产普鲁兰降解酶菌株的分离筛选及酶学特性的研究  

Screening and Identification of Pulllulanase-producing Strain and Its Enzymatic Characteristics

作　　者：王儒恺[1] 冯碧薇[1] 范洁琼[1] 周峻岗[1] 吕红[1] 

机构地区：[1]复旦大学生命科学学院遗传工程国家重点实验室,上海200433

出　　处：《复旦学报（自然科学版）》2012年第3期290-299,共10页Journal of Fudan University：Natural Science

基　　金：国家高技术研究发展计划(863计划)资助项目(2007AA021302)

摘　　要：普鲁兰降解酶(pullulan degrading enzymes)能够专一水解普鲁兰多糖α-1,6-糖苷键以及特定位置的α-1,4-糖苷键,在工业、食品业、医疗保健等行业中具有重要的应用意义.本研究采用唯一碳源法从稻田土样中培养筛选到21株产普鲁兰降解酶的菌株.对菌株A1的鉴定及其产生的普鲁兰降解酶的分析表明,该菌株为气单胞菌属,其产生的酶属于Ⅰ型普鲁兰酶(Type I pullulanase,EC 3.2.1.41),专一水解普鲁兰多糖α-1,6-糖苷键.该酶的最适反应温度为60℃,最适反应pH为6.0,具有良好的温度稳定性和适应性;同时在pH为4.0时仍然保留50%的酶活.进一步对A1菌株产酶的发酵条件(pH、温度、时间)进行了优化,摇瓶中A1分泌表达Ⅰ型普鲁兰酶的水平达到21.2 U/mL.根据已报道的气单胞菌属普鲁兰酶基因的保守序列设计引物,克隆获得了4 053 bp的A1普鲁兰酶基因pluA1全长序列,编码1351个氨基酸.本研究结果为该酶的开发应用奠定了基础.Type 1 pullulanase （EC 3. 2. 1.41） typically hydrolyzes the 1, 6-a-D-glucosidic linkages in pullulan or starch and has wide industrial applications. In the present study, 21 pullulanase producing strains were isolated from rice soil from Baoshan District, Shanghai, using a selective medium in which pullulan was used as the sole carbon source. Among these strains, the strain A1 was tentatively identified as Aeromonas sp. Pullulanase isolated from strain A1 was classified as type I pullulanase. The optimal reaction conditions for the enzyme were 60＂（2 and pH 6.0, respectively. The characteristic of PIuA1 meets the demand of industrial development that 50% enzyme activity was remained when reacted in a pH 4. 0 condition, The fermentation conditions were then optimized to obtain higher enzyme activity and finally the enzyme activity of pullulanase produced by the strain A1 approached 21.2 U/mL. Around 4 035 bp of pullulanase gene （p/uA1）, coding for 1 351 amino acids, was successfully amplified from Aeromonas sp. A1 genome.

关 键 词：气单胞菌 普鲁兰降解酶 Ⅰ型普鲁兰酶 酶学性质 

分 类 号：S154.3[农业科学—土壤学] Q556.2[农业科学—农业基础科学]