GpdA-promoter-controlled production of glucose oxidase by recombinant aspergillus niger using nonglucose carbon sources

The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion of GOD excreted into the culture fluid reached similar high levels (≅ 90%) by using either glucose or xylose as substrate, whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD.     

Discriminating three biothiols by using one fluorescent probe

A 1,1′-bi-2-naphthol (BINOL) based dialdehyde (R)-1 is found to exhibit selective fluorescent response towards cellular thiols, _l-Cys, _l-Hcy, and GSH. (R)-1 reacts with _l-Trp to form a Schiff base 2 which quenches the emission of _l-Trp at λ?=?340. Coordination of 2 with Zn(ll) leads to greatly enhanced emission at λ?=?530?nm. When the in situ generated 2 from the combination of the solution of (R)-1 (1.0?×?10^?5?M)?+?Trp (2?equiv) +Zn(OAc)₂ (2 equiv) is treated with _l-Cys, _l-Hcy, and GSH, different fluorescent responses at three emission wavelengths, including 340, 421 and 530?nm, are observed. This allows the in situ generated 2 to be used as a ratiometric fluorescent probe to discriminate these three biothiols. NMR study of (R)-1 with _D- or _l-Cys in the presence of Zn(II) shows that the unique reactivity of Cys provides basis for the selective ratiometric fluorescent response. (R)-1?+?Zn(II) also exhibits enantioselective fluorescent response toward _D- and _l-Cys.     

A self-assembled tetrapeptide that acts as a “turn-on” fluorescent sensor for Hg2+ ion

The tetrapeptide (Bz-$\mathrm{\Delta }$Phe(p-NPh₂)-l-DOPA(protected)-l-Phe-l-Phe-OMe was designed to incorporate seven phenyl rings so that it’s conformation, self-assembly and application in Hg²⁺ ions sensing could be studied. Peptide molecules adopted an overlapping β-turn of type III/III conformation in crystals. The peptide showed a highly selective turn-on response towards mercuric ion over other metal ions with a 10-fold enhancement in fluorescence intensity. This intensity change coupled with the selectivity of the peptide towards mercury allowed us to demonstrate simple colorimetric dip sensing of Hg²⁺ ions. The technique provides a highly selective and effective way to detect Hg²⁺ ions. The peptide also self-assembled into nanospheres with diameter ranges from 100 to 500?nm. Mercuric ion coordination enabled these peptide nanospheres to aggregate into well-defined nanoparticles. The enhanced fluorescence upon Hg²⁺ addition demonstrates that peptide scaffolds can be exploited in the development of different selective sensors.     

Perimidine-based synthetic receptors for determination of copper(II) in water solution

Perimidine-based chelators 1 and 2 were prepared, and their structures were confirmed by ¹H and ¹³C NMR, MS spectroscopy and elemental analysis. These compounds were studied as specific synthetic receptors for the recognition of transition metal ions. They exhibited high affinity and selectivity towards Cu(II) ions. The conditional binding constants, linear dynamic range and detection limit were determined by UV–vis spectroscopy. These parameters demonstrated high potential of the prepared synthetic receptors for the recognition and determination of Cu(II) ions. The minimum detectable concentrations of Cu(II) ions for the synthetic receptors 1 and 2 were 270 and 75 nM (R ² = 0.9915 and 0.9964) in aqueous medium (water/DMSO; 99:1 (v/v)), respectively.     

Amphiphilic block and statistical copolymers with pendant glucose residues: Controlled synthesis by living cationic polymerization and the effect of copolymer architecture on their properties

