纳米纤维素催化氧化玉米淀粉基复合膜的制备

以酸水解制备的纳米纤维素作为催化剂制备次氯酸钠氧化淀粉,并将制备的氧化淀粉与不同添加量聚乙烯醇(PVA)/甘油(GL)共混,采用流延成膜法制备复合膜。通过红外光谱分析了复合膜的官能团变化,扫描电镜进行表面形貌分析,热重分析和差示扫描量热法表征复合膜的热稳定性。同时用静态接触角测量仪、近红外紫外分光光度计分别研究了复合膜的疏水性和透明度,用张力测试机检测了复合膜的力学性能。研究发现,复合膜中PVA/GL的添加量对复合膜的透明度、热稳定性、阻水性和力学性能均产生影响,在PVA添加量为15%(GL添加量9.375%)时,复合膜的透明度为0.67,接触角达最高值100°,拉伸应力为12.89 MPa,复合膜的性能较优。

Seepage-Stress-Damage Coupled Model of Coal Under Geo-Stress Influence

In the seepage-stress-damage coupled process,the mechanical properties and seepage characteristics of coal are distinctly different between pre-peak stage and post-peak stage.This difference is mainly caused by damage of coal.Therefore,in the process of seepage and stress analysis of coal under the influence of excavation or mining,we need to consider the weakening of mechanical properties and the development of fractures of damaged coal.Based on this understanding,this paper analyzes the influence of damage on mechanics and seepage behavior of coal.A coupled model is established to analyze the seepage-stress-damage coupled process of coal.This model implemented into COMSOL and MATLAB software to realize the numerical solving.Two examples are adopted to verify the correctness of the model and some useful conclusions are obtained.The numerical model establishes the relationship between microcosmic damage evolution and macroscopical fracture and simulates the whole process of coal from microcosmic damage to macroscopical fracture,and the dynamic simulation of fluid flow in this process.It provides a numerical tool for further research on the seepage-stress-damage analysis.

Development of surface displaying system for heterologous protein expression in Candida tropicalis

The purpose of this study was to assess the potential application of cell surface display in Candida tropicalis.Surface display gene cassettes were constructed using five anchoring proteins from Saccharomyces cerevisiae,three of which[(suppression of exponential defect protein,SED1),(cell wall protein 2,CWP2)and(delayed anaerobic protein 4,DAN4)]were reported to show higher activity of heterologous proteins thanα-agglutinin(AGα1).The performance of yeast-enhanced green fluorescent protein(yeGFP)was evaluated using laser scanning confocal microscopy and flow cytometry.The results showed that the three anchoring regions(SED1,CWP2 and AGα1)successfully displayed yeGFP on the cell wall.To investigate the effect of the three anchoring proteins on the surface display of Rhizopus oryzaeα-amylase(ROA1)and Aspergillus aculeatusβ-glucosidase(BGL1)in C.tropicalis,we constructed surface display gene cassettes for ROA1 and BGL1,respectively.The strains containing the anchoring proteins SED1 and CWP2 showed higher activity of ROA1 and BGL1 than the strains containing the anchoring protein AGα1.The highest ROA1 and BGL1 activities of strains with SED1 were 6.37 U/g CDW and 7.93 U/g CDW,respectively,which were sixfold and eightfold higher than those of strain with AGα1.In addition,we also optimized signal peptides.The results indicated that signal peptides have an impact on enzyme activity.

肠膜明串珠菌蔗糖磷酸化酶的酶学表征及在催化合成α-熊果苷中的应用

将来源于肠膜明串珠菌Leuconostoc mesenteroides ATCC 12291的蔗糖磷酸化酶(Sucrose phosphorylase,SPase)基因进行密码子优化后将其插入到pET-28a中构建表达载体pET-28a-spase,诱导大肠杆菌Escherichia coli BL21(DE3)/pET-28a-spase表达制得SPase粗酶液,将重组SPase纯化后进行酶学表征。结果表明,重组SPase的比酶活为213.98 U/mg,纯化倍数为1.47倍,酶活回收率达87.80%。该酶最适温度为45℃,最适pH为6.5,该酶对蔗糖的Km为128.8 mmol/L,Vmax为2.167μmol/(mL·min),kcat为39237.86 min^-1,利用重组SPase催化氢醌合成α-熊果苷,最优条件为:氢醌添加量为40 g/L,蔗糖/氢醌的摩尔比为5:1,重组SPase 250 U/mL。在25 mmol/L的吗啉乙磺酸(MES)缓冲液(pH 7.0)中,反应温度30℃,避光反应24 h后终止反应,再用500 U/mL的糖化酶40℃处理2.5 h。α-熊果苷产量达98 g/L,氢醌的转化率接近99%。综上所述,文中克隆并研究了重组SPase,并建立了其在α-熊果苷生产中的应用。

