Comparison of the Biochemical Properties and Roles in the Xyloglucan-Rich Biomass Degradation of a GH74 Xyloglucanase and Its CBM-Deleted Variant from Thielavia terrestris

Abstract

Xyloglucan is closely associated with cellulose and still retained with some modification in pretreated lignocellulose; however, its influence on lignocellulose biodegradation is less understood. TtGH74 from Thielavia terrestris displayed much higher catalytic activity than previously characterized fungal GH74 xyloglucanases. The carbohydrate-binding module 1 (CBM1) deleted variant (TtGH74ΔCBM) had the same optimum temperature and pH but an elevated thermostability. TtGH74 displayed a high binding affinity on xyloglucan and cellulose, while TtGH74ΔCBM completely lost the adsorption capability on cellulose. Their hydrolysis action alone or in combination with other glycoside hydrolases on the free xyloglucan, xyloglucan-coated phosphoric acid-swollen cellulose or pretreated corn bran and apple pomace was compared. CBM1 might not be essential for the hydrolysis of free xyloglucan but still effective for the associated xyloglucan to an extent. TtGH74 alone or synergistically acting with the CBH1/EG1 mixture was more effective in the hydrolysis of xyloglucan in corn bran, while TtGH74ΔCBM showed relatively higher catalytic activity on apple pomace, indicating that the role and significance of CBM1 are substrate-specific. The degrees of synergy for TtGH74 or TtGH74ΔCBM with the CBH1/EG1 mixture reached 1.22-2.02. The addition of GH10 xylanase in TtGH74 or the TtGH74ΔCBM/CBH1/EG1 mixture further improved the overall hydrolysis efficiency, and the degrees of synergy were up to 1.50-2.16.

Keywords: Thielavia terrestris; Xyloglucan; family 1 carbohydrate-binding module (CBM1); glycoside hydrolase family 74 (GH74) xyloglucanase; lignocellulosic biomass; synergism.

Figure 1

(a): The modularity of TtGH74 and TtGH74ΔCBM. SG, signal sequence; CD, catalytic domain module; CBM1, CBM1 module. (b): SDS-PAGE analysis of the purified TtGH74 and TtGH74ΔCBM and endo H-treated TtGH74 and TtGH74ΔCBM. Lane 1, molecular mass marker; Lane 2, TtGH74; Lane 3, endo H-treated TtGH74; Lane 4, TtGH74ΔCBM; and Lane 5, endo H-treated TtGH74ΔCBM.

Figure 2

The effects of temperature (a) and pH (b) on the enzyme activity and the effects of temperature (c) and pH (d) on the stability of TtGH74 and TtGH74ΔCBM.

Figure 3

MALDI-TOF MS analysis of the products generated by TtGH74 from tamarind seed XG. The red arrow shows the possible XG cleavage sites of TtGH74, and the black cross indicates that TtGH74 did not cleave at the reducing end side of L units in XLXG and XLLG.

Figure 4

The adsorption differences of TtGH74 and TtGH74ΔCBM on various celluloses and lignocelluloses (a), the adsorption of TtGH74 on cellulose with different proportions of XG (b) and analysis of their adsorption capacities on XG by electrophoresis in 7.5% native polyacrylamide gel without xyloglucan (c) or with 0.005 w/v xyloglucan (d). Lane 1, TtGH74; and Lane 2, TtGH74ΔCBM.

Figure 5

Hydrolysis of TtGH74 and TtGH74ΔCBM on different amounts of XG, TtGH74, TtGH74ΔCBM, EG1 and CBH1 hydrolyze PASC coated with different proportions of XG, respectively (a). The schematic diagram of the association pattern of XG and PASC (b). Orange cylinders represent PASC, and XG is represented by a blue line. The left diagram shows the low proportion of XG/PASC; the right diagram shows the high proportion of XG/PASC; and the XG forms accessible “loops” and “tails” on the PASC surface.

Figure 6

TtGH74 and TtGH74ΔCBM enzymatic hydrolysis of sulfuric-acid-pretreated corn bran (a), DES-pretreated corn bran (b) and DES-pretreated apple pomace (c) with different pretreatment times. The product yield was expressed in grams of reducing sugar produced by per kilogram of dry material (Reducing Sugars g/kg Dry Material, RS g/kg DM). The statistical differences between two groups of TtGH74 and TtGH74ΔCBM were determined using Student’s t-test analysis. Statistical significance is defined as *p < 0.05 and ** p < 0.01.

Figure 7

HPAEC-PAD (left) and MALDI-TOF MS (right) analysis of end-products generated by TtGH74 from sulfuric-acid-pretreated corn bran (a,b), DES-pretreated corn bran (c,d) and apple pomace (e,f). The blank in the HPAEC-PAD represents a control experiment without TtGH74.

Figure 8

Time courses of the enzymatic hydrolysis of sulfuric-acid-pretreated corn bran, DES-pretreated corn bran and apple pomace by TtGH74 or TtGH74ΔCBM. (a–d): 10, 20, 40 and 80 mg of sulfuric-acid-pretreated corn bran, respectively; (e–h): 10, 20, 40 and 80 mg of DES-pretreated corn bran, respectively; (i–l): 10, 20, 40 and 80 mg of DES-pretreated apple pomace, respectively. The product yield was expressed in grams of reducing sugar produced by per kilogram of dry material (Reducing Sugars g/kg Dry Material, RS g/kg DM).

Figure 9

The synergy between TtGH74, TtGH74ΔCBM, GH10 xylanase and the CBH1/EG1 mixture on sulfuric-acid-pretreated corn bran and DES-pretreated lignocellulose (a,b). Enzymatic hydrolysis verification of pretreated lignocellulose components treated by strong alkali (c,d). The product yield was expressed in grams of reducing sugar produced by per kilogram of dry material (Reducing Sugars g/kg Dry Material, RS g/kg DM).