Engineering <Emphasis Type="Italic">Pichia pastoris</Emphasis> for Efficient Production of a Novel Bifunctional <Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis> Invertebrate-Type Lysozyme

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of >?99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na⁺. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.     

Preparation of Porous Polylactide Microspheres and Their Application in Tissue Engineering

In this study,porous polylactide (PLA) microspheres with different structures were prepared through the multiple emulsion solvent evaporation method.By changing organic solvents (ethyl acetate and chloroform) and adding effervescent salt NH4HCO3 in the inner water phase,microspheres with porous capsular,matrix,microcapsular and multivesicular structures were prepared.The protein encapsulation and release,and the cell growth behavior of porous microspheres were further explored.Under the same inner water phase,microspheres prepared with chloroform had higher protein encapsulation efficiency and less protein release rate as compared with those prepared with ethyl acetate.Cell experiments showed that the relatively rough surface of microspheres prepared with chloroform was more favorable for the cell growth in comparison with the smooth surface of microspheres prepared with ethyl acetate.This study shows a simple and effective method to control the protein release and cell growth behaviors of polymer microspheres by tuning their porous structure.     

Au促进Pt/WO3催化甘油氢解制1,3-丙二醇

近年来, 以生物质为基础的生物柴油得到了迅速发展, 导致了粗甘油过剩. 通过甘油选择性氢解制1,3-丙二醇, 进而被用于合成高价值的聚对苯二甲酸丙二醇酯被认为是最具工业应用潜力的反应之一, 对于提高生物柴油的利用效率有着极其重要的意义. 但由于反应空间位阻和热力学上的限制给甘油氢解制1,3-丙二醇带来了很大的挑战, 因此需要设计高活性的金属-酸双功能催化剂以降低甘油第二个C-O键断裂的活化能和减少其他副反应的发生. 目前Pt-W和Ir-Re双功能催化剂可高选择性制得1,3-丙二醇, 但仍需较严苛的反应条件实现对氢气的活化和解离. 本课题组曾将准单原子/单原子Pt高度分散于具有大量氧空位和酸位点的WOx载体上, 十分有利于甘油选择性氢解制1,3-丙二醇反应; 在Au-Pt/WOx催化剂中添加Au可促进B酸产生, 进而提高了甘油转化率和1,3-丙二醇的选择性.为了进一步研究Au对Pt/WOx催化剂结构和催化性能的影响, 本文利用CTAB辅助吸附法制备了Au/WO3, 再浸渍Pt制得Pt/Au/WO3双金属催化剂. 在甘油选择性氢解制1,3-丙二醇反应中, 所制催化剂表现出比Au-Pt/WOx更好的催化活性, 1,3-丙二醇时空收率为0.078 g1,3-PDO/(gcat·h), 是后者的1.95倍. 值得一提的是, Au-Pt/WOx催化剂在低压时活性较高, 而Pt/Au/WO3催化剂活性则在压力的升高而提高; 另外反应温度的升高导致副产物正丙醇的选择性上升, 1,3-丙二醇的选择性降低. 因此, 适宜的反应条件为155℃和5 MPa. 与Pt/WO3和Pt/WOx相比, Pt/Au/WO3表现出了更优异的催化性能, 其1,3-丙二醇的时空收率是Pt/WO3的2.36倍和Pt/WOx的4倍.为了探究Au的掺入对Pt/WOx催化剂性能的影响, 通过XRD, TEM, H2-TPR和XPS等技术对催化剂进行了深入表征. 结果表明, 与Pt/WO3相比, Pt/Au/WO3-600催化剂的XRD衍射峰向小角度偏移, 其原因是Au3+离子半径(0.85 ?)比W6+的(0.60 ?)大, Au3+以取代晶格W6+形式进入WO3晶格中; 对H2-TPR前300 ℃耗氢量的计算可知:Pt/WO3可被还原至Pt/WO2.96, 而Pt/Au/WO3可被还原至Pt/Au/WO2.91. 因此与Pt/WO3相比, Pt/Au/WO3表面氧空位更加丰富. TEM和XPS表征可知, 添加0.1 wt%Au后, 促进了更低价态的Pt均匀分散在WO3载体上, 其平均粒径为2.36 nm.综上所述, Au的掺杂改变了Pt/Au/WO3双金属催化剂的结构, 不仅降低了Pt和W的还原温度, 削弱了Pt和W之间的相互作用, 也促进了更低价态的Pt均匀分散于WO3载体上, 使得Pt/Au/WO3双金属催化剂在甘油氢解制1,3-丙二醇反应中具有更为优异的活性和产物选择性. 该催化剂有望被广泛运用于其他生物质平台化合物加氢脱氧的反应中.     

