• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Applied Microbiology >Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides
【24h】

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

机译：用于增加酸或酰胺产量的荧光假单胞菌EBC191芳基乙腈酶变体的构建和应用

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens , a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with ( R , S )-mandelonitrile and ( R , S )-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived ( S )-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to ( S )-mandeloamide or ( S )-mandelic acid with high yields and enantiopurities.
机译：荧光假单胞菌EBC191的芳基乙腈酶与之前研究的芳基乙腈的区别在于其在扁桃腈转换过程中的低对映体特异性以及在此反应过程中形成的大量酰胺。在来自荧光假单胞菌的腈水解酶的序列中，半胱氨酸残基（Cys163）存在于催化活性半胱氨酸残基的直接附近（朝向氨基末端），这在细菌腈水解酶中是相当独特的。因此，该半胱氨酸残基在来自荧光假单胞菌EBC191的腈水解酶中交换为存在于其他腈水解酶同源位置上的各种氨基酸残基。以（R，S）-扁桃腈和（R，S）-2-苯基丙腈为底物，分析了这些突变对反应特异性和对映体特异性的影响。获得的突变体在其酰胺形成能力方面显示出显着差异。 Cys163交换为天冬酰胺或谷氨酰胺残基，导致形成的酰胺量大大增加。相反，丙氨酸或丝氨酸残基的取代减少了形成的酰胺的量。新发现的突变与先前鉴定的突变相结合，这也导致酰胺形成增加。因此，构建了除了突变Cys163Asn外还具有在酶的C末端缺失和/或修饰的Ala165Arg的变体。与仅携带单个突变的突变体相比，这些构建体显示出增加的酰胺形成能力。编码质粒的酶变体的重组质粒可形成大量的扁桃酰胺或由扁桃腈形成几乎化学计量的扁桃酸，这些重组质粒可用于转化表达植物来源的（S）-羟基腈裂解酶的大肠杆菌菌株。以这种方式获得的全细胞生物催化剂以高收率和对映体纯度将苯甲醛加氰化物转化为（S）-扁桃酰胺或（S）-扁桃酸。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号