Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement.

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.

Enhancing the stress resistance of nitrile hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and ß- subunits ligation.

BACKGROUND: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and ß-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. METHODS AND RESULTS: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of ß-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of ß-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. CONCLUSION: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.

Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.

Two conserved second-sphere ßArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase ßR52A, ßR52K, ßR157A, ßR157K and ReNHase ßR61A) were successfully expressed and purified. Apart from the PtNHase ßR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase ßR mutant enzymes were between 1.8 and 12.4 s-1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ∼10 to ∼40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase ßR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere ßR residues in the proposed subunit swapping process and post-translational modification of the α-subunit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.

N-terminal loops at the tetramer interface of nitrile hydratase act as "hooks" determining resistance to high amide concentrations.

Nitrile hydratase (NHase) has been extensively utilized in industrial acrylamide production. However, the vulnerability to high concentrations of acrylamide limits its further application. Herein, we redesigned the N-terminal loop at the tetramer interface of a thermophilic NHase from Pseudonocardia thermophila JCM3095 (PtNHase), and its catalytic activity, resistance to high acrylamide concentrations, and thermostability were improved. Amino acid residues located in the N-terminal loop of the tetramer interface that are responsible for enhancing the resistance to high acrylamide concentrations were identified via static structural analysis and molecular dynamics simulations. A variant library was used to fine-tune the tetramer interface. Variant αL6T exhibited 3.5-fold greater resistance to 50% (v/v) acrylamide, whereas its activity was 1.2-fold higher than that of the wild-type (WT) enzyme, revealing no activity-stability trade-off. Compared to the use of Escherichia coli harboring the WT enzyme, the use of E. coli harboring αL6T increased the acrylamide concentration from 398.1 g/L to 500 g/L. Crystal structure-guided analysis of αL6T and molecular dynamics simulations revealed that increased enzyme surface hydration and the introduction of positive cross-correlation into the N-terminal loop of the tetramer interface caused the two loop regions to hook to each other, thus improving the resistance to high acrylamide concentrations.

A Proterozoic microbial origin of extant cyanide-hydrolyzing enzyme diversity.

In addition to its role as a toxic environmental contaminant, cyanide has been hypothesized to play a key role in prebiotic chemistry and early biogeochemical evolution. While cyanide-hydrolyzing enzymes have been studied and engineered for bioremediation, the extant diversity of these enzymes remains underexplored. Additionally, the age and evolution of microbial cyanide metabolisms is poorly constrained. Here we provide comprehensive phylogenetic and molecular clock analyses of the distribution and evolution of the Class I nitrilases, thiocyanate hydrolases, and nitrile hydratases. Molecular clock analyses indicate that bacterial cyanide-reducing nitrilases were present by the Paleo- to Mesoproterozoic, and were subsequently horizontally transferred into eukaryotes. These results present a broad diversity of microbial enzymes that could be optimized for cyanide bioremediation.

Systematic engineering of Escherichia coli for efficient production of nicotinamide riboside from nicotinamide and 3-cyanopyridine.

Nicotinamide riboside (NR), a key biosynthetic precursor of NAD+, is receiving increasing attention because of its role. In this study, a whole-cell catalysis method to efficiently synthesize NR was established. First, the performance of 5'-nucleotidase (UshA) from Escherichia coli was confirmed to have high catalytic activity to synthesize NR. Then, the endogenous NR degradation pathway was detected, and the genes (rihA, rihB, and rihC) involved in NR degradation were knocked out, which enabled NR biosynthesis. In addition, the important role of the signal peptide of UshA in NR transport had been confirmed. Subsequently, nitrile hydratase was introduced to achieve the conversion of 3-cyanopyridine to NR. Finally, the NR titer reached 25.6 and 29.8 g/L with nicotinamide and 3-cyanopyridine, respectively, as substrates in a 5-L bioreactor, the efficient biosynthesis of NR in E. coli by using nicotinamide and 3-cyanopyridine.

