Xylose Degradation Research Articles

GroEL-GroES solubilizes abundantly expressed xylulokinase in Escherichia coli

The aims of the present work were to solubilize the abundantly expressed recombinant xylulokinase in Escherichia coli and to develop a reliable xylulokinase assay. Three mutants of xylulokinase of Bacillus megaterium that were expressed at high level but formed insoluble protein in E. coli BL21(DE3)pLysS were selected for solubility study. The solubility of xylulokinase increased eight to 77-fold after introduction of molecular chaperones GroEL-GroES into the host. This investigation reports that GroEL-GroES minimizes the formation of insoluble protein in three highly expressed recombinant xylulokinases and an improved xylulokinase assay. Commercial production of bioethanol is critically dependent on the development of an efficient and low-cost process of enzymatic conversion of xylan, a major component in lignocellulose biomass, to xylulose-5-phosphate, which can then be channelled into pentose phosphate pathway and metabolized to ethanol. The improved intracellular xylulokinase activity is expected to facilitate the xylose degradation.

Novel xylose dehydrogenase in the halophilic archaeon Haloarcula marismortui.

During growth of the halophilic archaeon Haloarcula marismortui on D-xylose, a specific D-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to D-xylose, D-ribose was oxidized at similar kinetic constants, whereas D-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)-coding for a 39.9-kDA protein-was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui.

A modellistic view of the kinetics of metabolic processes: differences in the glucose and xylose degradation pathway

Abstract In vivo NMR spectroscopy and sugars selectively enriched with 13 C were used to follow the step-by-step metabolic kinetics of glucose and xylose by an isolated strain (G11) of Klebsiella planticola. The experimental results were then used to develop a model based on non-linear differential equations totally consistent with the experimental data. During the fitting procedure, a set of kinetic constants, each related to a specific metabolic path, were calculated.

A comparative kinetic study of acidic hemicellulose hydrolysis in corn cob and sunflower seed hull

The kinetics of dilute acid catalyzed hemicellulose hydrolysis in corn cob and sunflower seed hull was investigated with sulfuric acid concentrations of between 0·2 and 1 N at temperatures of 98–130°C. Batch hydrolysis data for two different lignosellulosic biomasses were correlated with a general kinetic model which assumed that hemicellulose was composed of two fractions with each fraction reacting according to first-order rates. When the concept of fast- and slowly-hydrolyzing hemicellulose was combined with a first-order-rate model for xylose degradation a complete kinetic model was developed. The respective rate constants were determined as functions of temperature and sulfuric acid concentration by a non-linear regression analysis. The results indicated that the kinetic parameters involved in hydrolysis reactions were dependent on substrate material. The experimental and model results were in a satisfactory agreement.

Hybrid process for the conversion of lignocellulosic materials.

Because of the recalcitrant nature of lignocellulosic materials, it is important to pretreat the biomass in order to obtain a suitable material for the bioconversion. In this study, two different types of pretreatments were performed. The first experiment used a 2-gal Parr reactor operated at 140, 150, 160, and 170 degrees C with sulfuric acid concentrations varying from 0.5 to 2%. A second pretreatment was performed with a two-stage low-temperature process. The first-stage pretreatment was performed at 100 or 120 degrees C with sulfuric acid concentrations of 0.5, 2, and 5% followed by a second-stage pretreatment at 120 degrees C with 2% acid concentration. The best residues for enzymatic hydrolysis and simultaneous saccharification and fermentations (SSF) came from the higher temperature pretreatment with the Parr reactor. However, a large portion of the xylose fraction was degraded to furfural and glucose was degraded to HMF. On the contrary, the two-stage low temperature pretreatment resulted in a very low percentage of xylose degradation, and no glucose degradation. The residues from this two-stage pretreatment performed satisfactorily toward the production of ethanol by SSFs. This study discusses the results obtained from these experiments.

Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus.

The xylose isomerase (xylA) and the xylulose kinase (xylB) genes from Streptomyces rubiginosus were isolated, and their nucleotide sequences were determined. The xylA and xylB genes encode proteins of 388 and 481 amino acids, respectively. These two genes are transcribed divergently from within a 114-nucleotide sequence separating the coding regions. Regulation of the xyl genes in S. rubiginosus was examined by fusing their promoters to the Pseudomonas putida catechol dioxygenase gene and integrating the fusions into the minicircle integration site on the S. rubiginosus chromosome. The expression of catechol dioxygenase was then measured under a variety of conditions. The results indicated that transcription of the xyl genes was induced by D-xylose and repressed by glucose. Data from quantitative S1 mapping were consistent with this conclusion and suggested that xylA had one and xylB had two transcription initiation sites. The transcription initiation site of xylA was 40 bp upstream of the coding region. The two transcription initiation sites of xylB were 20 and 41 bp 5' of its translation initiation codon. Under control of appropriate regulatory elements, the cloned xyl genes are capable of complementing either Escherichia coli xylose isomerase- or xylulose kinase-deficient strains. The deduced amino acid sequence of the S. rubiginosus xylA protein is highly homologous to sequences of other microbial xylose isomerases.

