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, 69 (8), 4359-66

Cloning of a Nitrilase Gene From the Cyanobacterium Synechocystis Sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

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Cloning of a Nitrilase Gene From the Cyanobacterium Synechocystis Sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

Ute Heinemann et al. Appl Environ Microbiol.

Abstract

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.

Figures

FIG. 1.
FIG. 1.
Proposed mechanism for the nitrilase reaction (30).
FIG. 2.
FIG. 2.
Demonstration of a nitrilase activity in E. coli(pDHE22) which harbors the recombinant nitrilase from Synechocystis sp. strain PCC6803. The reaction mixture contained, in a total volume of 5 ml, 50 mM Na-K phosphate buffer, 5 mM benzonitrile, and resting cells of E. coli(pDHE22) corresponding to an optical density at 546 nm of 4. Aliquots (50 μl each) were removed after different time intervals, the reaction was terminated by the addition of 5 μl of 1 M HCl, and cells and precipitated protein were removed by centrifugation (2 min, 16,600 × g). The conversion of benzonitrile to benzoate was determined by HPLC.
FIG. 3.
FIG. 3.
Hydrolysis of fumarodinitrile and decanoic acid dinitrile by the purified nitrilase from Synechocystis sp. strain PCC6803. The reactions were performed in 1 ml of Na-K phosphate buffer (pH 7.0) containing 1 mM (♦) or 3 mM (•) fumarodinitrile (left panel) or decanoic acid dinitrile (right panel) and 80 μg of purified enzyme. Aliquots (100 μl each) were removed after different time intervals, the reactions were terminated by the addition of 10 μl of HCl (1 M), and precipitated protein was removed by centrifugation (2 min, 16,600 × g). The concentration of ammonia was subsequently determined by using the Spectroquant test.
FIG. 4.
FIG. 4.
Hydrolysis of benzonitrile by the purified nitrilase from Synechocystis sp. strain PCC6803 in the presence of different concentrations of various organic solvents. The reaction mixtures contained 19 μg of the purified (His-tagged) nitrilase in 250 μl of 50 mM Na-K phosphate buffer (pH 7.0) plus 0 to 200 μl of the solvents indicated. These enzyme preparations were incubated for 10 min at room temperature. Subsequently, benzonitrile (10 mM) was added, and the reaction mixtures were shaken at 30°C. The reactions were stopped after 30 min by the addition of 10% (vol/vol) 1 M HCl. Precipitated protein was removed by centrifugation (2 min, 16,000 × g), and the concentration of benzoate formed was determined in the aqueous phase by HPLC. The enzyme preparation formed under these conditions in a purely aqueous phase 0.7 mM concentration of benzoate (100%). The relative amounts of benzoate (BA) formed in correlation to the control experiment in a purely aqueous phase are presented. DMSO, dimethyl sulfoxide; DMF, dimethyl formamide, MTBE, methyl tert-butyl ether; DIPE, diisopropyl ether.
FIG. 5.
FIG. 5.
Conversion of naphthalenecarbonitrile by the purified nitrilase from Synechocystis sp. strain PCC6803 in the presence of different volumes of hexadecane. The reaction mixtures contained 580 μg of the purified (His-tagged) nitrilase in 200 μl of 50 mM Na-K phosphate buffer (pH 7.0) plus 10 mM naphthalenecarbonitrile. Different volumes of hexane were added (0% [vol/vol] [•], 20% [vol/vol] [○], 40% [vol/vol] [▪], or 60% [vol/vol] [□]), and the reaction mixtures were shaken at 30°C in a Thermomixer at 1,000 rpm. The reactions were stopped after different time intervals by the addition of 10% (vol/vol) 1 M HCl. Precipitated protein was removed by centrifugation (2 min, 16,000 × g), and the concentration of ammonia formed was determined in the aqueous phase by using the Spectroquant ammonia test.
FIG. 6.
FIG. 6.
Dendrogram resulting from pairwise alignments of amino acid sequences of different nitrilases. The following sequences with their relevant NCBI registration numbers were used: Alcaligenes faecalis JM3 (D13419); C. testosteroni (L32589); Klebsiella pneumoniae (ozaenae) (J03196); Pseudomonas fluorescens EBC 191 (C. Kiziak, unpublished data); R. rhodochrous K22 (D12583); R. rhodochrous J1 (D12583); Synechocystis sp. strain PCC 6803 (D64005); Nit4 from Arabidopsis thaliana (U09961) and Nicotina tabacum (D63331); and Nit1, Nit2, and Nit3 from Arabidopsis thaliana (Y07648). The sequences were aligned by using the program CLUSTAL X1.8, the dendrograms calculated by bootstrap neighbor joining, and the phylogenetic tree drawn by using the program TreeView 1.6.6, with the standard parameters.

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