A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.     

Evaluation of dynamic modulus measurement for C/C-SiC composites at different temperatures

The determination of elastic properties at application temperature is fundamental for the design of fibre reinforced ceramic composite components. An attractive method to characterize the flexural modulus at room and high temperature under specific atmosphere is the nondestructive Resonant Frequency Damping Analysis (RFDA). The objective of this paper was to evaluate and validate the modulus measurement via RFDA for orthotropic C/C-SiC composites at the application temperature. At room temperature flexural moduli of C/C-SiC with 0/90° reinforcement were measured under quasi-static 4-point bending loads and compared with dynamic moduli measured via RFDA longitudinally to fibre direction. The dynamic modulus of C/C-SiC was then measured via RFDA up to 1250°C under flowing inert gas and showed an increase with temperature which fitted with literature values. The measured fundamental frequencies were finally compared to those resulting from numerical modal analyses. Dynamic and quasi-static flexural moduli are comparable and the numerical analyses proved that bending modes are correctly modeled by means of dynamic modulus measured via RFDA. The nondestructive RFDA as well as the numerical modeling approach are suitable for evaluation of C/C-SiC and may be transferred to other fibre reinforced ceramic composite materials.     

Prediction of Glass Forming Ability Through High Throughput Calculation

In this work, high throughput calculation (HTC) is used to identify composition regions with good glass forming ability (GFA) in the Al-Cu-Zr, Cu-Ni-Zr, Cu-Ti-Zr, Cu-Ni-Ti-Zr, Al-Cu-Ni-Ti-Zr, Mg-Ca-Cu, and Mg-Ca-Ni systems. The predicted composition regions agree well with those observed by experiments in the ternary systems, while less satisfactory agreement is found in the Cu-Ni-Ti-Zr quaternary and the Al-Cu-Ni-Ti-Zr quinary systems. The possible causes that lead to the deviation in the higher order systems are discussed. The major advantage of the HTC method used in this study is that it is simple and can be easily applied to multicomponent systems. The color maps of liquidus temperature and solidification range obtained by HTC provide a valuable guidance to the experimentalists, thus they can focus on the composition regions with high potential of forming bulk metallic glasses, avoid unnecessary trial-and-error test and save time and cost. This approach can also be combined with other criteria to filter compositions with better GFA.     

AI and robotics in the European restaurant sector: Assessing potentials for process innovation in a high-contact service industry

Electronic Markets - The restaurant technology market is rapidly evolving and is transforming the restaurant business as a significant sector of tourism and hospitality. Enabled by artificial...     

Electric transport properties of rare earth doped YbxCa1-xMnO3 ceramics (part I: Optimization of ceramic processing)

A comprehensive study was performed in order to find the effect of different calcination and sintering conditions on the physical properties of calcium manganite ceramics in dependence of temperature T and partial pressure of oxygen p(O₂). The eventual formation of oxygen vacancies during sintering was investigated and the results were confirmed by monitoring the release of oxygen using a ZrO₂ oxygen sensor. The phase transition behavior was studied using thermogravimetric analysis (TGA) in a wide range of p(O₂) ≈ 10^?1 MPa down to 10^-19 MPa at high temperatures accompanied by dilatometry- and XRD-measurements. Furthermore, the present study reveals for the first time a way for reducing and preventing crack formation that may occur during sintering. The present systematic research provides essential fundamental information before performing electrical measurements necessary in order to understand important factors about charge carriers and electrical transport mechanism.     

From competitive advantage to social welfare

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A Novel Technology for Natural Gas Conversion by Means of Integrated Oxidative Coupling and Dry Reforming of Methane

A novel process concept for the oxidative coupling of methane followed by the oligomerization to liquids has been developed within the frame of the EU integrated project OCMOL. This technology is based on process intensification principles via cutting‐edge structured microreactor technology. It is also a fully integrated industrial process through the re‐use and the recycling of by‐products, in particular CO₂, at every process stage. The focus of this contribution is on the reaction engineering aspects of the core steps, i.e., catalysts, kinetics and reactor design for the methane coupling and reforming.     

21世纪城市设计和开放空间规划中的浪漫主义精神

人类文明发展史上每一次价值观的改变,都会引起新的城市设计和开放空间规划思想和手法的产生.1 8世纪浪漫主义思想的产生,是文化思想发展史上一次重要的转折,它对同时期的景观设计领域,也产生了重要的影响.以往的园林,代表的是内部实体与外在形式创造性的统一,而21世纪的景观设计语言,必须为人与人之间或人与自然之间的相互关系,提供现实主义层面的反映.浪漫主义,是对应于21世纪人类价值观的设计思想和手法的一个探索性回答.     

共晶盐蓄冷球凝固过程的数值模拟研究

对共晶盐蓄冷球建立了物理和数学模型,利用FLUENT软件对其凝固过程进行了数值模拟研究,得到了温度场分布和相界面移动规律,分析了载冷剂温度和球径对凝固时间的影响。载冷剂温度越低,共晶盐蓄冷球的凝固时间越短,相界面移动速度越快;凝固时间与球径成正比。