Nitrilase Activity Research Articles

Biosynthesis of Benzoylformic Acid from Benzoyl Cyanide with a New Bacterial Isolate of Brevibacterium sp. CCZU12-1

Brevibacterium sp. CCZU12-1 with high nitrilase activity could effectively hydrolyze benzoyl cyanide into benzoylformic acid. After the culture optimization, the preferred carbon sources, nitrogen sources, and inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (2.5 g/L), and ε-caprolactam (2.0 mM), respectively. After the reaction optimization, the optimum reaction temperature, reaction pH, organic cosolvent, and metal ion were 30 °C, 7.0, ethanol (2%, v/v), and Ca(2+) (0.1 mM), respectively. At biotransformation of 120-mM benzoyl cyanide for 24 h, the yield of benzoylformic acid reached 91.8%. Moreover, the microbial nitrilase from Brevibacterium sp. CCZU12-1 could hydrolyze various nitriles, and it significantly exhibited high nitrilase activity against benzoyl cyanide, 3-cyanopyridine, and α-cyclohexyl-mandelonitrile.

Increased β-Cyanoalanine Nitrilase Activity Improves Cyanide Tolerance and Assimilation in Arabidopsis

Plants naturally produce cyanide (CN) which is maintained at low levels in their cells by a process of rapid assimilation. However, high concentrations of environmental CN associated with activities such as industrial pollution are toxic to plants. There is thus an interest in increasing the CN detoxification capacity of plants as a potential route to phytoremediation. Here, Arabidopsis seedlings overexpressing the Pseudomonas fluorescens β-cyanoalanine nitrilase pinA were compared with wild-type and a β-cyanoalanine nitrilase knockout line (ΔAtnit4) for growth in the presence of exogenous CN. After incubation with CN, +PfpinA seedlings had increased root length, increased fresh weight, and decreased leaf bleaching compared with wild-type, indicating increased CN tolerance. The increased tolerance was achieved without an increase in β-cyanoalanine synthase activity, the other enzyme in the cyanide assimilation pathway, suggesting that nitrilase activity is the limiting factor for cyanide detoxification. Labeling experiments with [¹³C]KCN demonstrated that the altered CN tolerance could be explained by differences in flux from CN to Asn caused by altered β-cyanoalanine nitrilase activity. Metabolite profiling after CN treatment provided new insight into downstream metabolism, revealing onward metabolism of Asn by the photorespiratory nitrogen cycle and accumulation of aromatic amino acids.

Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli, purification, characterization and use in continuous cyanide degradation

A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile. Vmax and Km for HCN were ca. 6.8mmolmin−1mg−1 protein and 109mM, respectively. Vmax for fumaronitrile and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3μmolmin−1mg−1, respectively) but Km was also lower (ca. 14.7 and 3.7mM, respectively). Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18–34 C-terminal aa residues. The enzyme exhibited the highest activity at 45°C and pH 8–9; it was unstable at over 35°C and at below pH 5.5. The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6h at 28°C. A reactor loaded with an excess of the catalyst was used to degrade 25mM KCN. A conversion rate of over 80% was maintained for 3 days.

Efficient production of (R)-(−)-mandelic acid using glutaraldehyde cross-linked Escherichia coli cells expressing Alcaligenes sp. nitrilase

Recombinant Escherichia coli cells expressing Alcaligenes sp. nitrilase were simply immobilized by direct cross-linking using glutaraldehyde. About 85 % of the total nitrilase activity was recovered under the optimal cross-linking conditions. The thermal stabilities of the cross-linked cells measured at 30, 40 and 50 °C were 4.5-, 5.3-, and 5.1-fold those of the free cells, respectively. The concentration of (R)-(-)-mandelic acid reached 280 mM after merely 2 h transformation with the immobilized cells using 300 mM mandelonitrile as substrate, affording an extremely high productivity of 510.7 g L(-1) d(-1). In addition, operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 15 cycles of batch reactions or 8 cycles of repeated fed-batch reactions. Therefore, the easy preparation and robust characteristics of the immobilized biocatalyst make it a very promising biocatalyst for high-performance and low-cost production of optically pure (R)-(-)-mandelic acid.

