中文摘要：

N-乙酰-β-D-氨基葡萄糖苷酶（N-acetyl-β-D-glucosaminidase,NAGase）是微生物几丁质酶系中不可缺少的一部分,近年来由于其在几丁质降解过程中的特殊作用而受到关注。笔者从凝结芽孢杆菌NL01基因组上克隆获得了1个假定的N-乙酰-β-D-氨基葡萄糖苷酶基因,长度为1 761 bp,编码586个氨基酸,蛋白质理论分子量为64.5 kDa,编码氨基酸序列与已报道的N-乙酰-β-D-氨基葡萄糖苷酶相似性仅为38%。将该基因克隆至表达载体pETDuet-1上,并在大肠杆菌BL21（DE3）中进行重组表达,结果显示重粗酶具有N-乙酰-β-D-氨基葡萄糖苷酶活力,经镍柱纯化获得纯酶的比酶活为74.3 U/mg。对重组酶酶学性质研究发现,该酶是一种新型耐高温N-乙酰-β-D-氨基葡萄糖苷酶。最适pH为5.5,最适温度为75℃,pH和热稳定性较好,在pH 3.5-7.5范围内比较稳定,在不高于65℃条件下热处理30 min后,酶活力可以保持在70%以上。

英文摘要：

Chitin, an unbranched homopolymer of 1,4-β-1inked N-acetyl-D-glucosamine （ GlcNAc）, is widely distributed and believed to be the second most abundant and renewable source in nature, next to cellulose. Chitinases, chitin- degrading enzymes are mainly composed of chitobiosidase, N-acetyl-β-D-glucosaminidase, β-D-glucosaminidase and chitosanase. As an indispensable member of the chitinolytic system and involved in chitin degradation, N-acetyl-β-D- glucosaminidase （NAGase） catalyzes the hydrolysis ofβ（1-4） linked N-acetylglucosaminyl residues from glycol-con- jugates at the non-reducing end, producing different lengths of GlcNAc. The NAGase is widely distributed among most types of living organisms and has numerous biological functions. Bacterial NAGase has a crucial physiological role in cell wall recycling, even in altering the function of the host cell. In recent years, NAGase has been extensively studied due to its significance in many fields. In this study, the Bacillus coagulans genome was used as the template to amplify the putative N-acetyl-β-D-glucosaminidase gene, whose length is 1 761 bp. It encodes 586 amino acids and the theoretical molecular weight mass is 64.5 kDa. The results of amino acid sequence analysis showed that the enzyme had a very low similarity with the reported N-acetyl-β-D-glucosaminidase, only 38%. The gene was cloned into pETDuet-I and expressed in Escherichia coli BL21 （DE3）. The crude enzyme had N-acetyl-β-D-glucosaminidase activity and the purified specific activity was 74.3 U/mg after Ni-NTA purification. The optimal pH and temperature is 5.5 and 75℃, respectively. Enzymological characteristics showed that the enzyme was stable in a range of pH from 3.5 to 7.5. The enzyme activity remained more than 70% after incubation for 30 rain below 65℃. A low-level inhibition of N-acetyl-β-D-glucosaminidase （15.1% ） was observed with Cu^2＋（ 2 mmol/L）, and a low-level promotion of N-acetyl-β-D-glucosaminidase （ 10. 7% ） and （ 13.5% ） was observed