Amphiphilic block and statistical copolymers of vinyl ethers (VEs) with pendant glucose residues were synthesized by the living cationic polymerization of isobutyl VE (IBVE) and a VE carrying 1,2:5,6‐di‐O‐isopropylidene‐D ‐glucose (IpGlcVE), followed by deprotection. The block copolymer was prepared by a two‐stage sequential block copolymerization, whereas the statistical copolymer was obtained by the copolymerization of a mixture of the two monomers. The monomer reactivity ratios estimated with the statistical copolymerization were r₁ (IBVE) = 1.65 and r₂ (IpGlcVE) = 1.15. The obtained statistical copolymers were nearly uniform with the comonomer composition along the main chain. Both the block and statistical copolymers had narrow molecular weight distributions (weight‐average molecular weight/number‐average molecular weight ∼ 1.1). Gel permeation chromatography, static light scattering, and spin–lattice relaxation time measurements in a selective solvent revealed that the block copolymer formed multimolecular micelles, possibly with a hydrophobic poly(IBVE) core and a glucose‐carrying poly(VE) shell, whereas the statistical copolymer with nearly the same molecular weight and segment composition was molecularly dispersed in solution. The surface properties of the solvent‐cast films of the block and statistical copolymer were also investigated with the contact‐angle measurement. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 459–467, 2001     

Microfluidic differential resistive pulse sensors

This study demonstrates an on-chip resistive pulse-sensing scheme with a design of symmetric mirror channels, which significantly reduces the noise and achieves better signal-to-noise ratio. Polystyrene particles of different sizes have been detected with the developed sensing scheme and a record low volume ratio of the particle to the sensing channel, or 0.0004%, has been detected with particles of 520 nm in diameter in a sensing aperture of 50x16x20 microm3. This volume ratio is about ten times lower than the lowest volume ratio reported in the literature including that specified for commercial Coulter counters.     

Research Advances in Ion Channel-based Electrochemical Sensing Techniques

During the past few years, the electrochemical sensing techniques based on ion channels have attracted considerable attention. Nowadays, these techniques have been widely used in DNA sequencing, measurement of molecular interactions, and detection of inorganic ions and biological species. Hence, in this review, the research progresses of the ion channel-based electrochemical techniques including amperometry, conductometry and potentiometry in chemical and biological sensing are addressed from the perspective of different electrochemical methods. The sensing mechanism and fabrication process of these sensing methods are mainly introduced. In addition, the further research orientations of the electrochemical sensing based on ion channels are prospected.     

一种新型酶生物燃料电池阳极构建及性能研究

通过酸化碳纳米管(CNTs)和β-环糊精(β-CD)之间的范德华力作用, 实现CNTs的β-CD功能化. β-CD具有内腔疏水、外壁亲水的环状结构, 其内腔容易与二茂铁(Fc)形成稳定的主客体包合结构, 实现Fc在碳纳米管上的高效固载; 再将CNTs-β-CD-Fc复合物与葡萄糖氧化酶(GOD)混合, 采用戊二醛实现酶分子间的交联, 形成GOD/CNTs-β-CD-Fc复合物, 然后将其涂覆到玻碳电极(GC)上, 得到一种新型的酶生物燃料电池阳极(GOD/CNTs-β-CD-Fc/GC). 采用同步热分析法、傅里叶变换红外光谱和透射电子显微镜对所制备的CNTs-β-CD-Fc复合物进行了表征, 采用循环伏安法研究了GOD/CNTs-β-CD-Fc/GC电极对葡萄糖氧化的催化性能. 结果表明: 在同等实验条件下, 没有固载Fc的GOD/CNTs- β-CD/GC电极基本无催化电流, 而GOD/CNTs-β-CD-Fc/GC电极表现出比GOD/CNTs-Fc/GC电极更为优越的电催化性能. 进一步以GOD/CNTs-β-CD-Fc/GC电极或GOD/CNTs-Fc/GC电极为酶阳极, 商用催化剂E-TEK Pt/C电极(E-TEK Pt/C/GC)为阴极, 构建葡萄糖/氧气生物燃料电池(EBFC), 结果表明前者的最大功率密度(33 μW·cm^-2, 0.18 V)几乎是后者的三倍(11.7 μW·cm^-2, 0.16 V). 通过记录开路电位随时间的变化研究了EBFC的稳定性, 以GOD/CNTs-β-CD-Fc/GC电极为阳极的EBFC在连续工作9 h后仍保留了92%的开路电位, 表明该电池具有良好的连续工作稳定性. 我们提出的这种新型生物燃料电池阳极的构造方法, 为构建高性能、高稳定性的葡萄糖/氧气EBFC提供了新的思路.