A nanobiocatalytic system based on the self-assembly of ferrous phosphate with a high regioselectivity nitrile hydratase.

Our previous study identified a novel nitrile hydratase (NHase) with remarkable biotransformation activity toward adipamide during the production of 5-cyanovaleramide (5-CVAM), an important intermediate of herbicide and chemical raw material. Nevertheless, free NHase will face harsh conditions if they are applied directly in industrial processes. In this study, we, therefore, prepared Fe3(PO4)2 hybrid nanoflowers for NHase immobilization based on the protein-inorganic hybrid self-assembly by establishing a novel and facile method. The results showed that the NHase@Fe3(PO4)2 nanoflowers had significantly enhanced tolerance to the temperature ranging from 40°C to 60°C when compared with free NHase. The catalytic activity of NHase@Fe3(PO4)2 nanoflowers remained high in extreme pH environments such as weak acid (pH 5) and strong alkali (pH 10) environments. In addition, the storage stability and reusability of encapsulated NHase were also superior to that of free NHase. NHase@Fe3(PO4)2 nanoflowers had a notable feature of high substrate tolerance. We found NHase@Fe3(PO4)2 nanoflowers still had 65% activity as the adiponitrile concentration increased up to 200 mmol L-1, whereas free NHase almost lost their catalytic activity when the adiponitrile concentration was just 100 mmol L-1. All of these results clearly demonstrated that ferrous phosphate nanocrystals might offer a novel strategy for 5-CVAM production with nanobiocatalytic systems.

Engineering enhanced thermostability into the Geobacillus pallidus nitrile hydratase.

Nitrile hydratases (NHases) are important biocatalysts for the enzymatic conversion of nitriles to industrially-important amides such as acrylamide and nicotinamide. Although thermostability in this enzyme class is generally low, there is not sufficient understanding of its basis for rational enzyme design. The gene expressing the Co-type NHase from the moderate thermophile, Geobacillus pallidus RAPc8 (NRRL B-59396), was subjected to random mutagenesis. Four mutants were selected that were 3 to 15-fold more thermostable than the wild-type NHase, resulting in a 3.4-7.6 â€‹kJ/mol increase in the activation energy of thermal inactivation at 63 â€‹°C. High resolution X-ray crystal structures (1.15-1.80 â€‹Å) were obtained of the wild-type and four mutant enzymes. Mutant 9E, with a resolution of 1.15 â€‹Å, is the highest resolution crystal structure obtained for a nitrile hydratase to date. Structural comparisons between the wild-type and mutant enzymes illustrated the importance of salt bridges and hydrogen bonds in enhancing NHase thermostability. These additional interactions variously improved thermostability by increased intra- and inter-subunit interactions, preventing cooperative unfolding of α-helices and stabilising loop regions. Some hydrogen bonds were mediated via a water molecule, specifically highlighting the significance of structured water molecules in protein thermostability. Although knowledge of the mutant structures makes it possible to rationalize their behaviour, it would have been challenging to predict in advance that these mutants would be stabilising.

"Toolbox" construction of an extremophilic nitrile hydratase from Streptomyces thermoautotrophicus for the promising industrial production of various amides.

Nitrile hydratase (NHase; EC 4.2.1.84) is widely used to synthesize the corresponding amides from nitriles, which is the most successful green biocatalyst. However, the limited acceptability of substrates and instability under harsh reaction conditions have hindered its widespread industrial application. Here, a gene encoding an extremophilic NHase from Streptomyces thermoautotrophicus (S.t NHase) was successfully overexpressed in Escherichia coli. The enzyme exhibited excellent thermostability, retaining >50 % of residual activity after heat treatment at 65 °C for 252 min. To further improve the catalytic performance of S.t NHase, semi-rational engineering of its substrate access tunnel was performed. A mutant ßL48D showed a specific activity of 566.18 ± 18.86 U/mg towards 3-cyanopyridine, which was 7.7 times higher than its parent enzyme (73.80 ± 5.76 U/mg). Molecular dynamics simulation showed that the introduction of aspartic acid into ßLeu48 resulted in a larger and more frequent opening of the substrate access tunnel entrance. On this basis, a "toolbox" containing various mutants on the substrate access tunnel was further established, whose catalytic activity towards various nitrile substrates was extensively improved, showing great potential for efficient synthesis of multiple high-value amides.

