A NOVEL BETA-N-ACETYLGLUCOSAMINIDASE FROM STREPTOMYCES-THERMOVIOLACEUS OPC-520 - GENE CLONING, EXPRESSION, AND ASSIGNMENT TO FAMILY 3 OF THE GLYCOSYL HYDROLASES

A beta-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviola ceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide se quence of the gene, which encodes NagA, revealed are open reading fram e of 1,896 bp, encoding a protein with an M-r of 66,329. The deduced p rimary structure of NagA was confirmed by comparison with the N-termin al amino acid sequence of the cloned beta-N-acetylglucosaminidase expr essed by S. lividans. The enzyme shares no sequence similarity with th e classical beta-N-acetylglucosaminidases belonging to family 20. Howe ver, NagA, which showed no detectable beta-glucosidase activity, revea led homology with microbial beta-glucosidases belonging to family 3; i n particular, striking homology with the active-site regions of beta-g lucosidases was observed. Thus, the above-mentioned results indicate t hat NagA Born S. thermoviolaceus OPC-520 is classified as a family 3 g lycosyl hydrolase. The enzyme activity was optimal at 60 degrees C and pH 5.0, and the apparent K-m and V-max values for p-nitrophenyl-beta- N-acetylglycosamine were 425.7 mu M and 24.8 mu mol min(-1) mg of prot ein(-1), respectively.