关键词: β-N-乙酰氨基葡萄糖苷酶, 解淀粉芽孢杆菌, 克隆, 酶学性质, 糖苷水解酶3家族

Abstract: In this study, a novel β-N-acetylglucosaminidase gene was cloned from Bacillus amyloliquefaciens YX-01 and successfully expressed in Escherichia coli BL21. The recombinant protein (defined as BaNagase) was purified by Ni-IDA affinity chromatography and analyzed. The results showed that it encoded 616 amino acids, with the highest similarity (76.97%) to the sequence of β-N-acetylglucosidase (AIY91451.1) from B. subtilis. According to the phylogenetic analysis and multiple sequence alignments, BaNagase belonged to glycoside hydrolase (GH) family 3. The recombinant BaNagase showed specific activity of 16.42 U/mg toward the substrate pNP-GlcNAc. Its optimal pH value was 6.0 and the optimal reaction temperature was 65 ℃. BaNagase showed good stability below 55 ℃ at which more than 70% of initial activity was still retained. BaNagase exhibited strong thermostability and strict substrate specificity. This study provides a theoretical basis for efficient preparation of N-acetylglucosamine.

Key words: β-N-acetylglucosaminidase, Bacillus amyloliquefaciens, cloning, characterization, glycoside hydrolase family 3