Influence of point mutations near the active site on the catalytic properties of fungal arylacetonitrilases from Aspergillus niger and Neurospora crassa

详细信息	查看全文 | 推荐本文 |

• 作者：Alena Pet艡í ; 膷ková ; ^a ; Olga Sosedov^b ; Stefanie Baum^b ; Andreas Stolz^b ; Ludmila Martí ; nková ; ^a ; ^{martinko@biomed.cas.cz}
• 关键词：Arylacetonitrilase variants ; Aspergillus niger ; Neurospora crassa ; (R ; S)-Mandelonitrile ; (R)-Mandelic acid ; (S)-Mandelamide ; (R ; S)-2-Phenylpropionitrile
• 刊名：Journal of Molecular Catalysis B ; Enzymatic
• 出版年：2012
• 期刊代码：55_13811177
• 类别：ce
• 出版时间：May, 2012
• 卷：77
• 期：Complete
• 页码：74-80
• 文件大小：577 K

摘要

Arylacetonitrilases from Aspergillus niger CBS 513.88 and Neurospora crassa OR74A (NitAn and NicNc, respectively) were expressed in recombinant Escherichia coli JM109. The respective whole-cell catalysts preferentially hydrolyzed (R)-enantiomers of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile. NitNc also formed significant amounts of mandelamide from (R,S)-mandelonitrile (40%of total product), mainly found as the (S)-enantiomer. At pH 4.5 and 5.0, the cells retained more than 40 and 60%of their nitrilase activity at pH 7, respectively. Nitrilase variants generated by site-directed mutagenesis carried amino acid replacements in the vicinity of the catalytically active cysteine residues (C162 and C167 in NitAn and NitNc, respectively). The conversions of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile by the nitrilase variants were compared to the wild-type nitrilases in terms of activity, enantioselectivity, and acid/amide ratio of the products formed. Thus the W168A variant of NitNc was identified, which formed significantly increased amounts of mandelamide and 2-phenylpropionamide, and which demonstrated an almost complete inversion of enantioselectivity for the conversion of (R,S)-2-phenylpropionitrile (from R- to S-selectivity).