Nucleotide and amino-acid sequences of a new-type pectate lyase from an alkaliphilic strain of Bacillus.
A pectate lyase (pectate transeliminase; EC 4.2.2.2), designated Pel-15E, was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-P15. The purified enzyme had a molecular mass of approximately 33 kDa, as determined by SDS/PAGE, and a pI of approximately pH 9.2. Pel-15E exhibited optimum activity at pH 10.5 and 50-55 degrees C in glycine/NaOH buffer. Pel-15E had an absolute requirement for Ca2+ ions for manifestation of the enzymatic activity and trans-eliminated poly(galacturonic) acid, most likely by endo-type cleavage. A gene for the enzyme, which was cloned using the shotgun method and sequenced, contained a 960-bp ORF encoding 320 amino acids. The mature enzyme (286 amino acids, 32 085 Da) from the deduced amino-acid sequence showed quite low homology to known Pels from various microorganisms with 16.1-20.4% identity. Furthermore, we were not able to find any conserved regions in the sequence of Pel-15E when aligned with the sequences of other enzymes from the established Pel superfamily. However, Pel-15E had some regions that were homologous to PelA from Azospirillum irakense with 39.8% identity. Based on their amino-acid sequence homology, Pel-15E and PelA appear to belong to a new class of Pel family, although the enzymatic properties of both enzymes were quite different.
Sawada
K
K
Tochigi Research Laboratories of Kao Corporation, Tochigi, Japan.
Ogawa
A
A
Ozawa
T
T
Sumitomo
N
N
Hatada
Y
Y
Kobayashi
T
T
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D011133:Polysaccharide-Lyases; C023911:pectate lyase