Jan Bach Kristensen

Cell-wall structural changes in wheat straw pretreated for bioethanol production.

BackgroundPretreatment is an essential step in the enzymatic hydrolysis of biomass and subsequent production of bioethanol. Recent results indicate that only a mild pretreatment is necessary in an industrial, economically feasible system. The Integrated Biomass Utilisation System hydrothermal pretreatment process has previously been shown to be effective in preparing wheat straw for these processes without the application of additional chemicals. In the current work, the effect of the pretreatment on the straw cell-wall matrix and its components are characterised microscopically (atomic force microscopy and scanning electron microscopy) and spectroscopically (attenuated total reflectance Fourier transform infrared spectroscopy) in order to understand this increase in digestibility.ResultsThe hydrothermal pretreatment does not degrade the fibrillar structure of cellulose but causes profound lignin re-localisation. Results from the current work indicate that wax has been removed and hemicellulose has been partially removed. Similar changes were found in wheat straw pretreated by steam explosion.ConclusionResults indicate that hydrothermal pretreatment increases the digestibility by increasing the accessibility of the cellulose through a re-localisation of lignin and a partial removal of hemicellulose, rather than by disruption of the cell wall.

Determining Yields in High Solids Enzymatic Hydrolysis of Biomass

As technologies for utilizing biomass for fuel and chemical production continue to improve, enzymatic hydrolysis can be run at still higher solids concentrations. For hydrolyses that initially contain little or no free water (10–40% total solids, w/w), the saccharification of insoluble polymers into soluble sugars involves changes of volume, density, and proportion of insoluble solids. This poses a new challenge when determining the degree of hydrolysis (conversion yield). Experiments have shown that calculating the yield from the resulting sugar concentration in the supernatant of the slurry and using the assumed initial volume leads to significant overestimations of the yield. By measuring the proportion of insoluble solids in the slurry as well as the sugar concentration and specific gravity of the aqueous phase, it is possible to precisely calculate the degree of conversion. The discrepancies between the different ways of calculating yields are demonstrated along with a nonlaborious method for approximating yields in high solids hydrolysis.

Pretreatment and enzymatic hydrolysis of wheat straw (Triticum aestivum L.) – The impact of lignin relocation and plant tissues on enzymatic accessibility

Wheat straw is a potential feedstock for bioethanol production. This paper investigates tissues from whole internode sections subjected to hydrothermal pretreatment at 185°C and subsequent enzymatic hydrolysis up to 144 h. Analyses revealed an increase in surface lignin as hydrolysis progressed, which could be coupled to the gradual decrease in hydrolysis rate over time. The data support the hypothesis of lignin extraction from the cell wall matrix during pretreatment and deposition as droplets upon cooling. These droplets are assumed to accumulate during enzymatic hydrolysis. Additionally, after 144 h of enzymatic hydrolysis the cortex had vanished, exposing the heavier lignified vascular tissue. Accumulation of lignin droplets and exposure of residual lignin could be part of the explanation for the decreasing hydrolysis rate. Flattening of macrofibrils after pretreatment together with more indentations on the surfaces was also observed, possibly caused by a proposed synergistic effect of cellobiohydrolases and endoglucanases.

The question of coherency in corporate branding – over time and across stakeholders

The paper investigates the successful establishment of a strong corporate brand with a particular emphasis on analysing the corporate branding literature’s assumptions about coherency. Successful corporate branding is claimed to imply a shared set of coherent statements about the company’s values towards its external and internal stakeholders over time. An empirical test is applied to the coherency assumption. First, the coherency of a corporate brand over time is investigated as it develops in the media. Secondly, the coherency between two stakeholders’ perceptions of the corporate brand, organisational members and the media is investigated. This research suggests there are three distinct types of coherencies in corporate branding strategies over time: statement coherency, interpretation coherency and uniqueness coherency. On the one hand, a strong corporate brand is characterised by tight coherency, as top management’s statements about values remain the same over time and towards different stakeholders, ie statement coherency. On the other hand, a strong corporate brand is simultaneously characterised by a loose, or even absent, coherency between stakeholders’ interpretations of top management’s statements as well as a lack of coherency in stakeholders’ interpretations of the corporate brand over time, ie interpretation coherency. Finally, a third coherency phenomenon is observed, ie stakeholders’ emphasis on changing topics over time, which they relate to the corporate brand. Stakeholders agree that these themes are unique features and hence the company is considered unique, ie the uniqueness coherency. The implications of multiple interpretations are discussed as well as changing interpretations in corporate branding. It is argued that statement coherency is a necessary element in successful corporate branding, and the viability of the ambition to develop interpretation coherency over time and across stakeholders in corporate branding is discussed from the point of view of allowing room for interpretation incoherency. Finally, the question of maintaining uniqueness coherency is discussed: for how long can a company represent “newness” in the eyes of its stakeholders – including itself? Implications for management are discussed.

Identification of catechols as histone–lysine demethylase inhibitors

Identification of inhibitors of histone–lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA‐based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity in the low μM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2‐OS cells.

High-throughput microarray profiling of cell wall polymers during hydrothermal pre-treatment of wheat straw.

Lignocellulosic plant material is potentially a sustainable source of fermentable sugars for bioethanol production. However, a barrier to this is the high resistance or recalcitrance of plant cell walls to be hydrolyzed. Therefore, a detailed knowledge of the structural features of plant cell walls that contribute to recalcitrance is important for improving the efficiency of bioethanol production. In this work we have used a technique known as Comprehensive Microarray Polymer Profiling (CoMPP) to analyze wheat straw before and after being subjected to hydrothermal pre‐treatments at four different temperatures. The CoMPP technique combines the specificity of monoclonal antibodies with the high‐throughput capacity of microarrays. Changes in the relative abundance of cell wall polysaccharides could be tracked during processing, and a reduction in xylan, arabinoxylans, xyloglucan, and mixed‐linked glucan epitopes was detected at the two highest temperatures of pre‐treatment used. This work demonstrates the potential of CoMPP as a complementally technique to conventional methods for analyzing biomass composition. Biotechnol. Bioeng. 2010; 105: 509–514.

Posttranslational modifications of the histone 3 tail and their impact on the activity of histone lysine demethylases in vitro.

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k cat/K m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.

Enzyme kinetic studies of histone demethylases KDM4C and KDM6A: Towards understanding selectivity of inhibitors targeting oncogenic histone demethylases

To investigate ligand selectivity between the oncogenic KDM4C and tumor repressor protein KDM6A histone demethylases, KDM4C and KDM6A were enzymatically characterized, and subsequently, four compounds were tested for inhibitory effects. 2,4‐dicarboxypyridine and (R)‐N‐oxalyl‐O‐benzyltyrosine (3) are both known to bind to a close KDM4C homolog and 3 binds in the part of the cavity that accommodates the side chain in position 11 of histone 3. The inhibition measurements showed significant selectivity between KDM4C and KDM6A. This demonstrates that despite very similar active site topologies, selectivity between Jumonji family histone demethylases can be obtained even with small molecule ligands.