摘要
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Mg^2+对该重组普鲁兰酶有激活作用,而Zn^2+、Mn^2+、Ni^2+、Fe^2+、Cu^2+、Co^2+、Ca^2+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 Mg^2+at a concentration of 10 mmol/L,but inhibited by Zn^2+,Mn^2+,Ni^2+,Fe^2+,Cu^2+,Co^2+,and Ca^2+to different extents.In this study,a recombinant strain with efficiently expressed pullulanase was constructed,which had a potential in industrial applications.
作者
苏红玉
崔堂兵
SU Hong-yu;CUI Tang-bing(School of Bioscience and Bioengineering,South China University of Technology,Guangzhou 510006,China)
出处
《现代食品科技》
EI
CAS
北大核心
2019年第7期107-113,35,共8页
Modern Food Science and Technology
