Characterization and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritima

Abstract

Pectin is a complex polysaccharide present in the cell wall of higher plants, where it forms a network by embedding the other cell wall polysaccharides cellulose and hemicellulose. The backbone of the pectin molecule mainly consists of (partly methylated) homogalacturonan, interspersed with rhamnogalacturonan units, which often contain sugar side chains composed of arabinan and galactan [1].Degradation of the pectin polymer occurs via a set of pectinolytic enzymes, which can roughly be divided into esterases, which remove ferulic acid, methyl or acetyl groups, and depolymerases. The latter can be classified into lyases (b-elimination) and hydrolases [2]. All hydrolases involved in degradation of pectin are classified as members of family 28 of the glycoside hydrolases, including the endopolygalacturonases, exopolygalacturonases and rhamnogalacturonases [3,4]. Although a handful of endopolygalacturonases, generally of fungal origin [5][6][7][8][9][10], and a single rhamnogalacturonase [11] An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105°C and a temperature optimum of 80°C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 UAEmg ). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.Abbreviations PelB, exopolygalacturonase B; PelA, exopectate lyase A; PGA, polygalacturonic acid; (GalpA) n , oligogalacturonate with degree of polymerization n; DP, degree of polymerization; HPSEC, high-performance size-exclusion chromatography; HPAEC, high-performance anion-exchange chromatography.

Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

“…This result verified another of our analyses (see Section 2.1 above). Until now, only two bacterial EPGs (PelB [27] and Pgu B [8]) had been reported in the literature, while other EPGs were isolated from eukaryotes [6,16] or soil metagenome samples [29]. This, the CbPelA obtained in this study, is only the third EPG to be isolated from bacteria.…”

“…The K m and V max were calculated as 0.3 mg·mL −1 and 386.8 U·mg −1 , respectively. The V max of CbPelA was lower than that of PelB [27] and PgaX [33], but higher than that of other EPGs (Table 1). …”

“…TS 47 [26]. However, only two thermophilic EPGs have been isolated from thermophilies [27–29]. One of the thermo-active EPGs that was identified was PelB cloned from T. maritime.…”

“…One of the thermo-active EPGs that was identified was PelB cloned from T. maritime. Its optimal active and melting temperatures are 80 and 105 °C, respectively [18,27]. Another thermophilic pectinase, PecJKR01, was isolated from a soil metagenome sample.…”

Purification and characterization of exo-type cellouronate lyase

“…This strain produces some lyases during the growth, and degrades cellouronate by cleaving glycoside bonds through b-elimination, resulting in the formation of depolymerized products having double bonds between C4 and C5 at the non-reducing termini. Also in the case of natural polyuronates such as alginate (Rehm 1998;Yoon et al 2000;Hashimoto et al 2000;Suzuki et al 2006;Ochiai et al 2006), pectin (Pissavin et al 1998;Shevchik et al 1999a, b;Benten et al 2000;Kluskens et al 2005;Klug-Santner et al 2006;Payasi et al 2006), xanthan (Hashimoto et al 1998(Hashimoto et al , 2001 and hyaluronan (Ponnuraj and Jedrzejas 2000;Oettl et al 2003), lyases predominantly take part in their enzymatic degradation. In a previous paper (Konno et al 2006), cellouronate lyase-I (CUL-I) was purified from the cell-free extracts of Brevundimonas sp.…”