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Nucleotide and amino‐acid sequences of a new‐type 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; EC, 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 ≈ 33 kDa, as determined by SDS/PAGE, and a pI of ≈ pH 9.2. Pel‐15E exhibited optimum activity at pH 10.5 and 50–55 °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.