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Nitrilases are promising biocatalysts to produce high‐value‐added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2‐chloronicotinonitrile (2‐CN), very few robust nitrilases have been reported for efficient production of 2‐chloronicotinic acid (2‐CA) from 2‐CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2‐CN was increased from 0.14 × 10−3 to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2‐CN was completely converted, resulting in 2‐CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2‐CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2‐CA with the nitrilase‐catalyzed route. [ABSTRACT FROM AUTHOR]

Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase (BaNITM0) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor (S)‐3‐cyano‐5‐methylhexanoic acid ((S)‐CMHA). However, BaNITM0 also catalyzed the hydration of IBSN to produce by‐product (S)‐3‐cyano‐5‐methylhexanoic amide. To obtain industrial nitrilase with vintage performance, we carried out engineering of BaNITM0 for simultaneous evolution of reaction specificity, enantioselectivity, and catalytic activity. The best variant V82L/M127I/C237S (BaNITM2) displayed higher enantioselectivity (E = 515), increased enzyme activity (5.4‐fold) and reduced amide formation (from 15.8% to 1.9%) compared with BaNITM0. Structure analysis and molecular dynamics simulations indicated that mutation M127I and C237S restricted the movement of E66 in the catalytic triad, resulting in decreased amide formation. Mutation V82L was incorporated to induce the reconstruction of the substrate binding region in the enzyme catalytic pocket, engendering the improvement of stereoselectivity. Enantio‐ and regio‐selective hydrolysis of 150 g/L IBSN using 1.5 g/L Escherichia coli cells harboring BaNITM2 as biocatalyst afforded (S)‐CMHA with >99.0% ee and 45.9% conversion, which highlighted the robustness of BaNITM2 for efficient manufacturing of pregabalin. [ABSTRACT FROM AUTHOR]

Nitrilases are widely distributed in nature and are able to hydrolyze nitriles into their corresponding carboxylic acids and ammonia. In industry, nitrilases have been used as green biocatalysts for the production of high value-added products. To date, biocatalysts are considered to be important alternatives to chemical catalysts due to increasing environmental problems and resource scarcity. This review provides an overview of recent advances of nitrilases in aspects of distribution, enzyme screening, molecular structure and catalytic mechanism, protein engineering, and their potential applications in industry. [ABSTRACT FROM AUTHOR]

Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi‐rational design was developed for the engineering of nitrilase. A chimera BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi‐rational design was executed on BaNIT to further generate variant BaNIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4‐fold increase in whole‐cell activity and the enantiomeric ratio (E) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes. [ABSTRACT FROM AUTHOR]

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L−1 day−1 and the space-time productivity of 143.2 mmol L−1 h−1 g−1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA. [ABSTRACT FROM AUTHOR]

A recirculating packed bed reactor (RPBR) was used for efficient production ofR-mandelic acid (R-MA) by kinetic resolution of racemicR,S-mandelonitrile (R,S-MN) using the recombinantE. colicells crosslinked with diatomite (DA)/glutaraldehyde (GA)/polyethyleneimine (PEI). The performance and productivity of RPBR were evaluated by several parameters, including cell load, substrate feeding rate, height diameter (H/D) ratio, reactor structures, and operation stability. The kinetic resolution process showed higher initial reaction rate (1.52 mM/min) and yield (100%) by recycling 100 mL of substrate solution (70 mM) through RPBR packed with 6.0 g immobilized cells at a substrate-feeding rate of 19 mL/min while the H/D ratio was 2.8. The immobilized cells were successfully applied into kinetic resolution ofR,S-MN in the RPBR for 50 batches with an average productivity of 4.12 g/L/h forR-MA with >99% of enantiomeric excess. [ABSTRACT FROM AUTHOR]

