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Nucleotide and aminoâacid sequences of a newâtype pectate lyase from an alkaliphilic strain of Bacillus
- Sawada, Kazuhisa, Ogawa, Akinori, Ozawa, Tadahiro, Sumitomo, Nobuyuki, Hatada, Yuji, Kobayashi, Tohru, Ito, Susumu
- European journal of biochemistry 2000 v.267 no.5 pp. 1510-1515
- Azospirillum irakense, Bacillus (bacteria), amino acid sequences, amino acids, calcium, culture media, enzyme activity, genes, ions, microorganisms, molecular weight, open reading frames, pH, pectate lyase, polyacrylamide gel electrophoresis, sequence homology, sodium hydroxide
- A pectate lyase (pectate transeliminase; ECâ188.8.131.52), designated Pelâ15E, was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSMâP15. The purified enzyme had a molecular mass of ââ33âkDa, as determined by SDS/PAGE, and a pI of ââpHâ9.2. Pelâ15E exhibited optimum activity at pHâ10.5 and 50â55âÂ°C in glycine/NaOH buffer. Pelâ15E had an absolute requirement for Ca2+ ions for manifestation of the enzymatic activity and transâeliminated poly(galacturonic) acid, most likely by endoâtype cleavage. A gene for the enzyme, which was cloned using the shotgun method and sequenced, contained a 960âbp ORF encoding 320 amino acids. The mature enzyme (286 amino acids, 32â085âDa) from the deduced aminoâacid sequence showed quite low homology to known Pels from various microorganisms with 16.1â20.4% identity. Furthermore, we were not able to find any conserved regions in the sequence of Pelâ15E when aligned with the sequences of other enzymes from the established Pel superfamily. However, Pelâ15E had some regions that were homologous to PelA from Azospirillum irakense with 39.8% identity. Based on their aminoâacid sequence homology, Pelâ15E and PelA appear to belong to a new class of Pel family, although the enzymatic properties of both enzymes were quite different.