Tài liệu Báo cáo khoa học: Characterization and mode of action of an exopolygalacturonase from the

Characterization and mode of action of an

exopolygalacturonase from the hyperthermophilic

bacterium Thermotoga maritima

Leon D. Kluskens1

, Gert-Jan W.M. van Alebeek2

, Jasper Walther1

, Alphons G.J. Voragen2

,

Willem M. de Vos1 and John van der Oost1

1 Laboratory of Microbiology, Wageningen University, the Netherlands

2 Laboratory of Food Chemistry, Wageningen University, the Netherlands

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 mole￾cule mainly consists of (partly methylated) homogalac￾turonan, 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, exo￾polygalacturonases and rhamnogalacturonases [3,4].

Although a handful of endopolygalacturonases, gener￾ally of fungal origin [5–10], and a single rhamnogalac￾turonase [11] have been the object of crystallization

Keywords

exopolygalacturonase; hydrolytic; mode of

action; pectin; thermostable

Correspondence

J. van der Oost, Laboratory of Microbiology,

Wageningen University, Hesselink van

Suchtelenweg 4, 6703 CT Wageningen,

the Netherlands

Fax: +31 317 483829

Tel: +31 317 483108

E-mail: [email protected]

(Received 28 July 2005, accepted 24 August

2005)

doi:10.1111/j.1742-4658.2005.04935.x

An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperther￾mophilic 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 monogalac￾turonate 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 UÆmg)1

). 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 tubin￾gensis, 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 phy￾siological 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.

5464 FEBS Journal 272 (2005) 5464–5473 ª 2005 FEBS