A New High-alkaline and High-molecular-weight Pectate Lyase from a Bacillus Isolate: Enzymatic Properties and Cloning of the Gene for the Enzyme

Abstract

A pectate lyase (Pel; pectate transeliminase: EC4.2.2.2.), designated Pel-15H, was found in an alkaline culture of Bacillus sp. strain KSM-P15 and purified to homogeneity by sequential column chromatographies. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 70,000 and the pI was around pH 4.6. Pel-15H randomly trans-eliminated polygalacturonate in the presence of Ca²⁺ ions, and the maximum activity was observed at pH 11.5 and at 55°C in glycine-NaOH buffer. The gene for Pel-15H was cloned and sequenced, and the structural gene contained a 2,031-bp open reading frame that encoded 677 amino acids including a possible 28-amino-acid signal sequence. The mature enzyme (649 amino acids, molecular weight 69,550) showed very low similarity to Pels from Bacillus with 12.7-18.2% identity. Interestingly, part of the amino acid sequence of Pel-15H had fairly high similarity only to an N-terminal half of PelL and a C-terminal half of PelX from Erwinia chrysanthemi 3937, and a C-terminal half of PelX from E. chrysanthemi EC16 (approximately 35% identity for all).

• pectate lyase
• trans-eliminase
• cloning
• alkaliphile
• Bacillus