Molecular cloning and expression of a novel mesophilic alkaline protease from Bacillus sp. L010 in Escherichia coli.

OBJECTIVE: Bacteria producing proteases were isolated and selected from the environment and the alkaline proteases with superior performance for commercial exploitation were screened. METHODS: The strain producing extracellular proteases was isolated by a casein plate and was identified by biochemical and morphological tests and by 16S rDNA sequence analysis. To acquire the open reading frame (ORF) of the protease, degenerate primers designing and genome walking method were used. The precursor and mature peptide of the protease were recombinant expressed in BL21 (DE3). After purification of the active protease, the characteristics and the catalytic ability were detected using synthetic peptide succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrates. RESULTS: Strain L010 isolated was named as Bacillus sp. L010 after identification. The ORF of the protease was 1149-bp long and encoded a protein of 382 amino acids comprised with a 30-residual signal peptide, a 77-residual propeptide, and a 275-residual mature protein, and the encoded protein was one of subtilisins-a member of serine proteases and designated as SprD. The precursor of SprD (pro-SprD) autoprocessed into active SprD mediated by the propeptide when pro-SprD was recombinant expressed in BL21 (DE3). The enzyme exhibited high catalytic efficiency (K(cat)/K(m)) towards synthesis substrates with optimal activity at 70 degrees C and pH 9 - 10. SprD was stable over a range of pH 7.0 to 10.0 and was thermal stable at 25 degrees C - 60 degrees C. CONCLUSION: The high stability of SprD towards alkaline conditions (pH 7 - 10) and under temperature 25 degrees C - 60 degrees C and the high catalytic efficiency suggested that the protease would find research value and potential applications.

Distribution of serotypes and virulence-associated genes in pathogenic Escherichia coli isolated from ducks.

The objective of the present study was to investigate the serotypes and virulence-associated genes of avian pathogenic Escherichia coli (APEC) isolated from duck colibacillosis cases. Two hundred and fifty-four APEC isolates from duck colibacillosis cases were serotyped and amplified for 12 known virulence-associated genes and the betA gene (encoding choline dehydrogenase) by polymerase chain reaction assays. One hundred and forty-three E. coli isolates from cloacal swabs of healthy ducks were also amplified for the same genes. A total of 53 O-serogroups were found in 254 APEC isolates, among which O93, O78 and O92 were predominant serogroups. Polymerase chain reaction results showed that Shiga-toxin-producing E. coli distributed in only 2.4% of ducks compared with 49.2% of the APEC isolates harbouring the irp2 gene, and 44.9% the fyuA gene, respectively. The ibeA gene was only present in 27 APEC isolates and was not found in healthy ducks. The rfaH gene was detected in 20.5% of APEC isolates, whereas 5.6% was found in healthy ducks. A total 79.5% of APEC isolates harboured the betA gene, which was significantly higher than in healthy ducks (16.1%), suggesting that betA may be associated with virulence.