NDLI: Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants, which form increased amounts of mandeloamide from mandelonitrile

Content Provider	Springer Nature Link
Author	Sosedov, Olga Stolz, Andreas
Copyright Year	2013
Abstract	The nitrilase from Pseudomonas fluorescens EBC191 was modified by introducing random mutations via error-prone PCR techniques in order to obtain nitrilase variants, which form increased amounts of mandeloamide from racemic mandelonitrile. A screening system was established and experimentally optimized, which allowed the screening of nitrilase variants with the intended phenotype. This system was based on the simultaneous expression of nitrilase variants and the mandeloamide converting amidase from Rhodococcus rhodochrous MP50 in recombinant Escherichia coli cells. The formation of increased amounts of mandeloamide from mandelonitrile by the nitrilase variants was detected after the addition of hydroxylamine and ferric iron ions by taking advantage of the acyltransferase activity of the amidase, which resulted in the formation of coloured iron(III)–hydroxamate complexes from mandeloamide. The system was applied for the screening of libraries of nitrilase variants and 30 enzyme variants identified, which formed increased amounts of mandeloamide from racemic mandelonitrile. The increase in amide formation was quantified by high-performance liquid chromatography and the genes encoding the relevant nitrilase variants sequenced. Thus, different types of mutations were identified. One group of mutants carried different deletions at the carboxy-terminus. The other types of variants carried amino acid exchanges in positions that had not been related previously to an increased amide formation. Finally, a nitrilase variant was created by combining two independently obtained point mutations. This enzyme variant demonstrated a true nitrile hydratase activity as it formed mandeloamide and mandelic acid in a ratio of about 19:1 from racemic mandelonitrile.
Starting Page	1595
Ending Page	1607
Page Count	13
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	98
Issue Number	4
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-05-22
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Nitrilase Nitrile hydratase Error-prone PCR Hydroxamate Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Improvement of the amides forming capacity of the arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed mutagenesis

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

Influence of different carboxy-terminal mutations on the substrate-, reaction- and enantiospecificity of the arylacetonitrilase from Pseudomonas fluorescens EBC191

Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5

Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis

A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry

Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants, which form increased amounts of mandeloamide from mandelonitrile

Similar Documents

Improvement of the amides forming capacity of the arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed mutagenesis

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

Influence of different carboxy-terminal mutations on the substrate-, reaction- and enantiospecificity of the arylacetonitrilase from Pseudomonas fluorescens EBC191

Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5

Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis

A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry

Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants, which form increased amounts of mandeloamide from mandelonitrile