代谢工程改造大肠杆菌合成羟基酪醇

羟基酪醇是重要精细化学品,作为天然抗氧化剂被广泛应用于食品、医药领域。利用合成生物学技术生产羟基酪醇具有重要意义。本文克隆并功能鉴定了来源于大肠杆菌EscherichiacoliBL21的羟化酶编码基因HpaBC,结果表明该酶的两个亚基均能成功表达并能催化酪醇生成羟基酪醇。通过CRISPR-Cas9技术将由tac启动子调控的HpaBC基因表达盒整合到前期构建的酪醇高产菌株YMG5A*R基因组中,同时删除副产物乙酸的合成途径,获得大肠杆菌代谢工程菌株YMGRD1H1。摇瓶发酵实验结果表明,重组菌株能够直接利用葡萄糖生产羟基酪醇,产量达到1.81g/L,同时发现几乎没有副产物积累。5L发酵罐规模的流加补料发酵实验表明,羟基酪醇最高产量达到2.95 g/L,是目前文献报道以葡萄糖从头合成羟基酪醇的最高水平。通过系统改造大肠杆菌,实现了羟基酪醇的大量合成,为进一步构建具有工业应用潜力的羟基酪醇细胞工厂奠定了基础,也为拓展芳香族化合物的微生物制造路线提供了有益的参考。

红球菌低分子量型腈水合酶的异源激活及激活子的结构域功能

腈水合酶激活子具有亚基自身交换伴随子或者金属离子伴随子的功能,能够辅助腈水合酶摄取金属离子,对于腈水合酶的活性表达必不可少。与腈水合酶自身相比,激活子的序列保守性低,研究其激活作用的特点,探索其结构与功能之间的关系,对于理解腈水合酶的成熟机制具有重要意义。将红球菌Rhodococcus rhodochrous J1低分子量型腈水合酶L-NHase分别与4种异源激活子组合共表达,测定异源激活子对L-NHase的激活作用,进一步对激活子进行序列分析和结构模拟,并研究关键结构域的功能。结果表明,4种异源激活子均能激活L-NHase,但激活后L-NHase的比酶活存在差异,激活子A对L-NHase的激活程度最高,激活后的L-NHase比酶活为出发酶的97.79%;激活子G对L-NHase的激活程度最低,激活后的L-NHase比酶活为出发酶的23.94%。激活子E和激活子G具有保守结构域TIGR03889,缺失其中部分序列会使两者的激活作用基本丧失。将激活子G的N端序列替换为激活子E的N端序列,并将激活子E的C端序列添加至激活子G的C端,能够使L-NHase的比酶活提高178.40%。激活子的激活作用具有普遍性和特异性,其保守结构域对激活作用至关重要,同时N端结构域和C端结构域也对激活作用产生重要影响。

Gas template-assisted spray pyrolysis： A facile strategy to produce porous hollow Co3O4 with tunable porosity for high-performance lithium-ion battery anode materials

Porous hollow Co3O4 microspheres have been synthesized from a mixed cobalt nitrate and urea solution through spray pyrolysis followed by calcination at 600 ℃ in air. This porous hollow Co3O4 is assembled by nanoparticles and exhibits variable porosity depending on the amount of gas in the system. In pyrolysis process, urea continuously decomposes into gaseous components, which act as a template to control the porous structure. The amount of gas escaping from precursor droplets can directly influence the porosity of the microspheres and the size of the nanoparticles controlled by the ratio of urea to cobalt nitrate. Electrochemical measurements show that the performance of the porous hollow Co3O4 microspheres is related to the porosity and size of the nanopartides. The sample with optimal porosity delivers a high first charge capacity of 1,417.9 mAh·g^-1 at 0.2C （1C = 890 mA·g^-1）, and superior charge cycle performance of 1,012.7 mAh.g-1 after 100 cycles. In addition, the optimized material displays satisfactory rate performance of 1,012.4 mAh.g-1 at 1C after 50 cycles and 881.3 mAh·g^-1 at 2C after 300 cycles. Superior charge/discharge capacity, excellent rate performance and high yield achieved in this study is promising for the development of high-performance Co3O4 anode materials for lithium-ion batteries.