High-Throughput Screening of Signal Peptide Library with Novel Fluorescent Probe.

Nitrile hydratase (NHase) is an excellent biocatalyst for the synthesis of amide compounds and is composed of two heterologous subunits. However, the secretory expression of NHase has been difficult to achieve because of its complex expression mechanism. In this work, a novel fluorescent probe Rho-IDA-CoII was synthesized by a one-pot method. Rho-IDA-CoII could specifically label His-tagged proteins in vitro, such as for staining in-gel, Western blot, and ELISA analysis. Furthermore, Rho-IDA-CoII combined with dot blots could quantitatively detect His-tagged proteins at between 1-10 pmol and perform high-throughput screening for the NHase signal peptide library. Recombinant Bacillus subtilis WB800/phoB-HBA with the extracellular expression of NHase was screened (ca. 6500 clones). After optimization of fermentation conditions, the NHase activity in the culture supernatant reached 17.34±0.16 U/mL. This is the first time that secretory NHase has been expressed in B. subtilis successfully.

Metabolism of Aldoximes and Nitriles in Plant-Associated Bacteria and Its Potential in Plant-Bacteria Interactions.

In plants, aldoximes per se act as defense compounds and are precursors of complex defense compounds such as cyanogenic glucosides and glucosinolates. Bacteria rarely produce aldoximes, but some are able to transform them by aldoxime dehydratase (Oxd), followed by nitrilase (NLase) or nitrile hydratase (NHase) catalyzed transformations. Oxds are often encoded together with NLases or NHases in a single operon, forming the aldoxime-nitrile pathway. Previous reviews have largely focused on the use of Oxds and NLases or NHases in organic synthesis. In contrast, the focus of this review is on the contribution of these enzymes to plant-bacteria interactions. Therefore, we summarize the substrate specificities of the enzymes for plant compounds. We also analyze the taxonomic and ecological distribution of the enzymes. In addition, we discuss their importance in selected plant symbionts. The data show that Oxds, NLases, and NHases are abundant in Actinobacteria and Proteobacteria. The enzymes seem to be important for breaking through plant defenses and utilizing oximes or nitriles as nutrients. They may also contribute, e.g., to the synthesis of the phytohormone indole-3-acetic acid. We conclude that the bacterial and plant metabolism of aldoximes and nitriles may interfere in several ways. However, further in vitro and in vivo studies are needed to better understand this underexplored aspect of plant-bacteria interactions.

Engineering of the thermophilic nitrile hydratase from Pseudonocardia thermophila JCM3095 for large-scale nicotinamide production based on sequence-activity relationships.

The green biocatalyst nitrile hydratase (NHase) is able to bio-transform 3-cyanopyridine into nicotinamide. As the NHase reaction is exothermic, an enzyme with high activity and stability is needed for nicotinamide production. In this study, we used sequence analysis and site-directed mutagenesis to generate a mutant of thermophilic NHase from Pseudonocardia thermophila JCM3095 with substantially enhanced activity and developed a powerful process for nicotinamide bio-production. The specific activity of αF126Y/αF168Y mutant was successfully increased by 3.98-fold over that of the wild-type enzyme. The half-life of such mutant was longer than 2 h, which was comparable to its parent enzyme. The relative activity of the αF126Y/αF168Y mutant after treatment with 1 M 3-cyanopyridine and 2 M nicotinamide was 73.2% and 63.7%, respectively, showing minor loss of its original stability. Structural analysis demonstrated that hydrogen bonds at the active site and α-ß subunit interface of the NHase contribute to the improved activity and the maintenance of stability. Escherichia coli transformant harboring the mutant NHase was used for nicotinamide bio-production, yielding a nicotinamide productivity of 251.1 g/(L·h), which is higher than the productivity obtained using other NHase-containing strains and transformants. The newly established variant is therefore a promising alternative for the industrial production of nicotinamides.

