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 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 Ca2+ 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 degrees C in glycine-NaOH buffer. Mn2+ and Sr2+ ions can serve as cofactors at almost the same level of Ca2+ 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 beta-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.
Kobayashi
T
T
Tochigi Research Laboratories of Kao Corporation 2606 Akabane, Ichikai, Haga, Tochigi, 321-3497, Japan. 113310@kastanet.kao.co.jp
Hatada
Y
Y
Higaki
N
N
Lusterio
D D
DD
Ozawa
T
T
Koike
K
K
Kawai
S
S
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000302:isolation & purification; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000302:isolation & purification*; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase