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In the chemical-biological synthesis route of gabapentin, immobilized Escherichia coli cells harboring nitrilase are used to catalyze the biotransformation of intermediate 1-cyanocyclohexaneacetonitile to 1-cyanocyclohexaneacetic acid. Herein, we present a novel cell immobilization method, which is based on cell adsorption using 75 g/L Escherichia coli cells and 6 g/L zeolite, cell crosslinking using 3 g/L polyethylenemine and biomimetic silicification using 18 g/L hydrolyzed tetramethylorthosilicate. The constructed "hybrid biomimetic silica particles (HBSPs)" with core-shell structure showed a specific activity of 147.2 ± 2.3 U/g, 82.6 ± 2.8% recovery of nitrilase activity and a half-life of 19.1 ± 1.9 h at 55 °C. 1-Cyanocyclohexaneacetonitrile (1.0 M) could be completely hydrolyzed by 50 g/L of HBSPs at pH 7.5, 35 °C in 4 h, providing 92.1 ± 3.2% yield of 1-cyanocyclohexaneacetic acid. In batch reactions, the HBSPs could be reused for 13 cycles and maintained 79.9 ± 4.1% residual activity after the 10th batch, providing an average product yield of 92.6% in the first 10 batches with a productivity of 619.3 g/L/day. In addition, multi-layer structures consisting of silica coating and polyethylenemine/glutaraldehyde crosslinking were constructed to enhance the mechanical strength of immobilized cells, and the effects of coating layers on the catalytic properties of immobilized cells was discussed. • Cells harboring nitrilase were encapsulated in the core-shell structure though silicification. • HBSPs showed 82.6% recovery of nitrilase activity and a half-life of 19.1 h at 55 °C. • The average product yield in the first 10 batches was 92.6% with a productivity of 619.3 g/L/day. • Multi-layer particles were constructed to explore the effect of coating layers. [ABSTRACT FROM AUTHOR]

The thermostability of nitrilase from different microorganisms varies widely and is closely related to conserved sequences. In this study, a semi-rational design based on sequence analysis was performed to improve the thermostability and specific activity of the regioselective nitrilase from Acidovorax facilis ZJB09122. Using thermostable nitrilases from thermophilic microorganisms as templates, consensus sites 151, 223, 250, 169 and 280 were found to have effects on the thermostability and enzyme activity. A triple mutant AcN-T151V/C223A/C250G (AcN-T) was obtained with 15.9% higher activity and 2.1-fold longer half-life at 50 °C compared with the parent AcN-M. The optimal reaction temperature was 55 °C and the product tolerance was also improved. It was found that site 250 close to the catalytic active center plays a key role in the thermostability of nitrilase, and this role is universal among homologous nitrilases. On the other hand, sites 151 and 223 mainly affect enzyme activity, and this role is not universal. [Display omitted] • Thermostability and activity of regioselective nitrilase A. facilis ZJB09122 was improved. • Mutant AcN-T151V/C223A/C250G has 15.9% higher activity and 2.1-fold longer half-life. • Semi-rational design was performed using consensus sequence analysis and conserved site substitution. • Site 250 have a critical and universal effect on the thermostability of homologous nitrilases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]