( R)-(−)-Mandelic acid ( R-MA) is widely used both as a versatile intermediate for pharmaceuticals and a resolving agent in chiral resolution processes. In the current study, to improve the stability of operation, recombinant Escherichia coli cells expressing nitrilase from Alcaligenes faecalis were immobilized for the enantioselective hydrolysis of ( R, S)-mandelonitrile to R-MA. Different immobilization methods including entrapment matrices, entrapment matrices cross-linked by cross-linking and polymerization agents, and direct cross-linking cells using glutaraldehyde (GA) or bionic silicon were investigated. To facilitate industrial solid-liquid separation, the direct cross-linking recombinant E. coli cells using diatomite/GA/polyethyleneimine with 135.95% relative activity compared with free cells was chosen using water as the reaction medium. The operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 28 cycles of batch reaction and the successive production of R-MA could reach 1.88 M. Moreover, the immobilized cells showed good storage stability with about 52% relative activity after storing for 30 days at 4 °C. Therefore, the immobilized biocatalyst is very promising for upscale production of optically pure R-MA with high performance and low cost. [ABSTRACT FROM AUTHOR]

Efficient recovery of of 1-CA from the bioreaction mixture was investigated using an ion-exchange (IEX) process. Among seven IEX resins examined, the anion resin 201×7 exhibited the highest adsorption capacity for 1-CA. The Freundlich model has the better description for 1-CA adsorption equilibrium than the Langmuir model. The adsorption kinetics showed that the pseudo-second order model was fitted very well. Dynamic adsorption and desorption experiments have been carried out in a fixed bed column to obtain optimal parameters for the recovery of 1-CA from the bioreaction mixture. Under the optimized conditions, 1-CA was recovered in yield of 91.3% with > 99% HPLC purity. [ABSTRACT FROM PUBLISHER]

The enantioselective hydrolysis of ortho-chloromandelonitrile with nitrilase is one of the most attractive approaches to prepare ( R)- ortho-chloromandelic acid. To date, efforts to develop this nitrilase-mediated process were plagued by either insufficient eep (enantiomeric excess of product) or low activity due to the steric hindrance from the ortho-substituted substrate. To improve the nitrilase potential for producing ( R)- ortho-chloromandelic acid, an enhancement of both activity and enantioselectivity towards sterically hindered nitriles would be highly desirable. Molecular docking of the ( R)- ortho-chloromandelonitrile into the active site of wild-type 2A6 nitrilase (nitA) allowed the identification of proximal nitA active site residues. Several residues (52, 132, 189 and 190) were selected as targets for single and double point mutation to improve nitA activity and enantioselectivity towards ortho-chloromandelonitrile. Targeted mutagenesis yielded several nitA variants with superior activity and enantioselectivity. The best mutant T132A/F189T exhibited a 4.37-fold higher specific activity (7.39 U/mg) towards ortho-chloromandelonitrile than the wild-type nitA. More importantly, the enantioselectivity ( E) was improved from 17.34 to >200, resulting in a highly enantiopure product. Molecular docking experiments further support the enhanced activity and enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of nitA are provided. The amino acids at sites 189 and 132 determine the activity and enantioselectivity towards ortho-chloromandelonitrile. With mutant T132A/F189T as a catalyst, a maximum of 450 mM of ( R)- ortho-chloromandelic acid was produced with a 90% conversion and >99% eep within 3 h. This is the first time that a high productivity of ( R)- ortho-chloromandelic acid of up to 671.76 g L−1 d−1 using a nitrilase-mediated approach is reported. The engineered T132A/F189T variant represents a promising and competitive biocatalyst for practical application in synthesizing ( R)- ortho-chloromandelic acid. [ABSTRACT FROM AUTHOR]

Abstract: The nitrilase activity of a newly isolate strain, Alcaligenes faecalis WBX11, which is capable of catalyzing the direct hydrolysis of iminodiacetonitrile to its corresponding dicarboxylic acid iminodiacetic acid, was significantly enhanced by low-energy ion beam implantation, a novel microbial breeding way. After ion implantation, nitrilase activity and the total biomass of the mutant were significantly improved by 45% and 75%, respectively, compared with the wild strain. The temperature and pH optimum of the variant nitrilase were 35°C and pH 7.5, respectively, basically consistent with those of the wild strain. The nitrilase was mesophilic because of the quick deactivation in temperature higher than 45°C. Nitrilase showed a broad substrate spectrum, especially on arylacetonitrile and aliphatic nitrile. Study on reaction kinetics showed that no obvious substrate inhibition was observed even though the concentration of the substrate was up to 250mM. All results indicate that the low-energy ion implantation is an effective way for microbial breeding and the resulting mutant may be suitable for industrial applications. [Copyright &y& Elsevier]

