Cloning of a Nitrilase Gene from the CyanobacteriumSynechocystissp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

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.

Requirement of the Nitrogen Starvation-Induced Protein Sll0783 for Polyhydroxybutyrate Accumulation in Synechocystis sp. Strain PCC 6803

“…Analysis of the surrounding gene region revealed that this gene appears as the first gene of a cluster which had been previously described in the context of the following gene, sll0784 (26). The sll0784 gene encodes a nitrilase which has already been characterized biochemically (14). The genes in the cluster occur in the following order: (i) a hypothetical protein (sll0783), (ii) a nitrilase (sll0784), (iii) a radical S-adenosylmethionine superfamily member (sll0785), (iv) a potential acetyltransferase (sll0786), (v) an AIR synthase-related protein (sll0787), (vi) a hypothetical protein (ssl1464), and (vii) a flavoprotein predicted to be involved in K ϩ transport (slr0801) (Fig.…”

Long-Term Response toward Inorganic Carbon Limitation in Wild Type and Glycolate Turnover Mutants of the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…Additionally, we found a similar strong decline in transcript abundances for the genes sll0783 to sll0787 (Table III). With exception of Sll0784, the nitrilase MerR (Heinemann et al, 2003), the functions of all other encoded proteins are unknown. By the action of nitrilases, which hydrolyze nitriles to the corresponding carboxylic acids and ammonium, many bacteria like Pseudomonas fluorescens EBC191 can use nitriles as nitrogen source (Kiziak et al, 2005).…”

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

“…Escherichia coli JM109 was used as the host strain for all plasmids. The cultivation and induction conditions for the recombinant strains used were described previously (16,20,40). Enzyme assays.…”

“…The construction of plasmids pIK7, pIK9, and pDHE22, which encode the nitrilases from Alcaligenes faecalis ATCC 8750, Pseudomonas fluorescens EBC191, and Synechocystis sp. strain PCC6803, respectively, was described previously (16,20,21). The gene for the rhodococcal nitrilase was amplified from a nitrileconverting Rhodococcus rhodochrous strain.…”