Enhancing thermostability and activity of sucrose phosphorylase for high-level production of 2-O-α-d-glucosylglycerol

Sucrose phosphorylase(SPase)can transfer the glucosyl group of sucrose to different compounds and has been widely used in industry.To overcome the low thermostability of the sucrose phosphorylase from Leuconostoc mesenteroides ATCC 12291(LmSP),a method named PROSS was used to construct mutants with increased thermostability.All variants were screened by measuring their residual activities after heating at 50℃.Then,a single point mutant and a combined mutant with improved thermostability and activity were obtained.The half-lives of mutants at 50℃ were approximately twice as high as those of the wild type.In addition,2-O-α-d-glucosylglycerol(αGG)was synthesized by the wild type and the two improved variants,and the reaction conditions were optimized.Under the conditions of glycerol concentration of 3.2 mol/L,sucrose concentration of 1.2 mol/L,and enzyme concentration of 40 U/mL at 37℃ for 60 h,the yield ofαGG reached the maximum,and the sucrose conversion rate of the wild type,the mutant V23L and the combined mutant V23L/S424R were 62.3%,70.7%and 76.3%,respectively.In this study,SPase mutants with higher activity and stability were obtained,and achieved high-level production ofαGG.

A CRISPR-Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola

The traditional homologous recombination(HR)gene-editing method faces problems such as low editing efficiency and absence of marker genes.CRISPR-Cas9-editing efficiency is high and has been widely used in bacteria and yeast.In com-parison with CRISPR-Cas9,CRISPR-Cas12a has many outstanding advantages.Here,we report an Acidaminococcus sp.BV3L6(As)Cas12a-based genome-editing method used for Starmerella bombicola.To demonstrate the high efficiency of the CCMGE system,we verified a counter-selectable marker in S.bombicola,orotidine 5’-phosphate decarboxylase(100%for URA3).We also tested the common gene UDP-glucosyltransferase(100%for UGTA)using a 300 bp donor containing hygromycin expression cassette.This toolkit was further extended to simultaneously edit two genes(18%for UGTA and leu)and three genes(13.8%for UGTA,leu and URA3).The system greatly reduces the screening time for such multi-site editing.Based on the CCMGE system,the PHA(polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase(PHAC).The PHA content and DCW reached 11.8%and 30.1 g/L,respectively.The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.

Analysis of female drivers’ ECG characteristics within the context of connected vehicles

Purpose–This study aims to analyze the differences of electrocardiograph(ECG)characteristics for female drivers in calm and anxious states during driving.Design/methodology/approach–The authors used various materials(e.g.visual materials,auditory materials and olfactory materials)to induce drivers’mood states(calm and anxious),and then conducted the real driving experiments and driving simulations to collect driver’s ECG signal dynamic data.Physiological changes in ECG during the stimulus process were recorded using PSYLAB software.The paired T-test analysis was conducted to determine if there is a significant difference in driver’s ECG characteristics between calm and anxious states during driving.Findings–The results show significant differences in the characteristic parameters of female driver’s ECG signals,including(average heart rate),(atrioventricular interval),(percentage of NN intervals>50ms),(R wave average peak),(Root mean square of successive),(Q wave average peak)and(S wave average peak),in time domain,frequency domain and waveform in emotional states of calmness and anxiety.Practical implications–Findings of this work show that ECG can be used to identify driver’s anxious and calm states during driving.It can be used for the development of personalized driver assistance system and driver warning system.Originality/value–Only a few attempts have been made on the influence of human emotions on physiological signals in the transportationfield.Hence,there is a need for transport scholars to begin to identify driver’s ECG characteristics under different emotional states.This study will analyze the differences of ECG characteristics for female drivers in calm and anxious states during driving to provide a theoretical basis for developing the intelligent and connected vehicles.