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

Abstract:

Abstract:［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 (Kcat/Km) towards synthesis substrates with optimal activity at 70℃ and pH 9－10．SprD was stable over a range of pH 7.0 to 10.0 and was thermal stable at 25℃ － 60℃.［Conclusion］The high stability of SprD towards alkaline conditions (pH 7 － 10) and under temperature 25℃－60℃ and the high catalytic efficiency suggested that the protease would find research value and potential applications.

Key words:Keywords: autoprocessing，Bacillus sp. L010，BL21 (DE3)，expression，PCR，SprD