Degradability of phenolic acid‐hemicellulose esters: A model system

AbstractA synthetic model system containing p‐coumaric and ferulic acids esterified to hemicellulose was used to study polysaccharide degradability. Oatspelts xylan was fractionated into Alinear, Blinear and Branched fractions prior to synthetic esterification with phenolic acids at treatment concentrations of 0, 25, 50, 75 and 100 g phenolic acid per kg hemicellulose. Concentrations of phenolic acids esterified to the hemicellulose fractions were determined by alkaline hydrolysis. In‐vitro dry matter disappearance (IVDMD) and degradability of hemicellulose neutral sugars were measured after 48 h ruminal fermentations. Esterification efficiency of the phenolic acids to the hemicellulose fractions was low (0.3 to 13.9%) and greater for p‐coumaric than ferulic acid (4.7 vs 3.1%, respectively). Concentration of esterified phenolic acids was negatively correlated with IVDMD for the Alinear and Bbranched fractions. Ferulic acid appeared to be more inhibitory to IVDMD than p‐coumaric acid. Generally the degradability of the side chain sugars of the hemicellulose fractions was negatively correlated with esterified phenolic acid concentrations. Xylose degradation was only correlated with esterified ferulic acid level in the Alinear fraction. The in‐vitro ruminal fermentations resulted in the degradation of the majority of the phenolic acid esters. Analysis of the synthetic phenolic acid‐hemicellulose esters by 13C NMR and FTIR was unable to prove the presence of monomeric phenolic acid esters. The presence of phenolic acid polyesters was unlikely because of the solubility of the synthetic phenolic acid‐hemicellulose esters. The neutral sugar degradation data suggest that esterification of the phenolic acids was limited to sugars with primary hydroxyl groups. While this model system was useful for studying cell wall degradation, future studies must employ model systems in which the chemical constituents being tested accurately model those found in nature.

Gastrointestinal function in patients with progressive systemic sclerosis.

In 24 patients with progressive systemic sclerosis (PSS) the pentagastrin-stimulated gastric acid secretion was determined to investigate if acid hypersecretion is associated with reflux-oesophagitis--the most common complication to oesophageal involvement in PSS. Gastro-oesophageal reflux was observed in 12, reflux-oesophagitis in 9 and oesophageal mycosis in 8 patients. Gastric acid secretion was increased in 13 (54%) patients and tended to be higher in patients with oesophagitis. Patients with reflux and increased acid secretion seemed to be free from oesophageal mycosis. Bacterial overgrowth and malabsorption are known complications to intestinal scleroderma and these items were investigated using non-invasive methods. Four patients had increased bile acid deconjugation, 3 had increased (14C)xylose degradation indicating bacterial overgrowth and 7 patients had decreased fat absorption in the triolein breath test. Nutritional status with respect to selenium, folate, cobalamin and fat-soluble vitamins was essentially normal.

Studies on Lactic Acid Fermentation

Resting cells of l. fermentum convert glyceraldehyde to equimolar lactic acid and neither the evolution of carbon dioxide nor the uptake of oxygen was observed. Glyceraldehyde-3-phosphate and 3-phosphoglycerate were identified as intermediates which were equally labeled with inorganic P32 in reaction systems, and the presence of triokinase was suggested. Resting cells of lactic acid bacteria oxidize mannitol only aerobically to produce lactic acid, accompanied by theoretical oxygen uptake. The presence of mannitol dehydrogenase in the extracts of heterofermenters as well as mannitokinase and mannitol-1-phosphate dehydrogenase in the extracts of homofermenters were demonstrated. DPN-dependence of both dehydrogenases and the reversibility of the reactions were ascertained. Mannitol dehydrogenase was partially purified from l. brevis, which is specific for mannitol and activated by cobalt ion. Pyruvate and acetaldehyde were detected as intermediates in the pathway of heterolactic fermentation in the presence of semicarbazide by resting cells of l. fermenti. Alcohol dehydrogenase in ammonium sulfate fraction catalyzes both DPN- and TPN- linked dehydrogenation of ethanol to acetaldehyde, and acetyl coenzyme A was reduced to acetaldehyde in the presence of either TPN or DPN. By coupling of xylose degradation with mannitol utilization or by the continuous supply of reduced methyl viologen, appreciable increase of ethyl alcohol was observed.