Nucleotide and amino‐acid sequences of a new‐type pectate lyase from an alkaliphilic strain of Bacillus

Abstract

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 < 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 8C in glycine/NaOH buffer. Pel-15E had an absolute requirement for Ca 2+ 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.

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits

“…Although the pure enzyme appears homogenous on SDS-PAGE and FPLC, the authors do not claim it as a totally pure preparation, since plant genomes contains several potential pectate lyase genes, which encode homologous proteins, which would be very difficult to separate by biochemical methods. The specific activity of the enzyme was 680 ± 50 pkat mg protein À1 , which was low compared to homogenous PEL from microbial sources (Crawford and Koattukuty, 1987;Liao et al, 1997;Benen et al, 2000;Sawada et al, 2000;Kluskens et al, 2003).…”

Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module

“…The catalytic module of Pel10A shows 30 and 36 % identities to pectate lyase A from A. irakense [14] and pectate lyase 15E from alkaliphilic Bacillus sp. strain KSM-P15 [19], respectively ( Figure 5). Structural and sitedirected mutagenesis studies of pectate lyases have indicated the importance of arginine in substrate charge neutralization, and acidic residues as proton donors during catalysis [28,29], and indeed family 10 pectate lyases possess six invariant arginine residues, four invariant aspartic acid residues and four invariant glutamic acid residues ( Figure 5).…”

“…The 35.8 kDa Cterminal module of Pel10A shows similarity to pectate lyase A from Azospirillum irakense [14] and pectate lyase 15E from the alkaliphilic Bacillus sp. strain KSM-P15 [19] (Figure 5). Both the A. irakense and the Bacillus enzymes are members of family A modular pectate lyase containing a carbohydrate-binding module The proposed ribosome-binding sequence is underlined.…”

Microbial pectate lyases: characterization and enzymological properties

“…and Ni 2? ions resulted in the recovery of the enzyme activity by 62, 36, 9 and 7%, respectively (Sawada et al 2000). Pel from banana can use Mn 2?…”