycosyl hydrolase family 16 (GH16) laminarinase module. The deduced amino acid sequence showed highest identity (34.9%) and similarity (45.1%) with laminarinase form Leeuwenhoekiella blandensi. The predicted molecular mass of mature protein was 74 kDa. His-tagged MzLam was overexpressed in Escherichia coli and purified as a fusion protein. Optimal temperature for rMzLam was 50°C, while optimal pH was 7.0. The rMzLam acitivity was enhanced by 20% with 25 mM of MnSO4. The substrate specific activitieysis of β-1,3-D-glucosidic linkages in glucan. Laminarinase coding genes have not been demonstrated in Mesoflavibacter speicies. Here, we first report a novel laminarinase gene from the Meso&#64258 avibacter zeaxanthinifaciens S86. Meso&#64258 avibacter zeaxanthinifaciens S86 was isolated from seawater of Chuuk State in Micronesia. The genome of M. zeaxanthinifaciens S86 was sequenced by Genome Sequencer-FLX (GS-FLX), a next generation sequencing (NGS) technology. A unique nucleotide sequence that showed homology to known laminarinase was identified by the BLAST algorithm. It was cloned and designated as MzLam. MzLam was 2106 bp which is encoding 702 amino acid residues and includes a glycosyl hydrolase family 16 (GH16) laminarinase module. The deduced amino acid sequence showed highest identity (34.9%) and similarity (45.1%) with laminarinase form Leeuwenhoekiella blandensi. The predicted molecular mass of mature protein was 74 kDa. His-tagged MzLam was overexpressed in Escherichia coli and purified as a fusion protein. Optimal temperature for rMzLam was 50°C, while optimal pH was 7.0. The rMzLam acitivity was enhanced by 20% with 25 mM of MnSO4. The substrate specific activitie