蜡样芽胞杆菌GXBC-3三个普鲁兰酶基因的表达及其酶学特性  

Gene expression and characterisation of three pullulanases from Bacillus cereus GXBC-3

作　　者：李美蓉[1,2] 汪小波[1,2] 黄英[1,2] 黄坚丽[1,2] 梁甲元[1,2] 黄日波[1,2,3] 杜丽琴[1,2] 韦宇拓[1,2] 

机构地区：[1]广西大学生命科学与技术学院,广西南宁530004 [2]亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004 [3]广西科学院,广西南宁530004

出　　处：《生物工程学报》2012年第4期466-475,共10页Chinese Journal of Biotechnology

基　　金：广西科学研究与技术开发计划(No.11107008-3)资助~~

摘　　要：探索获得优良的新型普鲁兰酶基因,丰富普鲁兰酶理论,对实现普鲁兰酶国产化具有重要意义。分析GenBank数据库中蜡样芽胞杆菌假定Ⅰ型、Ⅱ型普鲁兰酶基因序列,从实验室保藏的蜡样芽胞杆菌Bacilluscereus GXBC-3中克隆得到3个普鲁兰酶基因pulA、pulB、pulC,并分别导入大肠杆菌进行胞内诱导表达。纯化重组酶酶学性质研究表明重组酶PulA能水解α-l,6-和α-l,4-糖苷键,为Ⅱ型普鲁兰酶,以普鲁兰糖为底物时,最适反应温度及pH分别为40℃和6.5,比活力为32.89 U/mg;以可溶性淀粉为底物时,最适反应温度及pH分别为50℃和7.0,比活力为25.71 U/mg。重组酶PulB和PulC二者均只能水解α-l,6-糖苷键,为I型普鲁兰酶,以普鲁兰糖为底物时,其最适反应温度及pH分别为45℃、7.0和45℃、6.5,比活力分别为228.54 U/mg和229.65 U/mg。Exploring excellent new pullulanase genes, and enriching pullulanase theory are of great importance to realize the industrialization of pullulanase. Three genes, pulA, pulB and pulC, encoding pullulanases, were cloned from Bacillus cereus GXBC-3 by bioinformatics analyzing the open reading frame in Bacillus cereus, annotated as putative I and II pullulanases in the GenBank database. Characteristics of these recombinant enzymes were inducible intracellular expressed in Escherichia coli, the results showed PulA was typical II pullulanase. Recombinant PulA could hydrolyze α-1,4- and α-1,6-glycosidic bonds. Its specific activity was 32.89 U/rag with an optimum temperature of 40 ℃ and optimum pH 6.5 using pullulan as substrate. And for soluble starch substrate, its specific activity was 25.71 U/mg with an optimum temperature of 50℃ and optimum pH 7.0. PulB and PulC were I pullulanases and only hydrolyzed α-1,6-glycosidic bond. The specific activities, optimum temperature and optimum pH of PulB and PulC for pullulan substrate were 228.54 U/mg, 45℃, 7.0 and 229.65 U/rag, 45 ℃, 6.5, respectively.

关 键 词：蜡样芽胞杆菌GXBC-3 普鲁兰酶 克隆表达 酶学特性 

分 类 号：Q78[生物学—分子生物学]