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

Abstract

ABSTRACT A β- 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 M r of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β- N -acetylglucosaminidase expressed by S. lividans . The enzyme shares no sequence similarity with the classical β- N -acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-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 K m and V max values for p -nitrophenyl-β- N -acetylglucosamine were 425.7 μM and 24.8 μmol min −1 mg of protein −1 , respectively.