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

• Published in: Biochimica et biophysica acta Vol. 1427; no. 2; pp. 145 - 154
• Main Authors: Kobayashi, Tohru, Hatada, Yuji, Higaki, Norihiko, Lusterio, Decorosa D, Ozawa, Tadahiro, Koike, Kenzo, Kawai, Shuji, Ito, Susumu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 19.04.1999
• Subjects: Alkaliphile; Amino Acid Sequence; amino acid sequences; Bacillus; Bacillus (bacteria); Bacillus - enzymology; Bacillus - genetics; Bacterial Proteins; Bacterial Proteins - isolation & purification; Cloning; Cloning, Molecular; enzyme activity; enzymology; genetics; Hydrogen-Ion Concentration; isolation & purification; kinetics; Molecular Sequence Data; nucleotide sequences; Pectate lyase; Polysaccharide-Lyases; Polysaccharide-Lyases - genetics; Polysaccharide-Lyases - isolation & purification; Protein Structure, Secondary; Secondary structure prediction; Sequence Alignment; Substrate Specificity; Temperature; Trans-elimination; Pel, pectate lyase; Pectate lyase; Secondary structure prediction; Cloning; PGA, polygalacturonic acid; SDS, sodium dodecyl sulfate; p I, isoelectric point; IEF, isoelectric focusing; Alkaliphile; Trans-elimination; PAGE, polyacrylamide gel electrophoresis; Bacillus; kbp, kilobase pair(s); PCR, polymerase chain reaction
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(99)00017-3

A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from alkaliphilic Bacillus sp. strain KSM-P7, designated Pel-7, was purified to homogeneity. The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was close to or higher than pH 10.5. In the presence of Ca 2+ ions, Pel-7 trans-eliminated polygalacturonate in random manner to generate oligogalacturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 65°C in glycine-NaOH buffer. Mn 2+ and Sr 2+ ions can serve as cofactors at almost the same level of Ca 2+ ions. It also exhibited a protopectinase-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 Da) showed some conserved regions in Pel superfamily, although homology to amino acid sequences of known Pels with 27 to 32% identity. Furthermore, Pel-7 appears to have similar core structure of parallel β-helix and active site topology with other Pels as revealed by secondary structure prediction in the Pel proteins. These results suggest that Pel-7 is basically grouped into Pel superfamily although the enzymatic and molecular properties are different.