铜催化芳香醛和酮的氢硼化转化合成苄基硼酸酯类化合物（英文）

有机硼化合物广泛应用于合成化学、药物化学以及材料化学等领域,开发新颖实用的方法合成有机硼化合物是重要的研究领域.在各种有机硼化合物中,苄基硼酸酯有着一些特有的性质,例如活性相对较高,可以有效地当作苄基化试剂使用.目前已有多种合成苄基硼酸酯的方法,主要集中在苄基格氏试剂或者锂试剂的硼化反应,但是该方法底物兼容性较差,而且苄基格氏试剂或者锂试剂的制备比较困难.随着催化反应的发展,过渡金属(如Pd,Cu,Ni,Fe)催化苄基卤代物的硼化反应及芳基卤代物和1,1-二硼类化合物的偶联反应能够有效地合成这类化合物.一级苄醇在钯或铜的催化作用下也可以转化为苄基硼酸酯.苄基C–H键的催化硼化是潜在的构建苄基硼酸酯的高原子经济性的方法,但目前其选择性和反应活性仍不高.在无金属催化的条件下,对甲苯磺酰腙类化合物与HBpin或B_(2pin_2)发生1,2-金属迁移是合成苄基硼酸酯的有效方法.到目前为止,虽然有很多种合成苄基硼酸酯的方法,但仍无法满足其合成需求,因此开发新型的方法合成苄基硼酸酯具有重要的意义.本文开发了一种新型的铜催化芳香醛/酮类化合物的脱氧氢硼化转化体系.使用廉价易得的铜作为催化剂,叔丁醇钠或者叔丁醇钾作为碱,醇质子作为氢源,在100℃的条件下,芳香醛和芳香酮可直接转化成一级和二级苄基硼酸酯类化合物,该反应操作简单,反应体系可以兼容多种官能团,分离产率在21%–77%之间.反应机理方面,该转化有两种可能的过程,(1)反应体系中首先生成1,1-偕二硼化合物,该化合物在碱和EtOH的作用下发生脱硼质子解,最终转化成苄基单硼化合物;(2)醇质子转化成负氢物种,并与体系中的冄-OBpin硼酸酯生成四配位硼,发生1,2-迁移后得到目标产物.为了验证上述两种反应途径的可行性,我们进行了一系列的控制试验.首先合成了苯乙酮的1,1-二硼化合物,在催化量碱与当量醇的作用下,以99%的收率得到了脱硼质子解的产物,说明1,1-二硼化合物可以在反应体系中转化成苄基单硼化合物.以苯甲醛作为原料合成了冄-OBpin硼酸酯,首先将其投入到甲醇、叔丁醇钠和B_(2pin_2)的体系中,最终得到了47%的苄基单硼;同时将冄-OBpin硼酸酯投入到HBpin与叔丁醇钠的体系中,得到了57%的苄基单硼化合物,说明第二种反应过程通过1,2-迁移得到目标产物也是可行的.在当前的实验条件下,两种反应路径都是可能的.     

