Modification of the Secondary Binding Site of Xylanases Illustrates the Impact of Substrate Selectivity on Bread Making

Abstract

To investigate the importance of substrate selectivity for xylanase functionality in bread making, the secondary binding site (SBS) of xylanases from Bacillus subtilis (XBS) and Pseudoalteromonas haloplanktis was modified. This resulted in two xylanases with increased relative activity toward water-unextractable wheat arabinoxylan (WU-AX) compared to water-extractable wheat arabinoxylan, i.e., an increased substrate selectivity, without changing other biochemical properties. Addition of both modified xylanases in bread making resulted in increased loaf volumes compared to the wild types when using weak flour. Moreover, maximal volume increase was reached at a lower dosage of the mutant compared to wild-type XBS. The modified xylanases were able to solubilize more WU-AX and decreased the average degree of polymerization of soluble arabinoxylan in dough more during fermentation. This possibly allowed for additional water release, which might be responsible for increased loaf volumes. Altered SBS functionality and, as a result, enhanced substrate selectivity most probably caused these differences.

Assessing the impact of xylanase activity on the water distribution in wheat dough: A 1H NMR study

“…The wild type Bacillus subtilis Xyn A xylanase (XBS) was recombinantly expressed and purified according to Leys et al (2016).…”

“…WU-AX was incubated with 57 or 570 ppm XBS on WU-AX dm basis at 42% moisture for 30, 60 or 120 minutes (see 2.5). The two ratios of XBS enzyme to WU-AX substrate reflect on the one hand the ratio that yields the optimal functionality of XBS in bread making (low dosage) and on the other hand a ten-fold higher ratio (high dosage) (Leys, Pauly, Delcour, & Courtin, 2016). Both ratios are used throughout this study.…”

“…The carbohydrate contents after hydrolysis of flour, starch, purified WU-AX and WE-AX and aqueous extracts of xylanase treated WU-AX (see 2.2) were determined by gas chromatography as described in short by Leys et al (2016). The starch content was defined as 0.9 times the monosaccharide glucose content.…”

“…Courtin et al (1999) demonstrated that WU-AX negatively affects the bread volume, while WE-AX and solubilized AX (S-AX) with a high molecular weight , have a positive impact. It was hypothesized that the strong water retention capacity (WRC) of WU-AX would negatively affect the bread volume (Courtin et al, 1999;Leys, Pauly, Delcour, & Courtin, 2016). To alter AX functionality in bread making, endo-β-1,4-xylanases (EC 3.2.1.8), also referred to as endoxylanases or xylanases, are commonly used.…”

“…Especially xylanases with a high substrate selectivity are desired. These xylanases preferentially solubilize WU-AX to high molecular weight WE-AX, which has a lower WRC and a higher viscosity forming capacity compared to WU-AX (Leys et al, 2016).…”