Replacement of carbohydrate binding modules improves acetyl xylan esterase activity and its synergistic hydrolysis of different substrates with xylanase

Abstract

Background: Acetylation of the xylan backbone was a major obstacle to enzymatic decomposition. Removal of acetyl groups by acetyl xylan esterases (AXEs) is essential for completely enzymatic hydrolysis of xylan. Appended carbohydrate binding modules (CBMs) can promote the enzymatic deconstruction of plant cell walls by targeting and proximity effects. Fungal acetyl xylan esterases are strictly appended to cellulose-specific CBM1. It is still unclear whether xylan-specific CBMs have a greater advantage than CBM1 in potentiating the activity of fungal deacetylating enzymes and its synergistic hydrolysis of different substrates with xylanase.

Results: Three recombinant AXE1s fused with different xylan-specific CBMs, together with wild-type AXE1 with CBM1 and CBM1-deleted mutant AXE1dC, were constructed in this study. The optimal temperature and pH of recombinant AXE1s was 50 °C and 8.0 (except AXE1dC-CBM6), respectively. Cellulose-specific CBM1 in AXE1 obviously contributed to its catalytic action against substrates compared with AXE1dC. However, replacement of CBM1 with xylan-specific CBM4-2 significantly enhanced AXE1 thermostability and catalytic activity against soluble substrate 4-methylumbelliferyl acetate. Whereas replacements with xylan-specific CBM6 and CBM22-2 were more effective in enzymatic release of acetic acid from destarched wheat bran, NaClO₂-treated wheat straw, and water-insoluble wheat arabinoxylan compared to AXE1. Moreover, replacement with CBM6 and CBM22-2 also resulted in higher degree releases of reducing sugar and acetic acid from different substrates when simultaneous hydrolysis with xylanase. A good linear relationship exists between the acetic acid and reducing sugar release.

Conclusions: Our findings suggested that the replacement with CBM6 and CBM22-2 not only significantly improved the catalysis efficiency of AXE1, but also increased its synergistic hydrolysis of different substrates with xylanase, indicating the significance of targeting effect in AXE1 catalysis mediated by xylan-specific CBMs.

Keywords: Acetyl xylan esterase; Carbohydrate-binding module; Fusion enzyme; Synergism; Xylan; Xylan-specific.

Fig. 1

Schematic structures of five recombinant acetyl xylan esterases with and without CBMs. CD catalytic domain; linker, linker peptide, CBM1/4-2/6/22-2 family 1/4-2/6/22-2 carbohydrate-binding modules

Fig. 2

SDS-PAGE of purified rAXE1s and endo H-treated rAXE1s. The gel was stained with Coomassie Brilliant Blue and the samples were loaded in the following order (from left to right): M, protein markers, Lane 1–5, purified AXE1dC, AXE1, AXE1dC-CBM6, AXE1dC-CBM4-2, AXE1dC-CBM22-2; Lane 6–10, endoglycosidase-H-treated rAXE1s, and the bright bands near 26 kDa were endo H

Fig. 3

Biochemical properties of rAXE1s. a Optimum temperature; b Optimum pH; c Thermostability; d pH stability

Fig. 4

Binding specificities of rAXE1s to a Avicel, b destarched wheat bran, c NaClO₂-treated wheat straw and d water-insoluble wheat arabinoxylan (inAX). Lanes: M, protein markers; Lanes: 1, 3, 5, 7 and 9, unbound fractions in supernatant, and Lanes: 2, 4, 6, 8 and 10, fractions bound to substrates for AXE1dC, AXE1, AXE1dC-CBM6, AXE1dC-CBM4-2, and AXE1dC-CBM22-2, respectively

Fig. 5

Time course for the enzymatic release of acetic acid from a destarched wheat bran, b NaClO₂-treated wheat straw and c water-insoluble wheat arabinoxylan (inAX)

Fig. 6

Time course for the release of reducing sugar from a destarched wheat bran, b NaClO₂-treated wheat straw and c water-insoluble wheat arabinoxylan (inAX)

Fig. 7

Release of acetic acid from destarched wheat bran (WB), NaClO₂-treated wheat straw (WS) and water-insoluble wheat arabinoxylan (inAX) by simultaneous hydrolysis with XynII (X) and a AXE1dC (dC), b AXE1 (1), c AXE1dC-CBM4-2 (4-2), d AXE1dC-CBM6 (6) and e AXE1dC-CBM22-2 (22-2), respectively

Fig. 8

Linear relationships between release of acetic acid and reducing sugar from destarched wheat bran, NaClO₂-treated wheat straw and water-insoluble wheat arabinoxylan (inAX) after 24 h hydrolysis by combined rAXE1s and XynII