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 mutational frequency of 56%,the efficiency of mutation of 2.8 points/kb DNA,and the missense mutation rate of26.8%,was constructed with the capacity of 2×10~4 transformants 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 BAA28,displayed 37% increased activity.Sequencing results illustrated that mutant BAA28 had four separated mutations:T341 P,P348 L,T356 P,and P362 L and of which,T341 P and T356 P were in the random coil structure,P348 L was in the !-helix structure,and P362 L was in the "-fold structure.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 κcat/Kmwas increased by 92%.BAA28 mutant performed significantly improved catalysis and application properties in comparison to BAA,which is of potential application in BAA-overproducing strain improvement and industrial properties.