Cloning of a New Pectate Lyase GenepelCfromFusarium solanif. sp.pisi(Nectria haematococca,Mating Type VI) and Characterization of the Gene Product Expressed inPichia pastoris

Antibodies prepared against a pectate lyase (PLA) produced by a phytopathogenic fungusFusarium solanif. sp.pisi(Nectria haematococca,mating type VI) were previously found to protect the host against infection. The cDNA and gene (pelA) for PLA were cloned and sequenced. A new pectate lyase gene, pelC, was isolated from a genomic library of F. solani pisi with pelA cDNA as a probe. A 1.3-kb DNA fragment containing the pelC gene and its flanking regions was identified and sequenced. The coding region of pelC was amplified by reverse transcription–polymerase chain reaction using total RNA isolated from a pectin-induced F. solani pisi culture as template. The open reading frame of pelC was predicted to encode a 23.3-kDa protein of 219 amino acid residues, which shares 51% identity with PLA from F. solani pisi. No typical fungal leader peptide sequence could be identified at the N-terminus of the predicted protein sequence. The amplified pelC cDNA was expressed in Pichia pastoris yielding a pectate lyase C (PLC) with a molecular mass of 26.0 kDa and containing carbohydrates. PLC was purified to homogeneity using Mono Q anion-exchange chromatography. Purified PLC required Ca2+for its activity and showed optimal lyase activity at pH 9.5 and 55°C. Rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLC cleaved polygalacturonate chains in an endo fashion. Western blot using antibodies raised against PLA and PLC showed that PLC and PLA are immunologically related to each other.