A β- N-acetylhexosaminidase from Symbiobacterium thermophilum; gene cloning, overexpression, purification and characterization

Abstract

An ORF encoding the gene ( nahA)-related carbohydrate hydrolase was found in chromosomal DNA of the symbiotic thermophile, Symbiobacterium thermophilum. BLASTX results indicated that the product of this structural gene is β-glucosidase or β- N-acetylhexosaminidase. To investigate details of the nahA gene product, cloning of the gene, overproduction of the gene product in Escherichia coli, and purification and characterization of the resulting protein were conducted. The nahA gene was amplified by PCR using fragmented chromosomal DNA of S. thermophilum as a template, sequenced, and then ligated into the BamHI and NdeI sites of plasmid pET-25b(+) to construct the expression vector pST-BNAH-A for overproduction of the gene product. Results of studies on the hydrolytic activity of cell-free extracts against pNP-β-Glc, pNP-β-GlcNAc and pNP-β-GalNAc, obtained by disruption of cultured E. coli cells harboring pST-BNAH-A, suggested that the nahA gene product was β- N-acetylhexosaminidase. Comparison of the amino acid sequence of the recombinant protein with those of other β- N-acetylhexosaminidases indicated that the β- N-acetylhexosaminidase of S. thermophilum is a member of the 3-glycoside hydrolase family. The recombinant enzyme was purified to homogeneity from cell-free extract in an overall yield of 24%. This purified β- N-acetylhexosaminidase possessed thermostability, was stable in alkaline solution, and exhibited greater hydrolytic activity against chitin oligosaccharides than against pNP-β-GlcNAc.