Erratum to: Purification and characterization of heterologously expressed nitrilases from filamentous fungi

Two works cited in the previous article (Kaplan et al. 2006, 2011) were corrected (Kaplan et al. 2013a) or retracted (Kaplan et al. 2013b). The reason was the falsity of data (N-terminal aa sequence and theMALDI-TOF analysis) by KBwhose ethics of scientific workwas examined in 2012 by the Ethics Commission of the Charles University in Prague and the Institute of Microbiology of the Academy of Sciences of the Czech Republic. The Commission found scientific misconduct of KB in several cases. Therefore, we wish to correct the following parts of the previous article: Introduction: “The nitrilase studied by us in Aspergillus niger K10 was the first fungal nitrilase to be purified from both its native... and heterologous producer...” Actually, the enzymes purified from the native and heterologous producer were different. The enzyme natively expressed in this strain was purified and characterised (Kaplan et al. 2006) and its partial aa sequence was recently published (Kaplan et al. 2013a). The recombinant enzyme was recently examined for its substrate specificity in whole cells (Kaplan et al. 2013c), and was found to be a cyanide hydratase with a significant nitrilase activity. Materials and methods, Nitrilase expression: Construction of plasmid pOK101 referred toKaplan et al. 2011 (retracted article). Therefore, we wish to describe this method here: To construct plasmid pOK101, NdeI-HindIII fragment of the PCR product amplified from cDNA of Aspergillus niger K10 (forward primer 5′-GCC ATATGG CAC CMG TCT TRA AGA AGTACA A3′, M=A or C; R=A or G, and reverse primer, 5′-GCA AGC TTT TAC TAG TTC TCC GAATCC ACG GT-3′) was ligated into vector pET-30a(+) (Novagen). Results, Variability of fungal nitrilases: The statement that in the nitrilase fromA . niger K10 “both its amino acid sequence and its biochemical properties have been already known” referred to Kaplan et al. 2011 (retracted article). For substrate specificity of this enzyme please refer to Kaplan et al. 2013c (see also above). Results, Nitrilase in Aspergillus niger K10: The hypothesis that the difference of substrate specificities in the natively expressed and the recombinant enzymes were caused by misfolding of the latter referred to Kaplan et al. 2011 (retracted article). Actually, the two enzymes were different in terms of their primary structure (see above). The online version of the original article can be found at http://dx.doi.org/ 10.1007/s00253-011-3525-7.

Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine

Abstract Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH2 terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters Km and Vmax for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.

A New Isolate from Fusarium proliferatum (AUF-2) for Efficient Nitrilase Production

A fungal isolate from Fusarium proliferatum strain AUF-2 has been found to have a high nitrilase activity (≥ 1,000 U/l culture). The present work describes optimization of growth conditions and production medium to achieve maximum nitrilase production. The most effective carbon and nitrogen sources for nitrilase production were glucose and sodium nitrate, respectively. ε-Caprolactam was found to be the best inducer for maximum nitrilase production with 80 g/l wet cell biomass and 26 U/g nitrilase activity. An overall nitrilase activity of ≥ 2,000 U/l culture was obtained in this study, which is one of the best activities reported so far in any Fusarium strain. Chemo-profiling has shown that the strain is versatile in its ability to hydrolyze both aliphatic as well as aromatic nitriles. Efforts are being made to use the strain for biotransformation of pharmaceutical substrates.