通过SAPO-34分子筛笼中引入金属物种提升甲醇制烯烃反应中乙烯选择性

低碳烯烃(乙烯、丙烯)是化学工业极其重要的基本原料.甲醇制烯烃(MTO)反应是重要的烯烃生产石油替代路线.其中,磷酸硅铝类SAPO-34分子筛在MTO反应中表现出优异的低碳烯烃选择性.与丙烯相比,乙烯具有更高的经济附加值,因此提升MTO反应中乙烯的选择性有着重要的意义.本文采用传统离子交换法(CIE)、模板辅助离子引入法(TII)和醇相离子交换法(AIE)对SAPO-34分子筛进行金属Zn、Cu改性,利用多种表征手段对金属Zn、Cu改性SAPO-34分子筛的物理结构、化学组成、金属物种状态与分布、酸性及扩散性质等进行表征.首先,对金属Zn、Cu改性SAPO-34分子筛的物理结构和化学组成进行分析.X射线衍射表明,相比AIE法,CIE法和TII法改性基本保持SAPO-34分子筛的结晶度.X射线荧光分析表明,相比Co、Ni,金属Zn、Cu更易引入SAPO-34分子筛.N2物理吸附-脱附表明,CIE法改性能够保持SAPO-34分子筛的BET比表面积和微孔孔容.其次,考察了金属Zn、Cu改性SAPO-34分子筛中金属物种的状态.氢气-程序升温还原(H2-TPR)和X射线光电子能谱(XPS)结果表明,Zn物种主要以孤立态的Zn2+阳离子形式存在.H2-TPR、XPS、紫外-可见光谱和电子顺磁共振谱结果表明,Cu物种主要以孤立态的Cu2+阳离子以及部分CuO形式存在.继而对金属Zn、Cu改性SAPO-34分子筛中金属物种的分布进行表征.XPS表明,Zn阳离子改性的SAPO-34表层富硅、富Zn,呈类核壳结构;XPS和扫描式电镜-能量色散X射线光谱结果表明,Cu物种在Cu改性SAPO-34分子筛中均匀分布.进一步研究了金属Zn、Cu改性SAPO-34分子筛中酸性的变化.氨气-程序升温脱附和核磁共振氢谱结果表明,Zn、Cu改性SAPO-34酸性位点的酸量降低.最后,对金属Zn、Cu改性SAPO-34分子筛的扩散性质进行分析.智能重量分析表明,Zn、Cu阳离子的引入降低探针分子(乙烷、丙烷)的扩散系数,推断Zn、Cu阳离子的引入增加对MTO反应产物的扩散限制.热重表明,Zn阳离子改性SAPO-34分子筛反应初期积炭量略微增加.综上所述,Zn阳离子改性SAPO-34催化剂表层富硅、富Zn,呈现类核壳结构.Zn阳离子的引入增加对MTO反应产物的扩散限制,而且Zn阳离子的引入促进MTO反应初始阶段的碳沉积.因此,Zn阳离子改性SAPO-34分子筛显著增加MTO反应产物的扩散限制,对分子尺寸较大的反应产物的扩散限制更为明显,从而提高MTO反应初始阶段的乙烯选择性,增大乙烯/丙烯比.     

Synthesis of dihydrobenzoxazine pyrrolidone spirocyclic compounds

A simple and efficient synthetic strategy for the preparation of dihydrobenzoxazine spirocyclic compounds was reported via the cycloaddition reaction of o-succinimide-substituted benzaldehyde with nitrobenzene. The reactions can be conducted under very mild reaction conditions (room temperature) using 10?mol% of Cs₂CO₃ as catalyst. A series of dihydrobenzoxazine pyrrolidone spirocyclic compounds were afforded in moderate to good yields. This strategy provides a new direction for the synthesis and study on biological activities of dihydrobenzoxazine pyrrolidone spirocyclic compounds.     

Enhanced electrochemical performance of SnS nanoparticles/CNTs composite as anode material for sodium-ion battery

SnS/CNTs composite as anode for SIBs exhibits high reversible capacity, good cyclability as well as rate performance, which is superior to that of pure SnS. The enhanced electrochemical performance can be attributed to the adding of CNTs as a flexible and conductive structure supporter and the formation of SnS nanoparticles with small diameter.     

Simplified fabrication of high areal capacitance all-solid-state micro-supercapacitors based on graphene and MnO2 nanosheets

A universal simplified strategy was developed to fabricate all-solid-state planar micro-supercapacitors with high areal capacitance (~355 mF/cm²), based on interdigital patterned films of 2D pseudocapacitive MnO₂ nanosheets and electrochemically exfoliated graphene.     

A rapid and sensitive UPLC–MS/MS method for quantitative determination of arformoterol in rat plasma,lung and trachea tissues

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed and fully validated for determination of arformoterol in rat plasma, lung and trachea tissues.     

ZnIn2S4 flowerlike microspheres embedded with carbon quantum dots for efficient photocatalytic reduction of Cr(VI)

Development of efficient heterostructured photocatalysts that respond to visible light remains a considerable challenge. We herein show the synthesis of ZnIn₂S₄/carbon quantum dot hybrid photocatalysts with flowerlike microspheres via a facile solvothermal method. The ZnIn₂S₄/carbon quantum dot flowerlike microspheres display enhanced photocatalytic and photoelectrochemical activity compared with that of pure ZnIn₂S₄. With a content of only 0.5 wt % carbon quantum dots, 93% of Cr(VI) is reduced under visible-light irradiation at 40 min. As a co-catalyst, the carbon quantum dots improve the light absorption and lengthen the lifetime of charge carriers, consequently enhancing the photocatalytic and photoelectrochemical activity.