Expression of endo-1, 4-beta-xylanase from Trichoderma reesei in Pichia pastoris and functional characterization of the produced enzyme

<p>Abstract</p> <p>Background</p> <p>In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast <it>Pichia pastoris </it>can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes.</p> <p>Results</p> <p>The Xyn2 gene, which encodes the main <it>Trichoderma reesei </it>Rut C-30 endo-β-1, 4-xylanase was cloned into the pPICZαA vector and expressed in <it>Pichia pastoris</it>. The selected <it>P. pastoris </it>strains produced as 4,350 nkat/ml β-xylanase under the control of the methanol inducible alcohol oxidase 1 (<it>AOX1</it>) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60°C and it was active over a broad range of pH (3.0–8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50°C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent <it>K</it>_mand k_catvalues were 2.1 mg/ml and 219.2 S^-1, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity.</p> <p>Conclusion</p> <p>The <it>P. pastoris </it>expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, <it>P pastoris </it>is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.</p

Impact of dietary manganese on intestinal barrier and inflammatory response in broilers challenged with Salmonella Typhimurium

Growing concern for public health and food safety has prompted a special interest in developing nutritional strategies for removing waterborne and foodborne pathogens, including Salmonella. Strong links between manganese (Mn) and intestinal barrier or immune function hint that dietary Mn supplementation is likely to be a promising approach to limit the loads of pathogens in broilers. Here, we provide evidence that Salmonella Typhimurium (S. Typhimurium, 4 × 108 CFUs) challenge-induced intestinal injury along with systemic Mn redistribution in broilers. Further examining of the effect of dietary Mn treatments (a basal diet plus additional 0, 40, or 100 mg Mn/kg for corresponding to Mn-deficient, control, or Mn-surfeit diet, respectively) on intestinal barrier and inflammation status of broilers infected with S. Typhimurium revealed that birds fed the control and Mn-surfeit diets exhibited improved intestinal tight junctions and microbiota composition. Even without Salmonella infection, dietary Mn deficiency alone increased intestinal permeability by impairing intestinal tight junctions. In addition, when fed the control and Mn-surfeit diets, birds showed decreased Salmonella burdens in cecal content and spleen, with a concomitant increase in inflammatory cytokine levels in spleen. Furthermore, the dietary Mn-supplementation-mediated induction of cytokine production was probably associated with the nuclear factor kappa-B (NF-κB)/hydrogen peroxide (H2O2) pathway, as judged by the enhanced manganese superoxide dismutase activity and the increased H2O2 level in mitochondria, together with the increased mRNA level of NF-κB in spleen. Ingenuity-pathway analysis indicated that acute-phase response pathways, T helper type 1 pathway, and dendritic cell maturation were significantly activated by the dietary Mn supplementation. Our data suggest that dietary Mn supplementation could enhance intestinal barrier and splenic inflammatory response to fight against Salmonella infection in broilers

Cigarette Flavouring Regulation by Using Aroma-producing Microorganism Isolated from Maotai Daqu

The selected Moutai aromatic microorganisms and their metabolites were applied into the fermentation of tobacco leaves in order to improve the tobacco quality. The results showed that a variety of aromatic substances in Moutai, as well as the typical flavor substances commonly used in cigarettes, were detected in the fermented tobacco leaf extract. In view of the GC-MS results as well as the sensory smoking evaluation of tobacco leaf extracts under designed experimental conditions, the optimal parameters of stable single-strain fermentation process was at 40 ℃ for 10-15 days. The results of specific effects of different fermentation conditions on the content of aroma substances in different parts of tobacco leaves after fermentation, as well as the subsequent sensory evaluation, provided basic data for the improvement of tobacco fermentation and aroma flavoring technology, which was conducive to the development of new cigarettes

Cobalt-Dinitrogen Complexes with Weakened N-N Bonds

Article discussing research on cobalt-dinitrogen complexes with weakened N-N bonds

Mountain Landscape Preferences of Millennials Based on Social Media Data: A Case Study on Western Sichuan