Enhancing nitrilase production from Fusarium proliferatum using response surface methodology

The individual and interactive effects of three independent variables i.e. carbon source (glucose), nitrogen source (sodium nitrate) and inducer (ϵ-caprolactam) on nitrilase production from Fusarium proliferatum were investigated using design of experiments (DOE) methodology. Response surface methodology (RSM) was followed to generate the process model and to obtain the optimal conditions for maximum nitrilase production. Based on central composite design (CCD) a quadratic model was found to fit the experimental data (p<0.0001) and maximum activity of 59.0U/g biomass was predicted at glucose concentration (53.22 g/l), sodium nitrate (2.31 g/l) and ϵ-caprolactam (3.58 g/l). Validation experiments were carried out under the optimized conditions for verification of the model. The nitrilase activity of 58.3U/g biomass obtained experimentally correlated to the predicted activity which proves the authenticity of the model. Overall 2.24 fold increase in nitrilase activity was achieved as compared to the activity before optimization (26U/g biomass).

Biotransformation of iminodiacetonitrile to iminodiacetic acid by Alcaligenes faecalis cells immobilized in ACA-membrane liquid-core capsules

AbstractBiotransformation of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) was investigated with a newly isolated Alcaligenes faecalis ZJUTBX11 strain showing nitrilase activity in the immobilized form. To reduce the mass transfer resistance and to increase the toleration ability of the microorganisms to the toxic substrate as well as to enhance their ability to be reused, encapsulation of the whole cells in alginate-chitosan-alginate (ACA) membrane liquid-core capsules was attempted in the present study. The optimal pH and temperature for nitrilase activity of encapsulated A. faecalis ZJUTBX11 cells were 7.5°C and 35°C, respectively, which is consistent with free cells. Based on the Michaelis-Menten model, kinetic parameters of the conversion reaction with IDAN as the substrate were: K m = (17.6 ± 0.3) mmol L−1 and V max = (97.6 ± 1.2) μmol min−1 g−1 of dry cell mass for encapsulated cells and (16.8 ± 0.4) mmol L−1 and (108.0 ± 2.7) μmol min−1 g−1 of dry cell mass for free cells, respectively. After being recycled ten times, the whole cells encapsulated in ACA capsules still retained 90 % of the initial nitrilase activity while only 35 % were retained by free cells. Lab scale production of IDA using encapsulated cells in a bubble column reactor and a packed bed reactor were performed respectively.

Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools for Organic Synthesis

AbstractA high‐throughput screening of candidate nitrilases against 25 structurally diverse substrates allowed us to create a wide collection of 125 experimentally validated nitrilases. The enzymes were selected by genomic approach from 700 diverse prokaryotic species and one metagenome as representative of the nitrilase family diversity. The enzymatic screening of this collection expands the biocatalytic toolbox for chemical synthesis by providing a large number of tested nitrilases with their assigned substrates. Three examples illustrate the synthetic potential of our enzyme collection. The syntheses of carboxylic acid building blocks, a β‐substituted phenylpropanoic acid, a cyclic γ‐keto carboxylic acid and a mononitrile monocarboxylic acid, were achieved from the corresponding nitrile substrates, using three new nitrilases (two from Sphingomonas wittichii and one from Syntrophobacter fumaroxidans). Improvements of nitrilase activities through the optimization of reaction parameters and the preparative biocatalytic synthesis are presented for these three examples.

Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only <5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

Transformation of p-hydroxybenzonitrile to p-hydroxybenzoic acid using nitrilase activity of Gordonia terrae

Resting cells of Gordonia terrae with nitrilase (EC 3.5.5.1) activity were investigated for transformation of p-hydroxybenzonitrile to p-hydroxybenzoic acid. The maximum conversion was observed in 0.1 M potassium phosphate buffer, pH 8.0, using 40 mM substrate and resting cells corresponding to 0.70 Uml− 1 nitrilase activity at 35°C. A 500 mL fed batch reaction was designed for synthesis of p-hydroxybenzoic acid with six feedings of substrate at an interval of 1 h. A total of 14.4 g of p-hydroxybenzoic acid (> 98.7%) was obtained in 6 h with a productivity of 0.78 gh− 1g− 1DCW of G. terrae.

Трансформация 2- и 4-цианопиридинов свободными и иммобилизованными клетками нитрилгидролизующих бактерий

The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt 1 strain possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 strain containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5-4 times lower compared to 4-cyanopyridine and 1.6-2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt 1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on china clay was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carbonic acids.

