凝结芽孢杆菌N-乙酰-β-D-氨基葡萄糖苷酶基因的克隆鉴定及酶学性质

发布时间：2018-06-23 14:56

  本文选题：凝结芽孢杆菌 + N-乙酰-β-D-氨基葡萄糖苷酶　； 参考：《林业工程学报》2017年02期

【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 k Da,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。
[Abstract]:N-acetyl- 尾 -D-glucosaminidase (NAGase) is an indispensable part of microbial chitinase system. Recently, N-acetyl- 尾 -D-glucosaminidase (N-acetyl- 尾 -D-glucosaminidase) has attracted much attention in recent years because of its special role in chitin degradation. A hypothetical N- acetyl- 尾 -D- glucosaminidase gene was cloned from the genome of Bacillus coagulans NL01, with a length of 1 761 BP encoding 586 amino acids. The theoretical molecular weight of the protein is 64.5 kDa, and the similarity between the encoded amino acid sequence and the reported N-acetyl- 尾 -D- glucosaminidase is only 38. The gene was cloned into the expression vector pETDuet-1 and expressed in E. coli BL21 (DE3). The results showed that the heavy crude enzyme had the activity of N-acetyl- 尾 -D- glucosaminidase, and the specific activity of the purified enzyme was 74.3% Umg. The enzymatic properties of the recombinant enzyme were studied. It was found that the recombinant enzyme was a new type of high temperature resistant N acetyl-尾-D-glucosaminidase. The optimum pH was 5.5, the optimum temperature was 75 鈩，

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