Expression, characterization and mutagenesis of the gene encoding β-N-acetylglucosaminidase from Aeromonas caviae CB101

The gene encoding beta-N-acetylglucosaminidase (nagA1) from Aeromonas caviae CB101 was cloned, and its nucleotide sequence was determined. The open reading frame of the gene consisted of 2661 bp encoding a polypeptide of 887 amino acids including a putative signal peptide of 22 amino acids. The deduced amino acid sequence of nagA1 showed high similarities with beta-N-acetylglucosaminidase from various organisms, belonging to family 20 of glycosyl hydrolases. The nagA1 gene was expressed in Escherichia coli. The purified enzyme hydrolyzed N-acetylchitooligomers from N,N'-diacetylchitobiose to hexa-N-acetylchitohexaose with highest activity towards N,N'-diacetylchitobiose. The enzyme could also rapidly cleave p-nitrophenyl-N-acetyl-p-D-glucosaminide and p-nitrophenyl-N-acetyl-beta-D-galactosaminide. The hydrolytic activity of NagA1 was optimal at 50 degrees C and pH 8.0. Substitution of Glu538, Asp537 with Ala caused abolishing or largely decreasing of the enzyme activity, respectively, indicated that the predicted active sites Glu538, Asp537 were essential to the enzymatic activity of NagA1. (c) 2005 Elsevier Inc. All rights reserved.