解淀粉芽胞杆菌α-淀粉酶高活力突变体的创建与性质研究  

Construction and properties of Bacillus amyloliquefaciens α-amylase mutant of improved catalytic efficiency

作　　者：牛丹丹 靳晓 吴海洋[2] 刘晓光[2] 林娟 叶秀云 

机构地区：[1]福州大学生物科学与工程学院,福建省海洋酶工程重点实验室,福建福州350116 [2]天津科技大学生物工程学院,天津300457

出　　处：《食品与发酵工业》2017年第10期24-29,共6页Food and Fermentation Industries

基　　金：国家高技术研究发展计划(863计划)(2013AA102101);福建省教育厅产学研项目(JA15049);国家自然科学基金(31601407);福建省自然科学基金(2016J01157)

摘　　要：为进一步提高解淀粉芽胞杆菌α-淀粉酶(Bacillus amyloliquefaciensα-amylase,BAA)发酵生产与应用性能,对BAA编码基因实施了体外随机突变,建立了含有2×10~4个转化子的BAA突变库。BAA编码基因的突变频率为56%,基因突变效率为2.8/kb DNA,错义突变26.8%;以平板筛选法和摇瓶发酵法对BAA突变库进行筛选,得到6个酶活力显著提高的突变体,其中突变体BAA28的酶活力提高最多,提高达37%;进一步分析突变体BAA28序列,发现其4个氨基酸残基发生突变,分别是:T341P、P348L、T356P和P362L,其中T341P和T356P位于无规卷曲结构中,P348L位于α-螺旋结构中,P362L位于β-折叠结构中。进一步制备与纯化BAA28,并比较分析其酶学特征,与野生型BAA相比,BAA28的离子依赖性、热稳定性发生了显著变化;BAA28的比酶活提高了约31%,κcat/Km值提高了92%。突变体BAA28较BAA在催化与应用属性上有了显著提升,可应用于后续中温α-淀粉酶高产新菌种的构建并可显著改善其工业应用性能。Bacillus amyloliquefaciens α-amylase （BAA） is one of the most widely used industrial enzymes in the starch processing, food, brewing, fermentation, textile, papermaking, and medical industries. To further enhance fermentation level and application property, the mutant with significantly improved catalytic properties was constructed via in vitro molecular evolution and its nature was preliminarily illustrated. The BAA mutation library, with the muta- tional frequency of 56% , the efficiency of mutation of 2.8 points/kb DNA, and the missense mutation rate of 26. 8% , was constructed with the capacity of 2 - 104transformants by using error-prone PCR. The BAA mutants with enzymatic activity were screened using halo-plate assay and shaking flask fermentation test. Six mutants with improved activity were selected out and one of which, mutant BAA2$, displayed 37% increased activity. Sequencing results il- lustrated that mutant BAA28 had four separated mutations： T341 P, P348L, T356P, and P362L and of which, T341P and T356P were in the random coil structure, P348L was in the a-helix structure, and P362L was in the β-fold struc- ture. The BAA28 was further heterologously prepared and purified and its biochemical properties were examined in comparison to wild BAA. BAA28 displayed a remarkable change in ion-dependence and thermostability. Its specific activity was significantly increased with 1.31 folds of BAA and Kcat/Km was increased by 92%. BAA28 mutant per- formed significantly improved catalysis and application properties in comparison to BAA, which is of potential applica- tion in BAA-overproducing strain improvement and industrial properties.

关 键 词：中温α-淀粉酶 突变体 催化效率 

分 类 号：Q55[生物学—生物化学] TQ925.1[轻工技术与工程—发酵工程]