Nitrile Hydratases: From Industrial Application to Acetamiprid and Thiacloprid Degradation.

The widespread application of neonicotinoid insecticides (NEOs) in agriculture causes a series of environmental and ecological problems. Microbial remediation is a popular approach to relieve these negative impacts, but the associated molecular mechanisms are rarely explored. Nitrile hydratase (NHase), an enzyme commonly used in industry for amide production, was discovered to be responsible for the degradation of acetamiprid (ACE) and thiacloprid (THI) by microbes. Since then, research into NHases in NEO degradation has attracted increasing attention. In this review, microbial degradation of ACE and THI is briefly described. We then focus on NHase evolution, gene composition, maturation mechanisms, expression, and biochemical properties with regard to application of NHases in NEO degradation for bioremediation.

Proposed mechanism for post-translational self-modification of Co-NHase based on Co2+ diffusion limitation.

BACKGROUND: Nitrile hydratase (NHase), was an excellent biocatalyst for the synthesis of amide compounds. NHase was typical heterodimeric metalloprotein, required of the assistance of activator for active expressions. In this work, we found a special Co-NHase HBA from Caldalkalibacillus thermarum, which had the ability of post-translational self-modification and could incorporate Co2+ into the catalytic center in the absence of activator. METHOD AND RESULTS: We simulated the movement of Co2+ in silico and established a hypothetical model to predict the Co2+ incorporation efficiency (XCo ) of NHases. According to the simulation results, NHase mutants with different positive charge distribution were constructed. Compared with wild-type, the Co2+ incorporation efficiency of K1 (M10K) was increased by 2.1-fold from 0.36 to 0.76, and the specific activity was increased by 3.2-fold from 136.3 to 432.0 U mg-1 , while mutant K1H1 (M10K, D11H) and K2H2 (M10K, D11H, E20K, N21H) lost the ability of post-translation self-modification. CONCLUSIONS AND IMPLICATIONS: The interactions of positively charged residues near the catalytic center, such as lysine with strong electrostatic repulsive interaction, arginine with weak electrostatic repulsive interaction and histidine with metal affinity, could limit the free diffusion of Co2+ in NHase and affect the efficiency of post-translational self-modification. This work also provided an effective strategy for protein engineering of NHases and other metalloenzymes.

Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles.

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, ßLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant ßL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the ßHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.

Effect and mechanism analysis of different linkers on efficient catalysis of subunit-fused nitrile hydratase.

Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and under-explored. To further clarify the regular pattern of suitable type of linker for fusion proteins, one nitrile hydratase (NHase) was used as a target protein and subunit fusion strategy was carried out to improve its efficient catalysis. Subunit-fused variants with three different types of linkers were constructed and characterized. All variants exhibited higher stability than that of the wild type. The longer the linker was, the higher stability NHase showed, however, too long linker affected NHase activity and expression. Among the three types of linkers, the α-helical linker seemed more suitable for NHase than flexible or rigid linkers. Though it is not clear how the linkers affecting the activity, structure analysis indicated that the stability improvement is dependent on the additional salt bridge, H-bond, and the subunit interface area increasing due to the linker insertion, among which the additional salt bridge and interface area were more important factors. The results described here may be useful for redesigning other enzymes through subunit fusion.

Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation.

