多粘类芽孢杆菌新普鲁兰酶基因克隆及其在大肠杆菌中的表达

为了使新普鲁兰酶基因在大肠杆菌中表达并实现高密度发酵生产,采用PCR方法从多粘类芽孢杆菌基因组DNA中扩增出新普鲁兰酶基因Npu,测序结果表明Npu基因全长2106 bp,编码515个氨基酸,与其他多粘类芽孢杆菌新普鲁兰酶基因序相似性高达99%。将Npu基因连接到质粒PMD18T上,构建了PMD18T-Npu vecter重组载体。将该重组载体转化到大肠杆菌BL21中,该酶基因在大肠杆菌细胞中获得活性表达,并能将酶蛋白分泌到胞外。重组菌株BL21 PGEX 4T-1-Npu具有较好的遗传稳定性,连续传代培养10代,菌株所产酶总酶活性仍保持在715 U;纯化后的酶液于25 ℃保存6个月酶活性仍保持在5427 U,于4 ℃保存12个月酶活性仍保持在5390.5 U。这是首次对来源于类芽孢杆菌属(paenibacillus)的普鲁兰酶进行报道,由于Npu具有较好的水解淀粉支链的能力,因此其在淀粉加工业以及洗涤业上应用前景良好。

Cloning of Neopullulanase Gene from Paenibacillus polymyxa and Its Expression in Escherichia coli

The main purpose of this study was to express the neopullulanase gene of Paenibacillus polymyxa in Escherichia coli and to produce neopullulanase by high density fermentation. The neopullulanase gene npu was amplified from Paenibacillus polymyxa genomic DNA, the coding region contained 2106 bp in length and encoded 515 amino acids. The similarity of npu gene sequence with other neopullulanase genes of Paenibacillus polymyxa was 99%. The npu gene was successfully ligated with the plasmid PMD18T to construct the recombinant vector PMD18T-npu vecter, which was subsequently transformed into Escherichia coli BL21.After induced by methanol, npu gene was expressd in E. coli cells, and the enzyme could be secreted to the outside of E.coli cells. The recombinant strain BL21 PGEX 4t-1-npu had good genetic stability, and the total enzyme activity of the strain remained at 714 U after 10 generations of continuous culture.The enzyme activity of the purified enzyme solution remained at 5427 U after 6 months' storage at 25℃, and remained at 5390.5 U after 12 months' storage at 4℃. This is the first report of molecular characterization of an pullulanase from a strain of the genus paenibacillus. Npu is an attractive potential candidate for applications in starch processing and detergent industries due to its ability to effectively hydrolyze α-(1, 6) -linked branches in starch.