Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate

A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from a lkaliphilic Bacillus sp. strain KSM-P7, designated Pel-7, was purified to h omogeneity. The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric p oint was close to or higher than pH 10.5. In the presence of Ca2+ ions, Pel -7 trans-eliminated polygalacturonate in random manner to generate oligogal acturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 6 5 degrees C in glycine-NaOH buffer. Mn2+ and Sr2+ ions can serve as cofacto rs at almost the same level of Ca2+ ions. It also exhibited a protopectinas e-like activity, liberating soluble pectin and/or oligogalacturonides from cotton fibers. The pel gene was cloned and sequenced, and the deduced amino acid sequence of mature Pel-7 (302 amino acids, 33, 355 Dal showed some co nserved regions in Pel superfamily, although homology to amino acid sequenc es of known Pels with 27 to 32% identity. Furthermore, Pel-7 appears to hav e similar core structure of parallel beta-helix and active site topology wi th other Pels as revealed by secondary structure prediction in the Pel prot eins. These results suggest that Pel-7 is basically grouped into Pel superf amily although the enzymatic and molecular properties are different. (C) 19 99 Elsevier Science B.V. All rights reserved.