Mountain area is one of the most important modern tourist attractions, and unique mountain landscapes are highly appealing to millennials. Millennials post their travel photos and comments on social media, and these media messages can positively influence other millennials’ travel motivations. To fully understand the attraction of mountain tourist destinations to millennials, this study analyzed their landscape preferences using images posted on social media. As a case study, we analyzed the landscape resources in Western Sichuan Plateau Mountain Areas (WSPMA). We found that differences in genders, modes of transportation, and travel patterns of the millennials influenced their preferences for mountain landscapes. Our results broaden the current knowledge on mountain tourism from the perspective of millennials through social media data. Moreover, studying the landscape resources in WSPMA can facilitate the analysis of regional advantages. This will ultimately enhance tourism publicity and integrate various resources for tourism management and planning in more targeted and attractive ways

Mountain Landscape Preferences of Millennials Based on Social Media Data: A Case Study on Western Sichuan

Mountain area is one of the most important modern tourist attractions, and unique mountain landscapes are highly appealing to millennials. Millennials post their travel photos and comments on social media, and these media messages can positively influence other millennials’ travel motivations. To fully understand the attraction of mountain tourist destinations to millennials, this study analyzed their landscape preferences using images posted on social media. As a case study, we analyzed the landscape resources in Western Sichuan Plateau Mountain Areas (WSPMA). We found that differences in genders, modes of transportation, and travel patterns of the millennials influenced their preferences for mountain landscapes. Our results broaden the current knowledge on mountain tourism from the perspective of millennials through social media data. Moreover, studying the landscape resources in WSPMA can facilitate the analysis of regional advantages. This will ultimately enhance tourism publicity and integrate various resources for tourism management and planning in more targeted and attractive ways

An Ultrasonic Motor Using a Carbon-Fiber-Reinforced/Poly-Phenylene-Sulfide-Based Vibrator with Bending/Longitudinal Modes

In this study, a linear ultrasonic motor (USM) with carbon-fiber-reinforced/poly-phenylene-sulfide (PPS/CF) was developed and the feasibility of using PPS/CF to achieve a lightweight USM was tested. Here, anisotropic Young&rsquo;s moduli of PPS/CF possibly enhance the driving force when the slider&rsquo;s moving direction is orthogonal to the carbon-fibers&rsquo; filling direction. Further, PPS/CF&rsquo;s low density may help avoid excessive enhancement in weight. Initially, we measured anisotropic Young&rsquo;s moduli of PPS/CF, and determined the vibration modes, configuration, and dimensions of the PPS/CF vibrating body through finite element analysis. Subsequently, we fabricated a 45.7-mm-long 30-mm-diameter vibrator to form a linear motor. Finally, we evaluated the load characteristics of the PPS/CF-based motor and made comparisons with isotropic-material-based USMs. At 30.2 kHz frequency, the PPS/CF-based vibrator worked in the 2nd bending and 2nd longitudinal modes as predicted. The PPS/CF-based motor yielded the maximal thrust, no-load speed, and maximal output power of 392 mN, 1103 mm/s, and 62 mW, respectively. Moreover, the thrust force density and power density reached 20.3 N/kg and 3.2 W/kg, respectively, which were relatively high compared to those of the PPS- and aluminum-based USMs with the same vibration modes and similar structures. This preliminary study implies PPS/CF&rsquo;s feasibility for achieving lightweight USMs, and provides a candidate material for designing micro/meso USMs

Synthesis and reactivity of low-coordinate cobalt complexes and iron(I) alkyl complexes supported by β-diketiminate ligand

