Chemomechanical Simulation of Microtubule Dynamics With Explicit Lateral Bond Dynamics

We introduce and parameterize a chemomechanical model of microtubule dynamics on the dimer level, which is based on the allosteric tubulin model and includes attachment, detachment and hydrolysis of tubulin dimers as well as stretching of lateral bonds, bending at longitudinal junctions, and the possibility of lateral bond rupture and formation. The model is computationally efficient such that we reach sufficiently long simulation times to observe repeated catastrophe and rescue events at realistic tubulin concentrations and hydrolysis rates, which allows us to deduce catastrophe and rescue rates. The chemomechanical model also allows us to gain insight into microscopic features of the GTP-tubulin cap structure and microscopic structural features triggering microtubule catastrophes and rescues. Dilution simulations show qualitative agreement with experiments. We also explore the consequences of a possible feedback of mechanical forces onto the hydrolysis process and the GTP-tubulin cap structure.

Effect of plant tubulin kinetic diversification on microtubule lengths

Microtubules (MTs) are dynamic polymers vital for cellular physiology. Bulk tubulin polymerization is nucleation dependent, while individual filaments exhibit 'dynamic instability' driven by GTP hydrolysis rates. Although MTs assembled from well-studied animal brain tubulins have very comparable nucleation and GTP-hydrolysis rates, the kinetic rates of evolutionarily more distant species could diverge. Here we focus on a plant tubulin, the legume Vigna sp. (mung bean) to test the effect of kinetic diversification on MT polymerization. We activity purify tubulin from seedlings and find MT filaments are fewer and shorter than animal brain tubulin. We find mung tubulin polymerization kinetics is nucleation dependent with a high rate of GTP hydrolysis and a critical concentration lower than previously reported for tubulins. A computational model of the kinetics based on the relative influence of rates of nucleation and hydrolysis demonstrates increased rates of hydrolysis can affect MT filament numbers and their lengths, as compared to increasing nucleation rates. Our approach provides a framework to compare the effect of evolutionary diversification of MT nucleation and elongation.

Effect and optimization of NaOH combined with Fenton pretreatment conditions on enzymatic hydrolysis of poplar sawdust

In this study, the effects and mechanism of pretreatments of three types of chemical reagents combined with Fenton on poplar sawdust were studied and the optimization of enzymatic hydrolysis conditions was conducted using response surface methodology. The results showed that cellulase and hemicellulase had the best hydrolysis effect after NaOH-Fenton pretreatment, which were 63.73% and 29.29%, respectively. The optimal process of poplar substrate was to react in 1% NaOH at 100 ℃ for 1 h, then placed in the Fenton reaction system of 0.2 mmol Fe2+ and 25 mmol H2O2 for 7 h, and finally subjected to enzymatic hydrolysis for 72 h at 52 ℃, with a liquid-solid ratio of 33 and 15 μL/g of β-glycosidase. Under this condition, the enzymatic hydrolysis rates of cellulase and hemicellulase reached 86.65% and 43.9%, respectively. In conclusion, the combination of NaOH and Fenton pretreatment can effectively promote the enzymatic hydrolysis of poplar sawdust, which has great potential in the production of cellulosic ethanol.

Effect of integrated treatment on enhancing the enzymatic hydrolysis of cocksfoot grass and the structural characteristics of co-produced hemicelluloses

Background Cocksfoot grass (Dactylis glomerata L.) with high biomass yield and rich cellulose can be used to produce bioethanol as fuel additive. In view of this, ultrasonic and hydrothermal pretreatments followed by successive alkali extractions were assembled into an integrated biorefinery process applied on cocksfoot grass to improve its enzymatic hydrolysis. In this work, the effects of ultrasonic and hydrothermal pretreatments followed by sequential alkali extractions on the enzymatic hydrolysis of cocksfoot grass were investigated. In addition, since large amount of hemicelluloses were released during the hydrothermal pretreatment and alkali extraction process, the yields, structural characteristics and differentials of water- and alkali-soluble hemicellulosic fractions isolated from different treatments were also comparatively explored. Results The integrated treatment significantly removed amorphous hemicelluloses and lignin, resulting in increased crystallinity of the treated residues. A maximum saccharification rate of 95.1% was obtained from the cellulose-rich substrate after the integrated treatment. In addition, the considerable hemicelluloses (31.4% water-soluble hemicelluloses and 53.4% alkali-soluble hemicelluloses) were isolated during the integrated treatment. The released water-soluble hemicellulosic fractions were found to be more branched as compared with the alkali-soluble hemicellulosic fractions and all hemicellulosic fractions were mixed polysaccharides mainly composed of branched xylans and β-glucans. Conclusion The combination of ultrasonic and hydrothermal pretreatments followed by successive alkali extractions can dramatically increase the enzymatic saccharification rate of the substrates and produce considerable amounts of hemicelluloses. Detailed information about the enzymatic hydrolysis rates of the treated substrates and the structural characteristics of the co-produced hemicelluloses will help the synergistic utilization of cellulose and hemicellulose in cocksfoot grass.

pH-Stat Titration: A Rapid Assay for Enzymatic Degradability of Bio-Based Polymers

Bio-based polymers have been suggested as one possible opportunity to counteract the progressive accumulation of microplastics in the environments. The gradual substitution of conventional plastics by bio-based polymers bears a variety of novel materials. The application of bioplastics is determined by their stability and bio-degradability, respectively. With the increasing implementation of bio-based plastics, there is also a demand for rapid and non-elaborate methods to determine their bio-degradability. Here, we propose an improved pH Stat titration assay optimized for bio-based polymers under environmental conditions and controlled temperature. Exemplarily, suspensions of poly(lactic acid) (PLA) and poly(butylene succinate) (PBS) microparticles were incubated with proteolytic and lipolytic enzymes. The rate of hydrolysis, as determined by counter-titration with a diluted base (NaOH), was recorded for two hours. PLA was hydrolyzed by proteolytic enzymes but not by lipase. PBS, in contrast, showed higher hydrolysis rates with lipase than with proteases. The thermal profile of PLA hydrolysis by protease showed an exponential increase from 4 to 30 °C with a temperature quotient Q10 of 5.6. The activation energy was 110 kJ·mol−1. pH-Stat titration proved to be a rapid, sensitive, and reliable procedure supplementing established methods of determining the bio-degradability of polymers under environmental conditions.