Abstract: A nitrilase-producing strain ZJUTB06-99, capable of biotransforming acrylonitrile into acrylic acid, was newly isolated from soil samples. Based on the morphology, physiological tests, ATB system and its 16S rDNA sequence, strain ZJUTB06-99 was identified as Arthrobacter nitroguajacolicus. Optimal reaction conditions were investigated by the manipulation and measurement of various parameters including pH, temperature and certain cationic metals. The highest nitrilase activity was obtained when reaction was carried out at a pH of 6.5 phosphate buffer and in temperature of 40°C water bath. The nitrilase of A. nitroguajacolicus ZJUTB06-99 exhibited excellent thermostability. Nitrilase activity was strongly inhibited by Hg2+, Ag+ and Cu2+, but Ni2+ and Ca2+ increased enzyme activity to 163% and 158%, respectively. The investigation of substrates spectrum showed that A. nitroguajacolicus ZJUTB06-99 exhibited the highest nitrilase activity towards aromatic nitriles such as phenylacetonitrile. However, no detectable activity was recorded when any of the tested amides were used as substrates. [Copyright &y& Elsevier]

Abstract: Resting cells of Rhodococcus sp. G20 were used for the transformation of β-aminopropionitrile to β-alanine, an important beta amino acid. A 23 central composite experimental design was performed with the purpose of optimizing the β-alanine production in a bubble column reactor with 200mL working volume using response surface methodology (RSM). The individual and interactive effects of three independent variables (cells loading, substrate concentration, airflow rate) on β-alanine production were investigated. A quadratic polynomial predictive model was obtained after statistical analysis to predict the optimum biotransformation conditions. The optimum bioconversion conditions of β-aminopropionitrile in a batch operation for β-alanine production were as follows: cells loading of 16.50gww/200mL, substrate concentration of 1.29% (v/v), and airflow rate of 86.56L/h, under which an overall 40.6% increase in productivity of β-alanine was obtained. The influences of the temperature and pH on the conversion were also studied, and the optimums were 30°C and pH 7.5. The measured activation energy (E a) was found to be 22,199J/mol, thus indicating the presence of diffusional resistance. [Copyright &y& Elsevier]

Surface-crosslinked hydrogel particles encapsulating E. coli cells harboring nitrilase were constructed and used as a durable biocatalyst in the synthesis of gabapentin intermediate. E. coli cells (50 g/L) expressing nitrilase were encapsulated in 2% (w/v) alginate hydrogel, 0.5%(w/v) glutaraldehyde and 1%(w/v) polyethyleneimine were sequentially used as crosslinker. The constructed alginate particles with GA&PEI composite crosslinking had a similar catalytic capacity compared to equivalent free cells, which could completely hydrolyze 1.0 M substrate 1-cyanocyclohexaneacetonitrile within 4 h at pH 7.5 and 35 °C, and have a specific activity of 134.6 U/g, an optimum temperature at 55 °C and a corresponding half-life up to 19.1 ± 1.9 h. The particles showed excellent storage stability, reusability and mechanical strength, which retained 90.3% activity after 2-month storage at 4 °C, and 83.2% activity after 14-day storage at 35 °C. In batch reactions, 50 g/L particles could be reused for maximum 32 cycles, maintained 63.5% residual activity and 50.7% particle integrity, providing an average product yield of 94.6% in the first 30 batches and a productivity of 1467.03 g/L/day. Besides, the stability and catalytic activity of the particles were almost unaffected in several organic co-solvents and in purified water, showing their good industrial potential. [Display omitted] • Surface-crosslinked hydrogel particles encapsulating E. coli cells were prepared as biocatalysts. • The particles have similar catalytic capacity compared to equivalent free cells. • High activity and strength enable it to completely hydrolyze 1 M substrate within 4 h. • The particles retained 90.3% activity after 2 months of storage and could be reused up to 32 times. [ABSTRACT FROM AUTHOR]

