Development of improved enzymes for the hydrolysis of the other major carbohydrate polymer present in lignocellulosic biomass is also of commer­cial interest, particularly to those utilizing neutral or alkaline pretreatments that leave much of the hemicellulose intact. To develop these enzymes, an industrial residue of the wheat starch industry was used as a model sub­strate. In Europe, wheat is one of the major substrates for production of fuel ethanol. Processing of wheat starch for glucose results in a by-product stream (vinasse) consisting mainly of the wheat endosperm cell wall ma­terial and leftover yeast cells following the fermentation of the starch. The hemicellulose by-product is approximately 33 wt % carbohydrates of which approximately 66 wt % is arabinoxylan. Arabinoxylans consist of a linear backbone of P-1,4-linked D-xylopyranosyl units that are partially substituted with arabinofuranosyls. The major portion of the arabinoxylan in indus­trial wheat fermentation residues is water-soluble [39], the water-insoluble arabinoxylan is quantitatively more abundant in cell walls isolated directly from unprocessed wheat endosperm [40]. Arabinoxylans are hydrolyzed to monosaccharides by acid treatment or by enzymatic hydrolysis. The enzy­matic hydrolysis is usually preferred because it allows for a more specific and controlled modification and fewer undesirable by-products, making it more suitable for microbial fermentation using organisms that can metabolize both xylose and arabinose [41].

The enzymatic degradation of arabinoxylans requires both side-group cleaving and depolymerizing enzymes. Cleavage of the side chains re­quires the action of several accessory enzyme activities, including a-L — arabinofuranosidases, a-glucuronidases, ferulic acid esterase, and acetyl — esterases [41,42]. Depolymerization requires endo-1,4-|3-xylanases that result in unbranched xylooligosaccharides, including xylotriose and xylobiose, and в-xylosidases that cleave xylobiose and attack the non-reducing ends of short chain xylooligosaccharides to liberate xylose [41].

The hydrolysis of arabinoxylan is critical for improved utilization of wheat hemicellulose in the ethanol industry. Three Novozymes cellulolytic and hemicellulolytic enzyme preparations, Celluclast 1.5 L, Ultraflo L, and Vis — cozyme L were tested in various combinations for their ability to liberate arabinose and xylose from water-soluble wheat arabinoxylan. The substrate was medium viscosity water-soluble wheat arabinoxylan from Megazyme (Bray). The three different enzymes were evaluated individually and also in 50 : 50 combinations to look for possible synergistic effects. Reactions were carried out at pH 5 and 50 °C followed by analysis of arabinose, galactose, glu­cose, xylose, xylobiose, and xylotriose by high-performance anion exchange chromatography (HPAEC) [43]. The molecular weight and distribution of water-soluble wheat arabinoxylan and hydrolyzates were determined by high — performance size exclusion chromatography (HPSEC).

In those reactions containing the individual enzyme preparations, the lev­els of arabinose and xylose increased with increasing enzyme dosage and time. Ultraflo L was superior to Celluclast 1.5 L and Viscozyme L in releasing the arabinose from the water-soluble wheat arabinoxylan, meaning that Ul — traflo L must contain a significant amount of a-L-arabinofuranosidase. Cellu­clast 1.5 L was the best enzyme preparation for liberating xylose, resulting in 26 wt % of the available xylose. Ultraflo L released 16 wt % while Viscozyme L released less than 1.5 wt %. In a mixture of 50 : 50 Celluclast 1.5 L and Ultra — flo L there was no interaction among the arabinose-releasing side activities since the same amount of arabinose was obtained as when the two individual enzyme preparations were used and then the arabinose total was combined. The Viscozyme L preparation exhibited a weak antagonistic effect with Ul — traflo L and Celluclast 1.5 L since the amount of arabinose actually decreased compared to that observed with the individual enzyme preparations. The re­sults indicated that the arabinose-releasing side activities of Viscozyme L had the same activity as those demonstrated by Ultraflo L and Celluclast 1.5 L. Another possibile but less likely explanation is the Viscozyme L contained a — L-arabinofuranosidase inhibitors [43]. The 50 : 50 mixture of Celluclast 1.5 L and Ultraflo L produced an increase in the release of xylose compared with the sum of the individual enzyme preparations (Fig. 8). The mixture released 59 wt % of the available xylose, which was 32 wt % more than the theoret­ical addition of the individual enzyme preparations alone. Combination of Ultraflo L and Viscozyme L showed no such synergism, but incubation of Cel­luclast 1.5 L and Viscozyme L showed a weak synergistic effect in liberating some of the xylose from the wheat arabinoxylan.

