A Novel b-N-Acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: Gene Cloning, Expression, and Assignment to Family 3 of the Glycosyl Hydrolases

A b-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned b-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical b-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable b-glucosidase activity, revealed homology with microbial b-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of b-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-b-N-acetylglucosamine were 425.7 mM and 24.8 mmol min mg of protein, respectively.