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2009.The strategy of using exposed metal centers to efficiently perform chemical transformations is shared by enzymes, metal surfaces and synthetic homogeneous catalysts. Three-coordinate complexes of the transition metals are rare, because it is difficult with only three ligand donors to reach preferred configurations with 16 or 18 electrons in the metal bonding and nonbonding orbitals. By using extremely bulky β-diketiminate ligands, chemists have been able to stabilize three-coordinate iron centers. However, there has been much less attention to the area of diketiminate cobalt chemistry, which promised to be equally exciting. This thesis mainly describes the synthesis and reactivity of low-coordinate cobalt dinitrogen, hydride, fluoride complexes supported by β-diketiminate ligand LtBu. The final chapter discusses the synthesis of unusual iron(I) alkyl complexes of LtBu and their catalytic suitability for aryl-alkyl cross coupling reactions. In Chapter II, three low-coordinate cobalt dinitrogen complexes were synthesized, structurally characterized and compared to their iron analogues. Despite the identical ligand environment, there are significant differences between the cores of the two LtBuMNNMLtBu (M = Fe, Co) compounds: the Co-N bonds are longer than the Fe-N bonds, and the N-N bond is significantly shorter in the Co complex than in the Fe complex. These data suggest that π-backbonding is not as important in the Co complex. Because of the exact correspondence of ligands, it is possible to unambiguously conclude that LtBuCo is poorer at weakening N2 than LtBuFe at this oxidation level. In contrast to the differences between formally cobalt(I) and iron(I) species above, the formally cobalt(0) compounds have metrical parameters that are similar to their Fe analogues, with N-N distances of 1.21-1.22 Å. These are the longest N-N distances observed in any cobalt dinitrogen complex. In Chapter III, two rare low-coordinate cobalt hydride complexes were synthesized from LCoCl and KHBEt3. These isolable cobalt hydride complexes are highly reactive with dinitrogen, to give cobalt dinitrogen complexes. Such a transformation might be an important method of binding N2 to a transition metal complex in a catalytic system because the coordination and activation of the substrate N2 have been demonstrated to occur in a facile manner. It is also notable that there is a connection to the biological fixation of dinitrogen. It is known that the nitrogenase enzyme produces at least 1 equiv of H2 gas in the conversion of N2 to NH3. Although it is still not clear whether this arises from a necessary step in this reaction or from a side-reaction, there is a similarity to the formation of cobalt hydride complexes because H2 is also eliminated in the latter reaction. Chapter IV describes the reaction of LtBuCoCH3 with Me3SnF to give a rare example of partially fluorinated cobalt tin fluoride complex [LtBuCo(μ-F)2]2SnMe2·2Et2O and its transformation to a four-coordinate cobalt fluoride pyridine adduct LtBuCoF(py). The reason for their lack of catalytic hydrodefluorination activity was examined. Chapter V describes the synthesis of two unusual iron(I) alkyl complexes of LtBu, [KLtBuFeCH3]2 and KLtBuFePh·2Et2O. Their catalytic suitability for aryl-alkyl cross coupling reactions are discussed

Pathological Impairment, Cell Cycle Arrest and Apoptosis of Thymus and Bursa of Fabricius Induced by Aflatoxin-Contaminated Corn in Broilers

This study aimed to evaluate the comparative effects of aflatoxin-contaminated corn on the thymus and bursa of Fabricius (BF) in chickens by detecting histopathological lesions, cell cycle phase distribution and apoptosis. A total of 900 COBB500 male broilers were randomly allocated into five groups. The experiment lasted for six weeks and the five dietary treatments consisted of uncontaminated corn (control), 25% contaminated corn, 50% contaminated corn, 75% contaminated corn and 100% contaminated corn groups. The gross changes showed the decreased size of the thymus and BF, as well as the pale color of the BF in the broilers after aflatoxin contaminated diet exposure. There were more nuclear debris in the thymus and BF of birds in the 50%, 75%, and 100% contaminated corn groups, but the pathological impairments of the BF were more obvious than those of the thymus, which showed as more obvious lymphocyte depletion and the proliferation of reticulocytes and fibroblasts. At 21 days of age, the percentage of thymocytes and BF cells in the G2M phase was increased in a dose-dependent manner in the four AFB-contaminated corn groups. However, at 42 days of age, dietary AFB1 induced cell cycle perturbation at the G0G1 phase in thymocytes, but at the G2M phase in BF cells. The increased percentage of apoptotic cells in the thymus and BF were similarly observed in the AFB groups. According to these results, the severity of histopathological lesions may be correlated with the different sensitivity of the two central immune organs when exposed to AFB; different arrested cell cycle phases suggest that different mechanisms may be involved in the lesions of the thymus and BF, which need to be further researched