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标题：Cloning of the Gene of β-N-Acetylglucosaminidase from Lactobacillus casei ATCC 27092 and Characterization of the Enzyme Expressed in Escherichia coli
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作者：Mio SENBA ; Nobuhiro KASHIGE ; Yukihiko NAKASHIMA 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2000
卷号：23
期号：5
页码：527-531
DOI：10.1248/bpb.23.527
出版社：The Pharmaceutical Society of Japan
摘要：The gene encoding β-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 K_m value for p-nitrophenyl-N-acetyl-β-1, 4-D-glucosamine (PNP-GlcNAc) of r-GlcNAcase (6.4 μM) implied that the affinity of r-GlcNAcase for the substrate was 200-fold higher than that of the original ones.
关键词：β-N-acetylglucosaminidase;β-N-acetylglucosaminidase gene;Lactobacillus casei;Escherichia coli;cloning;expression