Reversibility of substrate adsorption for the cellulases Cel7A, Cel6A, and Cel7B from Hypocrea jecorina.

Adsorption of cellulases on the cellulose surface is an integral part of the catalytic mechanism, and a detailed description of the adsorption process is therefore required for a fundamental understanding of this industrially important class of enzymes. However, the mode of adsorption has proven intricate, and several key questions remain open. Perhaps most notably it is not clear whether the adsorbed enzyme is in dynamic equilibrium with the free population or irreversibly associated with no or slow dissociation. To address this, we have systematically investigated adsorption reversibility for two cellobiohydrolases (Cel7A and Cel6A) and one endoglucanase (Cel7B) on four types of pure cellulose substrates. Specifically, we monitored dilution-induced release of adsorbed enzyme in samples that had previously been brought to a steady state (constant concentration of free enzyme). In simple dilution experiments (without centrifugation), the results consistently showed full reversibility. In contrast to this, resuspension of enzyme-substrate pellets separated by centrifugation showed extensive irreversibility. We conclude that these enzymes are in a dynamic equilibrium between free and adsorbed states but suggest that changes in the physical properties of cellulose caused by compaction of the pellet hampers subsequent release of adsorbed enzyme. This latter effect may be pertinent to both previous controversies in the literature on adsorption reversibility and the development of enzyme recycling protocols in the biomass industry.

Sinomenine pretreatment alleviates hepatic ischemia/reperfusion injury through activating Nrf-2/HO-1 pathway.

INTRODUCTION: Ischemia-reperfusion (IR) injury is induced by an interrupted blood flow and succeeding blood restoration, which is common in the operation of liver transplantation. Serious IR injury is a major reason leading to transplant failure. Hepatic IR is featured by excessive inflammatory response, oxidative stress, and apoptosis. Sinomenine (SIN) is derived from the herb Sinomeniumacutum and shows properties of anti-inflammation and antiapoptosis in multiple IR-induced organ injuries. However, the effect of SIN in hepatic IR has not been investigated. METHODS: This study aims to investigate impacts of SIN on hepatic IR and the involved signaling pathway. An in vivo rat model of syngeneic orthotopic liver transplantation was constructed to induce the hepatic IR injury. RESULTS: Results showed that SIN pretreatment provided a significant prevention against IR-induced hepatic injury as manifested by the downregulated activities of serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, the alleviatedoxidative stress as shown by increased activities of serum superoxide dismutase and glutathione peroxidase, and decreased serum level of malondialdehyde, the suppressed inflammatory responses as shown by downregulated serum tumor necrosis factor-α, interleukin (IL)-6, IL-8 levels, and upregulated IL-10 level, as well as attenuated apoptosis as shown by decreased protein expression of cleaved caspase-3 and -9. In line with these results, SIN pretreatment also alleviatedthe hepatic histopathological changes in IR rats and induced Nrf-2/HO-1 activation. The use of brusatol, a selective inhibitor for Nrf-2, effectively reversed SIN-induced above effects. CONCLUSIONS: Altogether, our results demonstrate that SIN might be a useful therapeutic drug for preventing hepatic IR-induced injury during clinical liver transplantation.

Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays.

In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.

Ischemic preconditioning ameliorates intestinal injury induced by ischemia-reperfusion in rats.

AIM: To evaluate preventative effects of ischemic preconditioning (IP) in a rat model of intestinal injury induced by ischemia-reperfusion (IR). METHODS: Male Sprague-Dawley rats (250-300 g) were fasted for 24 h with free access to water prior to the operation. Eighteen rats were randomly divided into three experimental groups: S group (n = 6), rats were subjected to isolation of the superior mesenteric artery (SMA) for 40 min, then the abdomen was closed; IR group (n = 6), rats were subjected to clamping the SMA 40 min, and the abdomen was closed followed by a 4-h reperfusion; IP group (n = 6) rats underwent three cycles of 5 min ischemia and 5 min reperfusion, then clamping of the SMA for 40 min, then the abdomen was closed and a 4-h reperfusion followed. All animals were euthanized by barbiturate overdose (150 mg/kg pentobarbital sodium, i.v.) for tissue collection, and the SMA was isolated via median abdominal incision. Intestinal histologic injury was observed. Malondialdehyde (MDA), myeloperoxidase (MPO) and tumor necrosis factor (TNF)-α concentrations in intestinal tissue were measured. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression, as well as nuclear factor (NF)-κB activity and expression in intestinal tissue were also determined. RESULTS: Compared with the IR group, IP reduced IR-induced histologic injury of the intestine in rats (2.00 ± 0.71 vs 3.60 ± 0.84, P < 0.05). IP significantly inhibited the increase in MDA content (5.6 ± 0.15 µmol/L vs 6.84 ± 0.18 µmol/L, P < 0.01), MPO activity (0.13 ± 0.01 U/L vs 0.24 ± 0.01 U/L, P < 0.01), and TNF-α levels (7.79 ± 2.35 pg/mL vs 10.87 ± 2.48 pg/mL, P < 0.05) in the intestinal tissue of rats. IP also markedly ameliorated the increase in ICAM-1 (204.67 ± 53.27 vs 353.33 ± 45.19, P < 0.05) and VCAM-1 (256.67 ± 58.59 vs 377.33 ± 41.42, P < 0.05) protein expression in the intestinal tissues. Additionally, IP remarkably decreased NF-κB activity (0.48 ± 0.16 vs 0.76 ± 0.22, P < 0.05) and protein expression (320.23 ± 38.16 vs 520.76 ± 40.53, P < 0.01) in rat intestinal tissue. CONCLUSION: IP may protect against IR-induced intestinal injury by attenuation of the neutrophil-endothelial adhesion cascade via reducing ICAM-1 and VCAM-1 expression and TNF-α-induced NF-κB signaling pathway activity.