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]

Nitrilase-catalyzed regioselective hydrolysis of 1-cyanocyclohexaneacetonitrile (1-CHAN) is a green and efficient approach for the preparation of 1-cyanocyclohexaneacetic acid (1-CHAA), a key precursor for the synthesis of gabapentin. Here, a mesoporous biosilica particles prepared by the ethyleneamine-mediated silicification have been used as carrier for the encapsulation of nitrilase from Acidovorax facilis (NitA). The silica-encapsulated NitA (NitA@silica) with triethylenetetramine as an initiator showed the highest immobilization efficiency (98.3%) and specific activity (672.6 U/g). Both free and encapsulated NitA were optimally active at 40 °C and pH 7.0, however, the encapsulated enzyme exhibited wider optimum temperature range, and enhanced thermal stability compared with the free enzyme. The kinetic parameters K m and V max for free and encapsulated NitA were calculated to be 141 mM and 9.97 mM min−1, and 280 mM and 9.02 mM min−1, respectively. The encapsulated NitA showed good reusability and retained about 94.2% of its initial activity even after 16 cycles of reaction. Also, the storage experiments revealed high activity maintenance of encapsulated NitA after 17-day storage at 4 °C. A preparative scale regioselective hydrolysis of 1-CHAN to 1-CHAA with encapsulated NitA as biocatalyst was carried out in a 2 L stirred bioreactor. The concentration of 1-CHAA reached 152 g/L after 8 h reaction and the conversion was 90.9%. These results showed that the encapsulation of NitA in ethyleneamine-mediated biosilica is an efficient and simple way for preparation of stable nitrilase and have a great potential for application in enzymatic production of carboxylic acids. • A novel method of nitrilase encapsulation on ethyleneamine-mediated biosilica particles is proposed. • Biosilica-encapsulated nitrilase exhibited extremely high immobilization efficiency and specific activity. • The performance of the encapsulated nitrilase was evaluated by the synthesis of 1-cyanocyclohexaneacetic acid. • The encapsulated nitrilase can keep its catalytic activity after being reused sixteen times. [ABSTRACT FROM AUTHOR]

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co 2+ stimulated nitrilase activity, whereas Cu 2+ , Zn 2+ and Tween 80 showed a strong inhibitory effect. The V max and k cat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the K m was decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN. [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]

1-Cyanocyclohexaneacetic acid (1-CHAA) is an important intermediate in the preparation of gabapentin. Regioselective biotransformation of 1-cyanocyclohexaneacetonitile (1-CHAN) into 1-CHAA, mediated by the immobilization of recombinant microbial nitrilase, is a novel and promising strategy for its production. In this study, a recombinant E. coli expressing Acidovorax facilis nitrilase was flocculated with polyethylenemine, followed by cross-linking with glutaraldehyde to obtain cross-linked cell aggregates (CLCAs). Under the optimum preparation conditions, the activity recovery of CLCAs reached 68%. Characterization of the CLCAs showed that the optimum pH was not affected by cross-linking, but cross-linking resulted in slightly different optimum temperature. The half-life of CLCAs at 45 °C was 266.5 h, which is 11.6-times higher than that of the free cells. Preparative-scale biotransformation of 1-CHAN to 1-CHAA at high substrate loading (up to 1.0 M) was performed using CLCAs as the biocatalyst in a 2L stirred reactor, resulting in an conversion of 91%, with a productivity of 244.5 g L −1 d −1 . The CLCAs also exhibited a high operational stability and even after 30 cycles of reaction, it retained >80% of the initial conversion. [ABSTRACT FROM AUTHOR]

