Int J Biochem Mol Biol 2012;3(4):384-391
Original Article
Exploring residues crucial for nitrilase function by site
directed mutagenesis to gain better insight into
sequence-function relationships
Shubhangi Kaushik, Utpal Mohan, UC Banerjee
Biocatalysis and Protein Engineering Group, Department of Pharmaceutical Technology, National Institute of Pharmaceutical
Education and Research, Sector 67, S.A.S. Nagar-160 062, Punjab, India
Received September 5, 2012; Accepted December 6, 2012; Epub December 24, 2012; Published December 30, 2012
Abstract: Nitrilases represent a very important class of enzymes having an array of applications. In the present
scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on
the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with
P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to
lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional
role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A,
Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate
recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and
pattern of nitrile hydrolysis. (IJBMB1209001).
Keywords: Nitrilase, catalysis, sequence identity, site directed mutagenesis, mutants, conserved residues
Address all correspondence to:
Dr. U.C. Banerjee
Biocatalysis and Protein Engineering Group
Department of Pharmaceutical Technology
National Institute of Pharmaceutical Education and Research
Sector 67, S.A.S. Nagar-160 062, Punjab, India.
Tel: +91 172 2214682–87; Fax: +91 172 2214692
E-mail: ucbanerjee@niper.ac.in
Original Article
Exploring residues crucial for nitrilase function by site
directed mutagenesis to gain better insight into
sequence-function relationships
Shubhangi Kaushik, Utpal Mohan, UC Banerjee
Biocatalysis and Protein Engineering Group, Department of Pharmaceutical Technology, National Institute of Pharmaceutical
Education and Research, Sector 67, S.A.S. Nagar-160 062, Punjab, India
Received September 5, 2012; Accepted December 6, 2012; Epub December 24, 2012; Published December 30, 2012
Abstract: Nitrilases represent a very important class of enzymes having an array of applications. In the present
scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on
the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with
P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to
lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional
role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A,
Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate
recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and
pattern of nitrile hydrolysis. (IJBMB1209001).
Keywords: Nitrilase, catalysis, sequence identity, site directed mutagenesis, mutants, conserved residues
Address all correspondence to:
Dr. U.C. Banerjee
Biocatalysis and Protein Engineering Group
Department of Pharmaceutical Technology
National Institute of Pharmaceutical Education and Research
Sector 67, S.A.S. Nagar-160 062, Punjab, India.
Tel: +91 172 2214682–87; Fax: +91 172 2214692
E-mail: ucbanerjee@niper.ac.in
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