Pectate lyase from Bacillus subtilis: molecular characterization of the gene, and properties of the cloned enzyme

Abstract

Pectate lyases (PL) initiate soft‐rot diseases in plants by cleaving pectin which is the major component of the plant cell wall. The present paper reports the first cloning and characterization of a pectate lyase (pel) gene from the Bacillus genus. This gene was isolated from a Bacillus subtilis genomic library constructed in pUC18 as vector and Escherichia coli as host. By Southern hybridization this gene was shown to be present in a single copy in the B. subtilis genome. The nucleotide sequence of a 1.6 kb‐pair HindIII restriction fragment, which confers pectate lyase activity to E. coli, indicated a 1,260 bp open reading frame encoding a 420 amino acid polypeptide which includes a 21 amino acid signal sequence. The 45,605 Da deduced protein displays homologies to PLs from Erwinia chrysanthemi. The B. subtilis PL cloned in E.coli was located in the periplasm. It was purified to homogeneity in a one‐step procedure from the E. coli periplasmic fluid after overproduction using the pT7 system. Biochemical properties of the purified enzyme were similar to those found for the PL isolated from B. subtilis extracellular media.