Sophie Levy-Schil

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa.

A Brevibacterium sp. R312 DNA fragment encoding the wide-spectrum amidase (EC 3.5.1.4) has been cloned and sequenced, using limited amino acid (aa) sequence information obtained from the purified enzyme. The deduced aa sequence showed more than 80% strict identity with the Pseudomonas aeruginosa aliphatic amidase, the product of the amiE gene, suggesting a horizontal transfer of the gene during evolution between Gram+ and Gram- bacteria.

Biotin biosynthetic pathway in recombinant strains of Escherichia coli overexpressing bio genes: evidence for a limiting step upstream from KAPA

The effect of the overexpression of the bioABFCD operon on the biotin biosynthetic pathway was investigated in an Escherichia coli K12 bioR mutant with a chromosomal deletion for the biotin operon. When transformed with a multicopy number plasmid containing bioABFCD, this strain synthetized 10,000 times more biotin than a wild-type E. coli strain. In order to further increase biotin production, the bioA and bioB operons were subcloned into plasmids with stronger promoters and in some cases optimal ribosome binding sites. The new constructions led to the accumulation of large amounts of soluble Bio proteins (although not BioC) but did not improve biotin production. In all the constructed strains, BioA, BioD, and BioB activities were greatly amplified but these activitie did not correlate with the level of protein syntthesis. These strains accumulated only low levels of vitamers, auggesting that the major limiting step for higher biotin production occurs upstream from the first intermediate of the Bio pathway we assayed (7,keto-8-aminopelargonic acid). As BioC overproduction was shown to impair cell growth, we could not determine if this early step of pathway was limiting.

Construction of a broad-host-range non-mobilizable stable vector carrying RP4 par-region

Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290. Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid.

Enzymatic hydrolysis of adiponitrile into 5-cyano valeric acid, an intermediate for nylon 6

Publisher Summary An industrial process must be economic and safe for the environment. The chemical hydrolysis of nitrile in acid is well known. Nearly all nitriles react with either basic or acid catalysts, but considerable quantities of inorganic salts are always produced as by-products. The only way to suppress these by-products is to produce the ammonium carboxylate under neutral pH and then to recover the ammonia by dissociation of the salt between the weak base and acid. It is possible to design an excellent catalyst for a bulk product such as caprolactam. The first advantage of the biocatalysis in this route is able to carry out this reaction at neutral pH. The second is to selectively obtain the α,ω-cyanoacid starting from the α,ω-dinitrile. No other type of catalysis can do this.