Cloning of the gene of beta-N-acetylglucosaminidase from Lactobacillus casei ATCC 27092 and characterization of the enzyme expressed in Escherichia coli.
The gene encoding beta-N-acetylglucosaminidase (GlcNAcase) of Lactobacillus casei ATCC 27092 was cloned and expressed in Escherichia coli. The gene consisted of 1581 nucleotides, and had a promoter, Shine-Dalgarno, and rho-independent type transcription termination sequences typical in bacteria. The protein deduced from the sequence consisted of 526 amino acids, and had a putative signal peptide of 14 amino acids and 5 possible asparagine-linked glycosylation sites. A conserved sequence was homologous to the 12 other hexosaminidases from different origins. The recombinant GlcNAcase (r-GlcNAcase) purified from the transformed E. coli had a MW of 39 kDa and lacked oligosaccharide chains. The isoelectric point and the optimum pH for the activity of r-GlcNAcase were similar to those of original GlcNAcases (o-GlcNAcase). However, the thermal stability was lower, and sensitivity to Cd2+, Fe2+, Cu2+ and sodium dodecyl sulfate (SDS) was higher than those of o-GlcNAcases, suggesting that the oligosaccharide moieties of the enzyme contribute to their stability. The Km value for p-nitrophenyl-N-acetyl-beta-1,4-D-glucosamine (PNP-GlcNAc) of r-GlcNAcase (6.4 microM) implied that the affinity of r-GlcNAcase for the substrate was 200-fold higher than that of the original ones.
Senba
M
M
Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
Kashige
N
N
Nakashima
Y
Y
Miake
F
F
Watanabe
K
K
D000118:Acetylglucosaminidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli; D006596:Hexosaminidases / Q000737:chemistry; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D004247:DNA; D006596:Hexosaminidases; D000118:Acetylglucosaminidase