Gabapentin has been widely used for the treatment of epilepsy and neuropathic pain in the world. In this work, an efficient and greener chemoenzymatic manufacturing process for gabapentin has been developed. The whole cells expressing engineered nitrilase were immobilized in diatomite cross-linked with polyethyleneimine and glutaraldehyde to improve their reusability and stability for the synthesis of gabapentin intermediate 1-cyanocyclohexaneacetic acid from 1-cyanocyclohexaneacetonitrile. The initial enzyme activity in the bioreactors at each batch biotransformation was maintained at a constant value by introducing fresh immobilized cells into the system to keep the high conversion (100%) and the same operation time (8 h) in each batch. The resulting 1-cyanocyclohexaneacetic acid was then converted to gabapentin efficiently by simple chemical steps. An E factor (exclude water) of 11.5 was calculated for the chemoenzymatic route relative to 55.7 for the mentioned chemical process. This new route dramatically improved process efficiency compared to the chemical process. [ABSTRACT FROM AUTHOR]

A nitrilase gene from Acidovorax facilis ZJB09122 was cloned and expressed in Escherichia coli BL21 (DE3). To improve the activity of this nitrilase, a key amino acid Phe168 was selected and mutated by site-directed mutagenesis, based on the homology modeling and previously described “hot spot” mutation. After mutation and screening, a mutant (Mut-F168V) with higher activity and stability was obtained. The nitrilase activity of Mut-F168V to hydrolyze 1-cyanocyclohexylacetonitrile was 39.52-fold compared with wild type A. facilis nitrilase (Wt-Acf-Nit). The values of K m and V max of Mut-F168V were markedly decreased to 1.89-fold and increased to 50.34-fold as compared to Wt-Acf-Nit, respectively. The biotransformation study showed that 1.0 M of 1-cyanocyclohexylacetonitrile could be regioselectively hydrolyzed to 1-(cyanocyclohexyl) acetic acid with 90% yield. The yield of 1-(cyanocyclohexyl) acetic acid by Mut-F168V was 66.19-fold compared to Wt-Acf-Nit after 1 h at the concentration of 1.0 M 1-cyanocyclohexylacetonitrile as substrate. The 1-(cyanocyclohexyl) acetic acid was subsequently isolated and characterized. The mutant (Mut-F168V) appears promising for potential applications for the industrial production of 1-(cyanocyclohexyl) acetic acid. [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]

Abstract: Bioconversion of (R,S)-mandelonitrile (R,S-MN) to prepare (R)-(−)-mandelic acid (R-MA) with nitrilase is an attractive method in industrial application. However, during this bioconversion by whole cells of Alcaligenes faecalis CCTCC M 208168, R-MA was found to inhibit its own production. To improve R-MA productivity, a new biocatalytic process of in situ product removal (ISPR) has been developed utilizing anion-exchange resin. To optimize the bioconversion of R-MA from R,S-MN, several anion-exchange resins were examined: HZ202, demonstrated several exciting features including high R-MA and low R,S-MN adsorbance. In batch biotransformation, ISPR by addition of HZ202 increased R-MA volumetric productivity to 0.285mmol/l/min. The kinetic models for enantioselective hydrolysis of R,S-MN by A. faecalis CCTCC M 208168 with ISPR were established, and they were well fitted to the experimental data of the reaction kinetics. In fed-batch biotransformation with ISPR was also performed. As compared to the conventional fed-batch mode, this approach allowed R-MA volumetric productivity and biocatalyst productivity to be increased from 0.083mmol/l/min and 55.1mmol/g to 0.281mmol/l/min and 185.5mmol/g, respectively. [ABSTRACT FROM AUTHOR]

Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the nitrilase superfamily have an underlying dimer structure. The true nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these nitrilases. The sequences of the spiral nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases. [Display omitted] • Solubility-enhancing peptide tags and linkers were designed and screened. • Nitrilase with GKGKG tag showed improved soluble expression, thermostability and activity. • A high space-time yield of 2006.5 g/L/d was reached in 1-CA production. [ABSTRACT FROM AUTHOR]