To further examine the synergistic affect between Celluclast 1.5 L and Ul — traflo L the amounts of xylobiose and xylotriose released during enzymatic hydrolysis were quantified using HPAEC for both individual and combined enzyme preparations. During the initial stage of incubation, Celluclast 1.5 L

liberated small amounts of both xylobiose and xylotriose, indicating the pres­ence of endo-1,4-^-xylanase activities. As hydrolysis continued, the released xylobiose and xylotriose was hydrolyzed to xylose, indicating the Cellu — clast 1.5 L contained one or more в-xylosidase activities.

Ultraflo L treatment resulted in continual liberation of both xylobiose and xylotriose. Ultraflo L showed a low release of free xylose indicating one or more endo-1,4-^-xylanase activities, but little в-xylosidase activity. The synergistic effect between Celluclast 1.5 L and Ultraflo L in releasing xylose is therefore likely to be a result of the action of a-L-arabinofuranosidase and endo-1,4-^-xylanase activities present in Ultraflo L and the в-xylosidase present in Celluclast 1.5 L [43].

Since a strong synergistic effect was observed with a 50 : 50 combination of Celluclast 1.5 L and Ultraflo L for the breakdown of arabinoxylan, a sec­ond study was conducted to look for similar effects and viscosity reduction in the fermentation residue, vinasse. The effects of enzyme dosage, optimal temperature, and pH were examined in hydrolysis of whole vinasse, vinasse supernatant, and washed vinasse sediment that was provided by Tate & Lyle, Amylum UK (Greenwich, UK). On whole vinasse, the enzyme-catalyzed re­lease of arabinose and xylose by the 50 : 50 combination of Ultraflo L and Cel­luclast 1.5 L decreased as the substrate concentration of the vinasse increased. The monosaccharide release also decreased when the substrate concentration of the vinasse increased. Release of arabinose and xylose from the vinasse sediment was very low. The release of arabinose from the whole vinasse var­ied from 40- 50 g arabinose per kilogram vinasse DM while xylose release was between 75-100 g xylose per kilogram vinasse DM after a 24 h hydrolysis. The

Ultraflo L:Celluclast 1.5 L mixture released 53-75 g arabinose and 75-115 gof xylose per kilogram of vinasse DM after a 24 h hydrolysis [44].

Significant viscosity reduction was obtained by enzyme-catalyzed degra­dation of arabinoxylans present in the fermentation residue stream, vinasse. However, there was limited hydrolysis of the insoluble arabinoxylans in the vinasse sediment. The efficiency of enzymatic degradation of the arabinoxylan in vinasse was dependent on enzyme dosing and substrate dry matter [44].

In an effort to narrow down the specific activities involved in the previous studies, the в-xylosidase from Celluclast 1.5 L was purified and used as a sup­plement to Ultraflo L enzyme preparation. When dosed at 0.25 g в-xylosidase protein per kilogram of arabinoxylan along with Ultraflo L, this enzyme mix released the same or more xylose as the enzyme mix consisting of 50 : 50 Ultraflo L and Celluclast 1.5 L (Fig. 9).

In order to determine the optimal enzyme mix for the hydrolysis of vinasse arabinoxylan, several recombinant enzymes were made and tested in various combinations. Genes were cloned and expressed in the fungal host A. oryzae. Based on our studies the optimal enzyme mix for vinasse hydrolysis consists of a-L-arabinofuranosidase from Meripilus giganteus, a-L — arabinofuranosidase II from Humicola insolens, and T. reesei в-xylosidase. A mixture of 25 : 25 : 50 of a-L-arabinofuranosidase from M. giganteus, a-L- arabinofuranosidase from H. insolens and в-xylosidase from T. reesei was determined to be optimal for maximizing arabinoxylan hydrolysis. The success of this work in identifying and exploiting synergism between hemicellulase component activities is currently being applied to other relevant lignocellulosic substrates that differ significantly in their hemicellulose composition.

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