本研究根据类芽孢杆菌的普鲁兰酶基因的保守区设计兼并引物，从Paenibacillus puldeungensis LK18中扩增出普鲁兰酶基因（pulA），将其连接至pET-28a(+)载体上，构建出重组表达载体pET-28a(+)-pulA，转入到Escherichia coli BL21(DE3)中，成功地表达了重组普鲁兰酶。结果表明：该普鲁兰酶基因全长1968 bp，编码655个氨基酸。通过Ni柱亲和层析纯化出重组蛋白，测定其比酶活为508.8 U/mg，分子量约为76.95 ku。该重组酶的最适反应温度为45 ℃，在35 ℃~40 ℃下保温120 min后剩余酶活达60%以上；最适作用pH为6.0，在pH 6.0~8.0条件具有较好的稳定性；10 mmol/L的K+和Mg2+对该重组普鲁兰酶有激活作用，而Zn2+、Mn2+、Ni2+、Fe2+、Cu2+、Co2+、Ca2+等对其有不同程度的抑制作用。本研究成功地构建出一株可高效表达普鲁兰酶的重组菌株，具备一定的工业应用价值。

In this study, the pullulanase gene (pulA) was amplified from Paenibacillus puldeungensis LK18 strain based on the design and primer of its conserved region, and linked to the expression vector pET-28a (+). The recombinant plasmid pET-28a (+)-pulA was transformed into Escherichia coli BL21 (DE3) leading to the successful expression of the recombinant pullulanase. The results showed that the pulA gene had a full length of 1968bp encoded 655 amino acids. The recombinant pullulanase was purified by Ni affinity chromatography and had a specific activity of 508.8 U/mg and molecular weight of 76.95 ku. The optimal temperature of the recombinant enzyme was 45 ℃ and 60% of its enzyme activity was retained after an incubation at 35~40 ℃ for 120 min. This enzyme had an optimal pH of 6.0 and high stability at a pH in the range of 6.0~8.0. The enzyme was activated by K+ and Mg2+ at a concentration of 10 mmol/L, but inhibited by Zn2+, Mn2+, Ni2+, Fe2+, Cu2+, Co2+, and Ca2+ to different extents. In this study, a recombinant strain with efficiently expressed pullulanase was constructed, which had a potential in industrial applications.