Cloning and expression of a pectate lyase gene from Bacillus alcalophillus NTT33

Abstract

A gene encoding a pectate lyase (Pel) was cloned from genomic library of Bacillus alcalophillus NTT33 and expressed in Escherichia coli cells. A about 3.5 kb DNA fragment containing the pelA gene was sequenced. An open reading frame (ORF) of 1011 nucleotides encoded a protein of 311 amino acids. The expressed enzyme had a molecular mass of approximately 35 kDa determined by SDS–PAGE, and an isoelectric point of approximately pH 5.3. The PelA exhibited optimum activity at pH 9∼10 and 45 °C in Tirs–HCl buffer. The enzyme required Ca 2+ ions for activity, and was strongly inhibited by Ba 2+ and Mn 2+. The mature enzyme from the deduced amino acid sequence showed quite low homology to known Pels from various microorganisms with 16–20% identity. We did not found any conserved regions in the sequence of the PelA compared with the sequences of other enzymes from the established Pel superfamily. However, this PelA was homologous with pectate lyase P385 with 50% identity. We presumed that the PelA belonged to polysaccharide lyase family 10.