Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants

Abstract

The efficient degradation of complex xylans needs collaboration of many xylan degrading enzymes. Assays for xylan degrading activities based on reducing sugars or PNP substrates are not indicative for the presence of enzymes able to degrade complex xylans: They do not provide insight into the possible presence of xylanase-accessory enzymes within enzyme mixtures. A new screening method is described, by which specific xylan modifying enzymes can be detected.

Fermentation supernatants of 78 different fungal soil isolates grown on wheat straw were analyzed by HPLC and MS. This strategy is powerful in recognizing xylanases, arabinoxylan hydrolases, acetyl xylan esterases and glucuronidases.

No fungus produced all enzymes necessary to totally degrade the substrates tested. Some fungi produce high levels of xylanase active against linear xylan, but are unable to degrade complex xylans. Other fungi producing relative low levels of xylanase secrete many useful accessory enzyme component(s).

Introduction

In order to convert lignocellulosic biomass to ethanol, a whole array of enzymes is needed to degrade such a complex structure to monomers (Carvalheiro et al., 2008). An important part of cereal and wood hemicellulose is the class of xylans, comprising a backbone of β-1,4-xylopyranosyl units containing various side groups like, amongst others, 4-O-methyl-α-d-glucopyranosyl uronic acid, ferulic acid, arabino-furanose and O-acetyl groups (Ebringerova and Heinze, 2000). The composition of the lignocellulose determines the enzymes required for complete degradation of the substrate to monomers.

Wheat arabinoxylan (WAX) and Eucalyptus xylan hydrolysate (EXH) have been used before to identify and characterize an array of xylan degrading enzymes (Christov et al., 2000, Pouvreau et al., 2011, Tenkanen and Siika-Aho, 2000), since these substrates contain the most abundant substituents present in cereal lignocellulosic xylans (Gírio et al., 2010), except for ferulic acid (Vidmantiene et al., 2006). WAX contains mono and/or double α-l-arabinosylated xylopyranosyl units through O-3 and/or O-2 (Ebringerova and Heinze, 2000). The degradation of WAX requires, next to the presence of endo-xylanases (EC 3.2.1.8), the activity of AXH-m (releasing mono substituted arabinose: EC 3.2.1.55), AXH-d3 (releasing double substituted arabinose from position O-3: EC 3.2.1.55) or β-xylosidase (EC 3.2.1.37) within the fermentation supernatants (Gírio et al., 2010, Van Laere et al., 1997). The soluble EXH consists of O-acetyl-(4-O-methylglucurono)xylooligosaccharides (Christov et al., 2000). The acetyl substituents are closely associated with the 4-O-methylglucuronic acid (Evtuguin et al., 2003). The 4-O-methylglucuronic acid substituent may be substituted at O-2 with α-d-galactose (Shatalov et al., 1999). The degradation of EXH is only successful in the presence of acetyl xylan esterases (EC 3.1.1.72), endo- and exo-xylanases and α-glucuronidases (EC 3.2.1.131) (Christov et al., 2000, Tenkanen and Siika-Aho, 2000). α-Glucuronidases are enzymes that are able to hydrolyze the α-1,2-linkage between 4-O-methylglucuronic/glucuronic acid and xylose.

In the search for novel enzymes, in which the enzyme activity is often monitored using dyed substrates or via the formation of reducing end groups, no distinction can be made between different enzymes (Biely and Puchart, 2006, Ghatora et al., 2006). Next to these assays, proteomic approaches making use of genomic libraries will often result in the annotation of known enzymes instead of identifying real novel or desired enzymes (e.g. Wang et al., 2010).

Filamentous fungi are a good source of xylan degrading enzymes and their levels of enzyme excretion in the fermentation media are high, which makes them interesting for screening (Gírio et al., 2010, Handelsman et al., 1998, Polizeli et al., 2005). However, more precise assays are necessary to include a wide range of enzymes in such screening (Biely and Puchart, 2006).

In this paper a screening method is presented in which a range of xylan degrading enzymes in fungal fermentation liquids are identified. Analysis of the xylan digests was done by using HPAEC, HPLC and Maldi-TOF MS.