Tominaga Tatsuya, Hatakeyama Yoshinori
Saitama Industrial Technology Center North Institute, 2-133 Suehiro, Kumagaya, Saitama 360-0031, Japan.
Appl Environ Microbiol. 2007 Aug;73(16):5292-9. doi: 10.1128/AEM.00558-07. Epub 2007 Jun 29.
Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.
片球菌素PA-1是IIa类细菌素的一种,对乳酸菌具有抗菌作用。为了开发一种改良版的片球菌素PA-1,构建了片球菌素PA-1与另一种IIa类细菌素肠球菌素A之间的相互嵌合体。嵌合体EP由片球菌素PA-1的C端一半与肠球菌素A的N端一半融合而成,对从酸败乳制品中分离出的乳酸乳球菌菌株显示出增强的活性。为了开发一种更有效的该嵌合体版本,构建了一个DNA改组文库,其中片球菌素PA-1 N端一半内的四个特定区域与其他10种IIa类细菌素的相应序列进行了改组。活性筛选表明,280个改组突变体中有63个具有抗菌活性。菌落覆盖活性测定表明,其中一个突变体(命名为B1)在乳酸乳球菌上产生了>7.8毫米的生长抑制圈,而亲本片球菌素PA-1没有产生任何抑制圈。此外,活性改组突变体对各种乳酸杆菌、片球菌和肉杆菌物种显示出增强的活性。序列分析表明,活性突变体具有新的N端序列;例如,在活性突变体B1中,亲本片球菌素PA-1序列(KYYGNGVTCGKHSC)变为TKYYGNGVSCTKSGC。这些新的改良DNA改组细菌素可作为食品添加剂用于抑制乳制品的酸败。