N-Acetyl-D-glucosamine (GlcNAc) is a valuable monosaccharide widely used in the medical, agricultural, biofuel, and food industries. Its efficient and environment-friendly production depends on the binary system of beta-N-acetylhexosaminidase (HEX) and chitinase. In the present study, a HEX of glycoside hydrolasefamily 20 was identified in Streptomyces alfalfae ACCC40021, and was overexpressed in Escherichia coli. The purified recombinant SaHEX showed maximal activities at 60 degrees C and pH 5.5, and retained stable up to 45 degrees C. The enzyme not only exhibited broad substrate specificity including p-nitrophenyl beta-N-acetylglucosaminide, p-nitrophenyl beta-N-acetylgalactosaminide, chitooligosaccharides and colloidal chitin, but also had higher specific activities (up to 1149.7 +/- 72.6 U/mg) towards natural and synthetic substrates. When combined with a commercial chitinase, it achieved a conversion rate of 93.7% from 1% of colloidal chitin to GlcNAc in 6 h, with the product purity of >98%. These excellent properties make SaHEX a potential enzyme candidate for the chitin conversion for various industrial purposes. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.