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| 摘要: |
| 采用基因工程方法对嗜热地芽胞杆菌(Geobacillus kaustophilus)DY115的普鲁兰酶基因pulA在大肠杆菌中进行了克隆表达。该基因ORF全长为2 157bp,编码718个氨基酸。重组PulA在大肠杆菌(Escherichia coli)BL21(DE3)中能够有效表达,经Ni-Sepharose亲和层析获得纯化的重组PulA蛋白。PulA最适作用温度为70℃,最适pH为8.0,在65℃和碱性条件下具有良好的热稳定性;K+和Mn2+对PulA活性有明显促进作用,Cu2+和Zn2+则强烈抑制PulA活性;PulA对普鲁兰糖水解能力最强,且其水解支链淀粉和糯米淀粉的能力明显高于直链淀粉;PulA可水解普鲁兰糖的α-(1,6)糖苷键生成麦芽三糖,属于I型普鲁兰酶。这是首次对来源于地芽胞杆菌属(Geobacillus)的高温碱性普鲁兰酶进行报道,由于PulA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。 |
| 关键词: 嗜热地芽胞杆菌 普鲁兰酶 异源表达 酶学性质 |
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| Cloning, expression and enzymatic characterization of a pullulanase gene from Geobacillus kaustophilus DY115 |
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CHEN Xiaoyao, DONG Binbin , ZHOU Meixian
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| Abstract: |
| A pullulanase gene pulA from Geobacillus kaustophilus DY115 was cloned and expressed in Escherichia coli by genetic engineering method. The ORF of pulA gene is 2 157 bp in length and encodes a polypeptide (PulA) of 718 amino acids. The recombinant PulA was effectively expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. PulA exhibited optimal activity at 70 ℃ and pH 8.0, and was thermostable at 65 ℃ under alkaline condition. The enzyme activity was obviously enhanced in the presence of K+ and Mn2+, while Cu2+ and Zn2+ ions strongly inhibited the activity. PulA showed the highest activity of hydrolysis on pullulan, and also exhibited distinctly higher activity toward amylopectin and glutinous rice starch than amylose. PulA could hydrolyze α-(1, 6)-glucosidic linkage of pullulan forming maltotriose, suggesting that it is a type I pullulanase. This is the first report of molecular characterization of an alkaline thermostable pullulanase from a strain of the genus Geobacillus. PulA 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. |
| Key words: Geobacillus kaustophilus DY115 pullulanase heterologous expression enzyme characterization |