Effect of Integrated Treatment on Enhancing the Enzymatic Hydrolysis of Cocksfoot Grass and the Structural Characteristics of Co-Produced Hemicelluloses

BackgroundCocksfoot grass (Dactylis glomerata L.) with high biomass yield and rich cellulose can be used to produce bioethanol as fuel additive. In view of this, ultrasonic and hydrothermal pretreatments followed by successive alkali extractions were assembled into an integrated biorefinery process applied on cocksfoot grass to improve its enzymatic hydrolysis. In this work, the effects of ultrasonic and hydrothermal pretreatments followed by sequential alkali extractions on the enzymatic hydrolysis of cocksfoot grass were investigated. Additionally, since large amount of hemicelluloses were released during the hydrothermal pretreatment and alkali extraction process, the yields, structural characteristics and differentials of water- and alkali-soluble hemicellulosic fractions isolated from different treatments were also comparatively explored.ResultsThe integrated treatment significantly removed amorphous hemicelluloses and lignin, resulting in increased crystallinity of the treated residues. A maximum saccharification rate of 95.1% was obtained from the cellulose-rich substrate after the integrated treatment. In addition, the considerable hemicelluloses (31.4% water-soluble hemicelluloses and 53.4% alkali-soluble hemicelluloses) were isolated during the integrated treatment. The released water-soluble hemicellulosic fractions were found to be more branched as compared with the alkali-soluble hemicellulosic fractions and all hemicellulosic fractions were mixed polysaccharides mainly composed of branched xylans and β-glucans.ConclusionThe combination of ultrasonic and hydrothermal pretreatments followed by successive alkali extractions can dramatically increase the enzymatic saccharification rate of the substrates and produce considerable amounts of hemicelluloses. Detailed information about the enzymatic hydrolysis rates of the treated substrates and the structural characteristics of the co-produced hemicelluloses will help the synergistic utilization of cellulose and hemicellulose in cocksfoot grass.

Paper Mill Sludge as a Source of Sugars for Use in the Production of Bioethanol and Isoprene

Paper mill sludge (PMS) solids are predominantly comprised of cellulosic fibers and fillers rejected during the pulping or paper making process. Most sludges are dewatered and discharged into landfills or land spread at a cost to the mill; creating large economic and environmental burdens. This lignocellulosic residual stream can be used as a source of sugars for microbial fermentation to renewable chemicals. The aim of this study was to determine the possibility of converting mill sludge to sugars and then fermentation to either isoprene or ethanol. Chemical analysis indicated that the cellulosic fiber composition between 28 to 68% and hemicellulose content ranged from 8.4 to 10.7%. Calcium carbonate concentration in the sludge ranged from 0.4 to 34%. Sludge samples were enzyme hydrolyzed to convert cellulose fibers to glucose, percent conversion ranged from 10.5 to 98%. Calcium carbonate present with the sludge resulted in low hydrolysis rates; washing of sludge with hydrochloric acid to neutralize the calcium carbonate, increased hydrolysis rates by 50 to 88%. The production of isoprene “very low” (190 to 470 nmol) because the isoprene yields were little. Using an industrial yeast strain for fermentation of the sludge sugars obtained from all sludge samples, the maximum conversion efficiency was achieved with productivity ranging from 0.18 to 1.64 g L−1 h−1. Our data demonstrates that PMS can be converted into sugars that can be fermented to renewable chemicals for industry.

Spatial patterns of biphasic ectoenzymatic kinetics related to biogeochemical properties in the Mediterranean Sea

Prokaryotic ectoenzymatic activity, abundance and heterotrophic production were determined in the Mediterranean Sea, within the epipelagic and the upper part of the mesopelagic layers. The Michaelis-Menten kinetics were assessed, using a range of low (0.025 to 1 µM) and high (0.025 to 50 µM) concentrations of fluorogenic substrates. Thus, Km and Vm parameters were determined for both low and high affinity systems for alkaline phosphatase (AP), aminopeptidase (LAP) and β-glucosidase (βGLU). Based on the constant derived from the high AP affinity system, in-situ hydrolysis rates of N-protein contributed of 48 % ± 30 % for the heterotrophic prokaryotic nitrogen demand within epipelagic waters and of 180 % ± 154 % within deeper layers. LAP hydrolysis rate was higher than bacterial N demand only within the deeper layer, and only based on the high affinity system. Although ectoenzymatic hydrolysis contribution to heterotrophic prokaryotic need was high in terms of N, but low in terms of C. Based on a 10 % bacterial growth efficiency, the cumulative hydrolysis rates of C-proteins and C-polysaccharides contributed to a small part of the heterotrophic prokaryotic carbon demand, on average 2.5 % ± 1.3 % in the epipelagic layers. This study notably points out the biases in current and past interpretation of the relative activities differences among the 3 tested enzymes, in regard to the choice of added concentrations of fluorogenic substrates. In particular, enzymatic ratios LAP/βGLU, as well as some trends with depth, were different considering activities resulting from the high or the low affinity system.