Glc4 backbone oligosaccharides are the end products of the reaction of XG with Cel9D. However, the enzyme produced XLLG more slowly than the other Glc4-backbone oligosaccharides (Additional file 1: Figure S1). We tested, whether the addition of the β-galactosidase Bga2B can increase the amount of Glc4 backbone oligosaccharides released by Cel9D. To ensure, that incubation conditions are applicable for both enzymes, we used a pH vs temperature activity profile (Fig. 1). Conditions suitable for both enzymes were visually determined to be pH 6.4 and 60 °C. To study the beneficial interaction between the two enzymes for XG hydrolysis, we first incubated a fixed amount of Cel9D (480 mU) with varying amounts of Bga2B (0–155 mU). With 31 mU Bga2B determined as the required amount, we varied the Cel9D concentration from 120 mU to 24 U to achieve a maximal reduction of Cel9D needed and achieved an 80% reduction in total enzyme load.
To test whether the addition of a β-galactosidase improves the XG degradation capability of Cel9D, 480 mU of Cel9D were incubated with increasing amounts of Bga2B (0–155 mU). As shown in Fig. 2, the reaction yield during the incubation interval doubled with the addition of only 3.1 mU Bga2B and increased further to sevenfold when 31 mU or more Bga2B was added. The smallest amount of Bga2B to produce even a slight increase in reaction yield was determined to be 1.55 mU. Negative controls using only Bga2B without Cel9D showed the release of galactose and no degradation of the polymeric XG backbone (data not shown).
In the next experiment, between 120 mU and 24 U of Cel9D were added to the reaction mix, with and without a constant addition of 31 mU of Bga2B (Fig. 3, gray bars and black bars respectively). In the absence of Bga2B, reaction yield correlated directly with Cel9D concentration. In contrast, in the presence of 31 mU Bga2B reaction yield reached nearly 100% using only 360 mU Cel9D. Therefore, the yield ratios between reactions with and without Bga2B were more pronounced at lower Cel9D concentrations, peaking at more than a 22-fold yield increase for the reaction containing 240 mU Cel9D.
Interestingly, even with the highest amount of Cel9D used in this study the XG backbone cleavage reaction could not be completed to the extent achievable with Bga2B addition. Using 24 U of only Cel9D (50 µg) resulted in an 85.3 ± 2.6% reaction yield. In contrast, only 360 mU of Cel9D (0.75 µg) produced a yield of 93 ± 2.7% when combined with 31 mU Bga2B (10 µg). Thus, in addition to the increased yield, the total amount of enzyme in the reaction was concomitantly reduced by nearly 80%. This may indicate that Cel9D is unable to efficiently degrade the parts of the XG substrate that contain a high abundance of double galactose substitutions (XLLG). This negative effect is alleviated by removing the side chains with the β-galactosidase. Bga2B was the only enzyme with β-galactosidase activity toward XG available in our lab. Screening for a more active β-galactosidase could further lower the amount of enzyme needed in the reaction.
All other endo- and xyloglucanases from C. thermocellum, apart from Cel9D, with specific activities toward XG in a similar range (Xgh74A, Cel5E, and Cel9/44 J), were analyzed for a stimulating effect of galactosyl moiety removal, but none exhibited an increase in reaction yield from the addition of Bga2B. With the addition of 31 mU Bga2B, Cel9D was the most effective XG degrading enzyme from C. thermocellum (Additional file 1: Figure S2). Additionally, an enzyme from Herbivorax saccincola GGR1T with activity toward XG was tested, which revealed that Cel9K from this recently isolated bacterium [30] acts in a manner similar to Cel9D (unpublished data). Therefore, this effect is not limited to Cel9D from C. thermocellum, but may be specific to a certain subset of GH9 endoglucanases. Finding the underlying structural associations that produce this dependency would be an interesting task for future studies to further define this subset of enzymes.
The removal of the galactose moieties from the side chains of the XG substrate is a prerequisite for its complete hydrolysis to monosaccharides [31, 32], which makes the beneficial effects of synergism between the β-galactosidase Bga2B and endoglucanase Cel9D observed in this study particularly useful. With tamarind XG as a substrate, the reaction using these two enzymes in appropriate amounts for a complete reaction results in only two major reaction products: the heptasaccharide XXXG and the monosaccharide galactose. Galactose could easily be removed via various approaches, such as nanofiltration or simulated moving bed chromatography, leaving the XXXG oligosaccharide as the major product. The selective production of one large oligosaccharide with a defined degree of polymerization implies the potential for industrial application, including use as a platform chemical. Additional properties, such as application as prebiotic food or feed supplement, should be evaluated. The addition of Bga2B increased the total product yield obtained using the model XG substrate as well as tamarind kernel powder (TKP, Additional file 1: Figure S3). TKP is a bulk substrate and a side product of the tamarind pulp industry and may serve as a cheap substrate for industrial applications.
In conclusion, our study demonstrates the first beneficial combination of two enzymes for the degradation of the hemicellulose XG. β-Galactosidase Bga2B addition during the endoglucanase Cel9D-catalyzed hydrolysis of XG boosted the reaction yield and lowered the total amount of enzyme needed. The effect described in this study could be applied in the valorization of substrates with high XG contents, such as TKP.