一株普鲁兰酶产生菌的筛选及其基因克隆和酶学特性研究

Isolation of a Pullulanase Producing Strain and its Gene Cloning and Enzymatic Properties

普鲁兰酶作为一种解支酶,能够特异地作用于多糖中的α-1,6糖苷键,将淀粉中高度分支的支链淀粉转变成直链淀粉。本文通过以普鲁兰糖为唯一碳源的功能平板筛选方法,从云南腾冲温泉淤泥中分离到1株产普鲁兰酶的菌株73号,经16S rDNA序列分析,该菌属于厌氧芽胞杆菌属(Anoxybacillus sp.)。通过普鲁兰酶的保守区片段扩增及序列比对,最终从该菌中克隆得到普鲁兰酶编码基因pul73,全长2121bp,编码706个氨基酸。将该基因在大肠杆菌BL21(DE3)中进行异源表达和纯化,获得酶蛋白Pul73最适温度50℃,最适pH6.0,在65℃有较好的热稳定性,保温30min后仍有72%的剩余酶活。50℃时Pul73对普鲁兰糖的Km为1.643mg/mL,其最大反应速度Vmax为1.34U/mg,kcat为535.8/s。

As a starch debranching enzyme, pullulanase can specifically hydrolyze α-1,6 glucosidic linkages in polysaccharide to change amylopectin into amylose. One strain （ No. 73 ） with the higher pullulanse activities was selected from the thermal spring in Tengchong, Yunnan Province. The strain was identified as Anoxybacillus sp. by 16S rDNA phylogenetie tree analysis. The full-length sequences of the pullulanase gene from Anoxybacillus sp. 73 was obtained by PCR amplification with the degenerate primers designed based on the conservative domains of pullulanase from Bacillaceae. The pullulanase gene pul73 contained 2 121 nueletides, comprising one open reading frame encoding a polypeptide of 706 amino acids. The pul73 gene was cloned and expressed in E. coli BL21 （DE3）. The recombinant protein Pu173 was purified by Ni-NTA column and characterized. The optimal pH and temperature of Pu173 were 6.0 and 50℃ respectively. The enzyme showed stability at 65℃ after incubating for 30min. The Kin, Vmax and kcat of the purified Pu173 with pullulan as the substrate were approximately 1. 643 mg,/mL, 1.34 U/mg and 535.8/s, respectively.