Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus.
A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.
Tochigi Research Laboratories, Kao Corporation, Japan. 113310@kastanet.kao.co.jp
T
T
Kobayashi
K
K
Koike
T
T
Yoshimatsu
N
N
Higaki
A
A
Suzumatsu
T
T
Ozawa
Y
Y
Hatada
S
S
Ito
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007526:Isoelectric Point; D008670:Metals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D007202:Indicators and Reagents; D008670:Metals; D010446:Peptide Fragments; D011133:Polysaccharide-Lyases; C023911:pectate lyase