Nitrile hydratase (NHase), an excellent bio-catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3°C) obtained by in silico screening in our study exhibited high flexibility of α-subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that ß-subunit played a key role in adjusting the flexibility of α-subunit and the different conformations of linker in "α5-helix-coil ring" supersecondary structure of ß-subunit can affect the interaction between ß-subunit and α-subunit. Mutant NHCTA1-α6 C with a random coil linker and mutant NHCTA1-αßγ with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1-α6 C and NHCTA1-αßγ (Tmα6C = 74.4°C, Tmαßγ = 65.6°C) verified that the flexibility of α-subunit adjusted by ß-subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial-important enzymes.

Biotransformation of insecticide flonicamid by Aminobacter sp. CGMCC 1.17253 via nitrile hydratase catalysed hydration pathway.

AIMS: This study evaluates flonicamid biotransformation ability of Aminobacter sp. CGMCC 1.17253 and the enzyme catalytic mechanism involved. METHODS AND RESULTS: Flonicamid transformed by resting cells of Aminobacter sp. CGMCC 1.17253 was carried out. Aminobacter sp. CGMCC 1.17253 converts flonicamid into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM). Aminobacter sp. CGMCC 1.17253 transforms 31·1% of the flonicamid in a 200 mg l-1 conversion solution in 96 h. Aminobacter sp. CGMCC 1.17253 was inoculated in soil, and 72·1% of flonicamid with a concentration of 0·21 µmol g-1 was transformed in 9 days. The recombinant Escherichia coli expressing Aminobacter sp. CGMCC 1.17253 nitrile hydratase (NHase) and purified NHase were tested for the flonicamid transformation ability, both of them acquired the ability to transform flonicamid into TFNG-AM. CONCLUSIONS: Aminobacter sp. CGMCC 1.17253 transforms flonicamid into TFNG-AM via hydration pathway mediated by cobalt-containing NHase. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that bacteria of genus Aminobacter has flonicamid-transforming ability. This study enhances our understanding of flonicamid-degrading mechanism. Aminobacter sp. CGMCC 1.17253 has the potential for bioremediation of flonicamid pollution.

Biotransformation of flonicamid and sulfoxaflor by multifunctional bacterium Ensifer meliloti CGMCC 7333.

Flonicamid is a novel, selective, systemic pyridinecarboxamide insecticide that effectively controls hemipterous pests. Sulfoxaflor, a sulfoximine insecticide, effectively controls many sap-feeding insect pests. Ensifer meliloti CGMCC 7333 transforms flonicamid into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM). Resting cells of E. meliloti CGMCC 7333 (optical density at 600 nm [OD600] = 5) transformed 67.20% of the flonicamid in a 200-mg/L solution within 96 h. E. meliloti CGMCC 7333 transforms sulfoxaflor into N-(methyl(oxido){1-[6-(trifluoromethyl) pyridin-3-yl] ethyl}-k4-sulfanylidene) urea (X11719474). E. meliloti CGMCC 7333 resting cells (OD600 = 5) transformed 89.36% of the sulfoxaflor in a 200 mg/L solution within 96 h. On inoculating 2 mL of E. meliloti CGMCC 7333 (OD600 = 10) into soil containing 80 mg/kg flonicamid, 91.1% of the flonicamid was transformed within 9 d (half-life 2.6 d). On inoculating 2 mL of E. meliloti CGMCC 7333 (OD600 = 10) into soil containing 80 mg/kg sulfoxaflor, 83.9% of the sulfoxaflor was transformed within 9 d (half-life 3.4 d). Recombinant Escherichia coli harboring the E. meliloti CGMCC 7333 nitrile hydratase (NHase)-encoding gene and NHase both showed the ability to transform flonicamid or sulfoxaflor into their corresponding amides, TFNG-AM and X11719474, respectively. These findings may help develop a bioremediation agent for the elimination of flonicamid and sulfoxaflor contamination.

Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability.

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/ßA20P/ßP38L/ßF118W/ßS130Y/ßC189N/ßC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.