Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.Fil: Senf, Deborah. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Ruprecht, Colin. Max Planck Institut für Kolloid und Grenzflächenforschung; AlemaniaFil: de Kruijff, Goswinus H. M.. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; Alemania. University Mainz. Institute of Institute of Organic Chemistry, Johannes Gutenberg; AlemaniaFil: Simonetti, Sebastián Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Max Planck Institut für Kolloid und Grenzflächenforschung; AlemaniaFil: Schuhmacher, Frank. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Seeberger, Peter H.. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Pfrengle, Fabian. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; Alemani

Fungi isolated from Miscanthus and sugarcane: biomass conversion, fungal enzymes, and hydrolysis of plant cell wall polymers.

BackgroundBiofuel use is one of many means of addressing global change caused by anthropogenic release of fossil fuel carbon dioxide into Earth's atmosphere. To make a meaningful reduction in fossil fuel use, bioethanol must be produced from the entire plant rather than only its starch or sugars. Enzymes produced by fungi constitute a significant percentage of the cost of bioethanol production from non-starch (i.e., lignocellulosic) components of energy crops and agricultural residues. We, and others, have reasoned that fungi that naturally deconstruct plant walls may provide the best enzymes for bioconversion of energy crops.ResultsPreviously, we have reported on the isolation of 106 fungi from decaying leaves of Miscanthus and sugarcane (Appl Environ Microbiol 77:5490-504, 2011). Here, we thoroughly analyze 30 of these fungi including those most often found on decaying leaves and stems of these plants, as well as four fungi chosen because they are well-studied for their plant cell wall deconstructing enzymes, for wood decay, or for genetic regulation of plant cell wall deconstruction. We extend our analysis to assess not only their ability over an 8-week period to bioconvert Miscanthus cell walls but also their ability to secrete total protein, to secrete enzymes with the activities of xylanases, exocellulases, endocellulases, and beta-glucosidases, and to remove specific parts of Miscanthus cell walls, that is, glucan, xylan, arabinan, and lignin.ConclusionThis study of fungi that bioconvert energy crops is significant because 30 fungi were studied, because the fungi were isolated from decaying energy grasses, because enzyme activity and removal of plant cell wall components were recorded in addition to biomass conversion, and because the study period was 2 months. Each of these factors make our study the most thorough to date, and we discovered fungi that are significantly superior on all counts to the most widely used, industrial bioconversion fungus, Trichoderma reesei. Many of the best fungi that we found are in taxonomic groups that have not been exploited for industrial bioconversion and the cultures are available from the Centraalbureau voor Schimmelcultures in Utrecht, Netherlands, for all to use

Heterologous expression and functional characterization of a GH10 endoxylanase from \u3ci\u3eAspergillus fumigatus\u3c/i\u3e var. \u3ci\u3eniveus\u3c/i\u3e with potential biotechnological application

Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-β-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60°C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMNGH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass

Purification and Characterization of Endoxylanases Cloned from Fibrobacter Succinogenes S85 and Expressed in Escherichia Coli HB101

The xylanase enzyme from Escherichia coli HB 101 containing the xylanolytic recombinant plasmid pBX6 was purified to homogeneity using ultrafiltration, DEAESepharose, CM-Sepharose and Sephadex 0-200 chromatography. Three xylanases, namely, Xyn A, Xyn BI and Xyn BII were obtained and were found to have the same molecular weight and optimum pH which were estimated to be 60.3 kDa and pH 7.0 respectively. The optimum assay temperature for both Xyn A and Xyn BI was 50°C, while for Xyn BII, it was 40°C. The xylanases were stable up to 45°C at pH 7.2 for 30 min. Approximately 80% of the enzyme activity was retained at the pH range of 5.0 to 8.0. The isoelectric point for Fraction A, Fraction BI and Fraction BII was 8.2, 8.5 and 5.5, respectively. The respective apparent K", and Vmax value on oat-spelt xylan was 12.2 mg/ml and 47.9 µmol xylose/min/mg protein for Xyn A; 10.8 mglml and 52.1 µmol xylose/min/mg protein for Xyn BI; 8.7 mg/ml and 54.2 µmol xylose/min/mg protein for Xyn BII. From the hydrolysis products of oat-spelt xylan analysed on thinlayer chromatography, the xylanases hydrolysed xylan through an endo-acting mechanism as no xylose, xylobiose or arabinose was detected. Thus, the xylanases were classified as an endoxylanase. The xylanases showed no activity toward carboxymethyl cellulose (CMC), crystalline cellulose (Avicel) and cellulose filter paper. The xylanases were not affected by potassium chloride, EDT A and EGTA at concentrations of 10 mM. Calcium chloride and magnesium chloride at the same concentrations enhanced the xylanase activities by 50%. Mercury chloride at 1.0 mM concentration completely inhibited the activities of all the purified xylanases. From zymogram analysis and characteristics of the xylanases investigated, multiplicity of xylanases in E. coli HB 101 (pBX6) was probably due to post-translational modification of a single gene product