CelAB, a multifunctional cellulase encoded by Teredinibacter turnerae T7902T, a culturable symbiont isolated from the wood-boring marine bivalve Lyrodus pedicellatus

Abstract

We characterized a multifunctional cellulase (CelAB) encoded by the endosymbiont Teredinibacter turnerae T7902(T). CelAB contains two catalytic and two carbohydrate-binding domains, each separated by polyserine linker regions. CelAB binds cellulose and chitin, degrades multiple complex polysaccharides, and displays two catalytic activities, cellobiohydrolase (EC 3.2.1.91) and beta-1,4(3) endoglucanase (EC 3.2.1.4).

FIG. 1.

(A) Structural features of CelAB. Domains are noted. Black box, type II secretion signal; striped boxes, polyserine linker regions. (B) Endoglucanase activity of CelAB. Rapid reduction in viscosity of a 5% CMC gel by K. lactis Δcts-1 celAB (solid line) culture supernatant compared to the K. lactis Δcts-1 background strain (dashed line) suggests endoglucanase activity. (C) Carbohydrate binding characteristics of CelAB. Immunoblot detection of CelAB indicates binding to Avicel, cellulose, and chitin but not xylan or curdlan. Lanes, 1, Avicel; 2, cellulose; 3, chitin; 4, xylan; 5, curdlan. Molecular mass markers (kDa) are indicated to the left.

FIG. 2.

Enzymatic characterization of CelAB. (A) Glycosylation of CelAB by the K. lactis Δcts-1 strain. Treatment of CelAB purified from the K. lactis Δcts-1 celAB strain with PNGase F resulted in a single protein band at the expected molecular mass. Lane 1, PNGase F-treated CelAB; lane 2, untreated CelAB. Molecular mass markers (kDa) are given to the left. (B) Temperature and pH optima for CelAB based on the hydrolysis of pNP-cellobioside. (C) Endoglucanase and cellobiohydrolase activity of CelAB demonstrated with coumarin-linked substrates. Lanes: 1 d-glucose-coumarin; 2, cellobiose-coumarin; 3, cellotriose-coumarin; 4, cellotetraose-coumarin; 5, cellopentaose-coumarin; 6, CelAB plus cellotetraose-coumarin; 7, CelAB plus cellopentaose-coumarin. (D) Cleavage site of coumarin-linked cellotetraose and cellopentaose produced by CelAB. CelAB produced cellobiose-coumarin from cellotetraose-coumarin (below left) indicative of cellobiohydrolase (CBH) activity and released cellotriose-coumarin and cellobiose-coumarin from cellopentaose-coumarin (below right), indicative of cellobiohydrolase and β-1,4 endoglucanase (ENG) activities. Open stars, coumarin; solid circles, glucose.