Improvement of nitrilase production from a newly isolated Alcaligenes faecalis mutant for biotransformation of iminodiacetonitrile to iminodiacetic acid

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.

Nocardia globerula NHB-2 nitrilase catalysed biotransformation of 4-cyanopyridine to isonicotinic acid.

Isonicotinic acid (INA) is an important pyridine derivative used in the manufacture of isoniazid (antituberculosatic drug) and other pharmaceutically important drugs. Nitrilase catalysed processes for the synthesis of pharmaceutically important acids from their corresponding nitriles are promising alternative over the cumbersome, hazardous, and energy demanding chemical processes. Nitrilase of Nocardia globerula NHB-2 (NitNHB2) is expressed in presence of isobutyronitrile in the growth medium (1.0% glucose, 0.5% peptone, 0.3% beef extract, and 0.1 % yeast extract, pH 7.5). NitNHB2 hydrolyses 4-cyanopyridine (4-CP) to INA without accumulation of isonicotinamide, which is common in the reaction catalysed via fungal nitrilases. The NitNHB2 suffers from substrate inhibition effect and hydrolysing activity up to 250 mM 4-CP was recorded. Complete conversion of 200 mM 4-CP to INA was achieved in 40 min using resting cell concentration corresponding to 10 U mL-1 nitrilase activity in the reaction. Substrate inhibition effect in the fed batch reaction (200 mM substrate feed/40min) led to formation of only 729 mM INA. In a fed batch reaction (100 mM 4-CP/20min), substrate inhibition effect was encountered after 7th feed and a total of 958 mM INA was produced in 400 min. The fed batch reaction scaled up to 1 L and 100% hydrolysis of 700 mM of 4-CP to INA at 35°C achieved in 140 min. The rate of INA production was 21.1 g h-1 mgDCW-1. This is the fastest biotransformation process ever reported for INA production with time and space productivity of 36 g L-1 h-1 using a bacterial nitrilase.

The nit1C gene cluster of Pseudomonas pseudoalcaligenes CECT5344 involved in assimilation of nitriles is essential for growth on cyanide

A proteomic approach was used to identify several proteins induced by cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344, two of them, NitB and NitG, encoded by genes that belong to the nit1C gene cluster. The predicted products of the nit1C gene cluster are a Fis-like σ(54) -dependent transcriptional activator (NitA), a nitrilase (NitC), an S-adenosylmethionine superfamily member (NitD), an N-acyltransferase superfamily member (NitE), a trifunctional polypeptide of the AIRS/GARS family (NitF), an NADH-dependent oxidoreductase (NitH) and two hypothetical proteins of unknown function (NitB and NitG). RT-PCR analysis suggested that nitBCDEFGH genes were co-transcribed, whereas the regulatory nitA gene was divergently transcribed. Real-time RT-PCR revealed that expression of the nitBCDEFGH genes was induced by cyanide and repressed by ammonium. The P. pseudoalcaligenes CECT5344 nit1C gene cluster was found to be involved in assimilation of free and organic cyanides (nitriles) as deduced for the inability to grow with cyanides showed by the NitA, NitB and NitC mutant strains. The wild-type strain CECT5344 showed a nitrilase activity that allows growth on cyanide or hydroxynitriles. The NitB and NitC mutants only presented low basal levels of nitrilase activity that were not enough to support growth on either free cyanide or aliphatic nitriles, suggesting that nitrilase NitC is specific and essential for cyanide and aliphatic nitriles assimilation.

