A plasmid borne, functionally novel glycoside hydrolase family 30 subfamily 8 endoxylanase from solventogenic Clostridium.

Glycoside hydrolase family 30 subfamily 8 (GH30-8) ß-1,4-endoxylanases are known for their appendage-dependent function requiring recognition of an α-1,2-linked glucuronic acid (GlcA) common to glucuronoxylans for hydrolysis. Structural studies have indicated that the GlcA moiety of glucuronoxylans is coordinated through six hydrogen bonds and a salt bridge. These GlcA-dependent endoxylanases do not have significant activity on xylans that do not bear GlcA substitutions such as unsubstituted linear xylooligosaccharides or cereal bran arabinoxylans. In the present study, we present the structural and biochemical characteristics of xylanase 30A from Clostridium acetobutylicum (CaXyn30A) which was originally selected for study due to predicted structural differences within the GlcA coordination loops. Amino acid sequence comparisons indicated that this Gram-positive-derived GH30-8 more closely resembles Gram-negative derived forms of these endoxylanases: a hypothesis borne out in the developed crystallographic structure model of the CaXyn30A catalytic domain (CaXyn30A-CD). CaXyn30A-CD hydrolyzes xylans to linear and substituted oligoxylosides showing the greatest rate with the highly arabinofuranose (Araf)-substituted cereal arabinoxylans. CaXyn30A-CD hydrolyzes xylooligosaccharides larger than xylotriose and shows an increased relative rate of hydrolysis for xylooligosaccharides containing α-1,2-linked arabinofuranose substitutions. Biochemical analysis confirms that CaXyn30A benefits from five xylose-binding subsites which extend from the -3 subsite to the +2 subsite of the binding cleft. These studies indicate that CaXyn30A is a GlcA-independent endoxylanase that may have evolved for the preferential recognition of α-1,2-Araf substitutions on xylan chains.

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyrosolvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark ß8-α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved ß8-α8 loop region of these enzymes influences xylan substrate specificity but not necessarily ß-1,4-xylanase function.

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum.

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5'UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.

Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava.

BACKGROUND: The aim of this study is determine the relative sensitivity of a panel of seven polyhydroxyalkanoate producing bacteria to a panel of seven lignocellulosic-derived fermentation inhibitors representing aliphatic acids, furans and phenolics. A further aim was to measure the polyhydroxybutyrate production of select organisms on lignocellulosic-derived monosaccharides arabinose, xylose, glucose and mannose. FINDINGS: We examined the sensitivity of seven polyhydroxyalkanoate producing bacteria: Azohydromonas lata, Bacillus megaterium, Bacillus cereus, Burkholderia cepacia, Pseudomonas olevorans, Pseudomonas pseudoflava and Ralstonia eutropha, against seven fermentation inhibitors produced by the saccharification of lignocellulose: acetic acid, levulinic acid, coumaric acid, ferulic acid, syringaldehyde, furfural, and hyroxymethyfurfural. There was significant variation in the sensitivity of these microbes to representative phenolics ranging from 0.25-1.5 g/L coumaric and ferulic acid and between 0.5-6.0 g/L syringaldehyde. Inhibition ranged from 0.37-4 g/L and 0.75-6 g/L with acetic acid and levulinic acid, respectively. B. cepacia and P. pseudoflava were selected for further analysis of polyhydroxyalkanoate production. CONCLUSIONS: We find significant differences in sensitivity to the fermentation inhibitors tested and find these variations to be over a relevant concentration range given the concentrations of inhibitors typically found in lignocellulosic hydrolysates. Of the seven bacteria tested, B. cepacia demonstrated the greatest inhibitor tolerance. Similarly, of two organisms examined for polyhydroxybutyrate production, B. cepacia was notably more efficient when fermenting pentose substrates.

Response surface methodology (RSM) to evaluate moisture effects on corn stover in recovering xylose by DEO hydrolysis.

Response surface methodology (RSM), based on a 2(2) full factorial design, evaluated the moisture effects in recovering xylose by diethyloxalate (DEO) hydrolysis. Experiments were carried out in laboratory reactors (10 mL glass ampoules) containing corn stover (0.5 g) properly ground. The ampoules were kept at 160 °C for 90 min. Both DEO concentration and corn stover moisture content were statistically significant at 99% confidence level. The maximum xylose recovery by the response surface methodology was achieved employing both DEO concentration and corn stover moisture at near their highest levels area. We amplified this area by using an overlay plot as a graphical optimization using a response of xylose recovery more than 80%. The mathematical statistical model was validated by testing a specific condition in the satisfied overlay plot area. Experimentally, a maximum xylose recovery (81.2%) was achieved by using initial corn stover moisture of 60% and a DEO concentration of 4% w/w. The mathematical statistical model showed that xylose recovery increases during DEO corn stover acid hydrolysis as the corn stover moisture level increases. This observation could be important during the harvesting of corn before it is fully dried in the field. The corn stover moisture was an important variable to improve xylose recovery by DEO acid hydrolysis.

Transcript analysis of genes encoding a family 61 endoglucanase and a putative membrane-anchored family 9 glycosyl hydrolase from Phanerochaete chrysosporium.

Phanerochaete chrysosporium cellulase genes were cloned and characterized. The cel61A product was structurally similar to fungal endoglucanases of glycoside hydrolase family 61, whereas the cel9A product revealed similarities to Thermobifida fusca Cel9A (E4), an enzyme with both endo- and exocellulase characteristics. The fungal Cel9A is apparently a membrane-bound protein, which is very unusual for microbial cellulases. Transcript levels of both genes were substantially higher in cellulose-grown cultures than in glucose-grown cultures. These results show that P. chrysosporium possesses a wide array of conventional and unconventional cellulase genes.