Related Experiment Video
Updated: Aug 9, 2026

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Engineering the residues on "A" surface and C-terminal region to improve thermostability of nitrilase
Zhe Xu1, Ting Cai1, Neng Xiong1
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China.
Abstract:
Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability. The best mutant AcN-T201F/Q339K/Q343K exhibited about 14-fold longer half-life at 45 °C. The result of homology modeling suggested that the site 201, which was located on the "A" surface (the dimer interaction), played an important role in the oligomerization of nitrilase and the stabilization of substrate binding pocket. The Phe substitution on site 201 was selected in protein engineering of nitrilase LNIT5, which also demonstrated an improvement of thermostability. In addition, lysine substitution on Q339 and Q343 which brought positive charges to the α helix in the C-terminal region stabilized the surface.
Related Concept Videos
Introduction to Mechanisms of Enzyme Catalysis
Preparation of Nitriles
Nitriles to Carboxylic Acids: Hydrolysis
Nitriles to Amines: LiAlH4 Reduction
As shown below, the mechanism involves three steps. Firstly, the hydride ion acting as a nucleophile attacks the nitrile carbon to form an anion. In the second step, a second equivalent of the hydride ion attacks the anion to...
Preparation of Amines: Reduction of Amides and Nitriles
Amides can be reduced to primary, secondary, and tertiary amines using catalytic hydrogenation, active metals like Fe,...
Preparation of Carboxylic Acids: Hydrolysis of Nitriles

