Molecular cloning, expression and characterization of alpha-amylase gene from a marine bacterium Pseudoalteromonas sp. MY-1.

A gene (amyA) encoding an extracellular alpha-amylase from a marine bacterium Pseudoalteromnonas sp. MY-1 was cloned and expressed in Escherichia coli. It comprised an open-reading-frame of 2,007 base pairs and encoded a protein of 669 amino acids with a predicted molecular weight of 73,770 daltons and a pI of 5.15. The entire amino acid sequence of amyA gene showed 86% similarity to the alpha-amylase preproprotein from Pseudoalteromonas haloplanktis. It consisted of a signal peptide, alpha-amylase catalytic domain and an amy C domain. The recombinant amylase was purified to homogeneity and biochemically characterized. The enzyme revealed maximum activity at pH 7.0 and 40 degrees C. The enzyme hydrolyzed soluble starch and some maltooligosaccharides to several oligosaccharides, and maltose was the common product from different substrates.

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1.

The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.