Influence of point mutations near the active site on the catalytic properties of fungal arylacetonitrilases from Aspergillus niger and Neurospora crassa

Arylacetonitrilases from Aspergillus niger CBS 513.88 and Neurospora crassa OR74A (NitAn and NicNc, respectively) were expressed in recombinant Escherichia coli JM109. The respective whole-cell catalysts preferentially hydrolyzed (R)-enantiomers of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile. NitNc also formed significant amounts of mandelamide from (R,S)-mandelonitrile (40% of total product), mainly found as the (S)-enantiomer. At pH 4.5 and 5.0, the cells retained more than 40 and 60% of their nitrilase activity at pH 7, respectively. Nitrilase variants generated by site-directed mutagenesis carried amino acid replacements in the vicinity of the catalytically active cysteine residues (C162 and C167 in NitAn and NitNc, respectively). The conversions of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile by the nitrilase variants were compared to the wild-type nitrilases in terms of activity, enantioselectivity, and acid/amide ratio of the products formed. Thus the W168A variant of NitNc was identified, which formed significantly increased amounts of mandelamide and 2-phenylpropionamide, and which demonstrated an almost complete inversion of enantioselectivity for the conversion of (R,S)-2-phenylpropionitrile (from R- to S-selectivity).

Application of a Recombinant Escherichia coli Whole‐Cell Catalyst Expressing Hydroxynitrile Lyase and Nitrilase Activities in Ionic Liquids for the Production of (S)‐Mandelic Acid and (S)‐Mandeloamide

AbstractThe conversion of benzaldehyde and cyanide into mandelic acid and mandeloamide by a recombinant Escherichia coli strain which simultaneously expressed an (S)‐hydroxynitrile lyase (oxynitrilase) from cassava (Manihot esculenta) and an arylacetonitrilase from Pseudomonas fluorescens EBC191 was studied. Benzaldehyde exhibited a pronounced inhibitory effect on the nitrilase activity in concentrations ≥25 mM. Therefore, it was tested if two‐phase systems consisting of a buffered aqueous phase and the ionic liquid 1‐butyl‐1‐pyrrolidinium bis(trifluoromethanesulfonyl)imide (BMpl NTf2) or 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMim PF6) could be used for the intended biotransformation. The distribution coefficients of the substrates, intermediates and products of the reaction were determined and it was found that BMpl NTf2 and BMim PF6 were highly efficient as substrate reservoirs for benzaldehyde. The recombinant E. coli strain was active in the presence of BMpl NTf2 or BMim PF6 phases and converted benzaldehyde and cyanide into mandelic acid and mandeloamide. The two‐phase systems allowed the conversion of benzaldehyde dissolved in the ionic liquids to a concentration of 700 mM with product yields (=sum of mandelic acid and mandeloamide) of 87–100%. The cells were slightly more effective in the presence of BMpl NTf2 than in the presence of BMim PF6. In both two‐phase systems benzaldehyde and cyanide were converted into (S)‐mandeloamide and (S)‐mandelic acid with enantiomeric excesses ≥94%. The recombinant E. coli cells formed, in the two‐phase systems with ionic liquids and increased substrate concentrations, higher relative amounts of mandeloamide than in a purely aqueous system with lower substrate concentrations.

Production of Nitrilase by a Recombinant Escherichia coli in a Laboratory Scale Bioreactor

Effects of medium pH (uncontrolled and controlled), aeration rate and agitation intensity on the production of biomass and nitrilase by a recombinant Escherichia coli in a stirred-tank bioreactor are reported. The recombinant bacterium expressed the nitrilase gene of Alcaligenes faecalis . The initial pH of the culture medium had a strong influence on the growth of biomass and enzyme production. In batch fermentation process the growth and enzyme production were maximized at 37°C with an initial medium pH 7.0. The fermentation was influenced by oxygen transfer efficiency of the bioreactor and by the turbulence regimen. The optimal production conditions were an aeration rate of 0.4 vvm and an agitation speed of 400 rpm. Higher values of agitation speed and aeration rate proved detrimental to both biomass production and nitrilase activity. Under optimal conditions, the final dry biomass concentration was 6.9 g/L and the biomass specific enzyme activity was 58 U/g dry cells.

Biosynthesis of Iminodiacetic Acid from Iminodiacetonitrile by Immobilized Recombinant Escherichia coli Harboring Nitrilase

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl₂, and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase.