Characterization of Aldehyde Dehydrogenases Applying an Enzyme Assay with In Situ Formation of Phenylacetaldehydes

Herein, different dehydrogenases (DH) were characterized by applying a novel two-step enzyme assay. We focused on the NAD(P)⁺-dependent phenylacetaldehyde dehydrogenases because they produce industrially relevant phenylacetic acids, but they are not well studied due to limited substrate availability. The first assay step comprises a styrene oxide isomerase (440 U mg^?1 _protein) which allows the production of pure phenylacetaldehydes (>70 mmol L^?1) from commercially available styrene oxides. Thereafter, a DH of interest can be added to convert phenylacetaldehydes in a broad concentration range (0.05 to 1.25 mmol L^?1). DH activity can be determined spectrophotometrically by following cofactor reduction or alternatively by RP-HPLC. This assay allowed the comparison of four aldehyde dehydrogenases and even of an alcohol dehydrogenase with respect to the production of phenylacetic acids (up to 8.4 U mg^?1 _protein). FeaB derived from Escherichia coli K-12 was characterized in more detail, and for the first time, substituted phenylacetaldehydes had been converted. With this enzyme assay, characterization of dehydrogenases is possible although the substrates are not commercially available in sufficient quality but enzymatically producible. The advantages of this assay in comparison to the former one are discussed.     

Covalent Immobilization of Dehydrogenases on Carbon Felt for Reusable Anodes with Effective Electrochemical Cofactor Regeneration

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon‐felt‐based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD⁺), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD⁺ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD⁺ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches.     

An Improved Enzyme Assay for Molybdenum-Reducing Activity in Bacteria

Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, K _m for the laboratory-prepared 10:4-phosphomolybdate is 2.56 ± 0.25 mM (arbitrary concentration), whereas the apparent V _max is 99.4 ± 2.85 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent Michaelis constant or K _m for NADH as the electron donor is 1.38 ± 0.09 mM, whereas the apparent V _max is 102.6 ± 1.73 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent K _m and V _max for another electron donor, NADPH, is 1.43 ± 0.10 mM and 57.16 ± 1.01 nmol Mo-blue min⁻¹ mg⁻¹ protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V _max obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.     

Synthesis and Pharmacological Studies on a Cyclooligopeptide from Marine Bacteria

A natural proline‐rich tetrapeptide cyclo‐prolyl‐leucyl‐prolyl‐phenylalanyl was prepared employing solution‐phase method of peptide synthesis through coupling of dipeptide fragments Boc‐l‐Pro‐l‐Leu‐OH and l‐Pro‐l‐Phe‐OMe which utilizes diisopropylcarbodiimide (DIPC) as coupling agent and N‐methylmorpholine (NMM) as the base. Deprotection of linear tetrapeptide unit followed by its cyclization provided a cyclopeptide, identical in all aspects to the natural molecule. Pharmacological evaluation showed cytotoxic, antifungal and antihelmintic potential of synthesized peptide against Dalton's Lymphoma Ascites (DLA) and Ehrlich's Ascites Carcinoma (EAC) cell lines, pathogenic dermatophytes and earthworms.     

Two-Dimensional Tin Selenide (SnSe) Nanosheets Capable of Mimicking Key Dehydrogenases in Cellular Metabolism

While dehydrogenases play crucial roles in tricarboxylic acid (TCA) cycle of cell metabolism, which are extensively explored for biomedical and chemical engineering uses, it is a big challenge to overcome the shortcomings (low stability and high costs) of recombinant dehydrogenases. Herein, it is shown that two-dimensional (2D) SnSe is capable of mimicking native dehydrogenases to efficiently catalyze hydrogen transfer from 1-(R)-2-(R′)-ethanol groups. In contrary to susceptible native dehydrogenases, lactic dehydrogenase (LDH) for instance, SnSe is extremely tolerant to reaction condition changes (pH, temperature, and organic solvents) and displays extraordinary reusable capability. Structure–activity analysis indicates that the single-atom structure, Sn vacancy, and hydrogen binding affinity of SnSe may be responsible for their catalytic activity. Overall, this is the first report of a 2D SnSe nanozyme to mimic key dehydrogenases in cell metabolism.     

Catalase Measurement in Marine Biological Samples by Fluorometric Assay

Condenced aromatics in the environment are harmful to the organisms for its carcinogenesis. When it is metabolized in organism, several kinds of radicals and active oxygen intermediates such as superoxide radical anion (O₂^-) and hydrogen peroxide would produce. These active oxygen intermediates may cause DNA damage. Catalase (CAT), a major endogenous antioxidant, presents in all aerobes and has a crucial function in protecting living organisms against the unrestrained reactivity of H₂O₂. When the amount of H₂O₂ generated in the body increase due to the outside factors, the activity of the CAT will be induced to rise significantly. Thus the activity of CAT, as a biomarker, may reflect die exist of condensed aromatics in the environment. Now there are many methods to detect CAT, such as spectrophotometric methods, titration,electrochemical process, volumetric analysis, but they are not sensitive enough for direct detection of CAT in marine biological samples.     

海洋沉积物中不同结合态无机碳的测定

作为全球碳的重要源与汇的海洋沉积物,在碳循环中起着重要的作用。探讨沉积物中无机碳的行为对深入了解海洋碳循环具有重要意义。本研究根据沉积物中无机碳在不同溶剂中的溶解能力不同,利用顺序浸提法,将沉积物中的无机碳分为：NaCl相(Ⅰ)、氨水相(Ⅱ)、氢氧化钠相（Ⅲ）、盐酸羟胺相（Ⅳ）和盐酸相(Ⅴ)。通过大量实验确定了不同相的最佳浸取条件。在Ⅰ～Ⅲ相,分别将浸取剂和样品加入塑料离心管中,将离心管密封后放在震荡器上震荡2h,然后离心分离,并用水洗涤残渣,将洗涤液并入浸取液,最后用容量法测定其中的无机碳;在Ⅳ,Ⅴ相,将第Ⅲ步残渣转移到锥形瓶中,加入浸取剂后用高纯N2将所产生的CO2吹出,并用饱和Ba(OH)2溶液吸收,最后用容量法测定无机碳。该方法具有较好的精密度。     

海洋石油降解细菌对石油烃降解效果的气相色谱法分析

用毛细管气相色谱法分析评价海洋石油降解细菌对石油烃的降解效果,结果表明,毛细管气相色谱法能够测定出石油降解细菌对石油烃的降解进程和对正构烷烃的总降解率,以及温度对降解速率的影响。石油降解细菌对正构烷烃有明显的降解作用,混合菌株的降解率明显高于单菌株的降解率;在20℃的条件下,经过21 d后,绝大部分的正烷烃被降解,总的降解率为94.93%。     

Alginate Lyases from Marine Bacteria: An Enzyme Ocean for Sustainable Future

The cell wall of brown algae contains alginate as a major constituent. This anionic polymer is a composite of β-d-mannuronate (M) and α-l-guluronate (G). Alginate can be degraded into oligosaccharides; both the polymer and its products exhibit antioxidative, antimicrobial, and immunomodulatory activities and, hence, find many commercial applications. Alginate is attacked by various enzymes, collectively termed alginate lyases, that degrade glycosidic bonds through β-elimination. Considering the abundance of brown algae in marine ecosystems, alginate is an important source of nutrients for marine organisms, and therefore, alginate lyases play a significant role in marine carbon recycling. Various marine microorganisms, particularly those that thrive in association with brown algae, have been reported as producers of alginate lyases. Conceivably, the marine-derived alginate lyases demonstrate salt tolerance, and many are activated in the presence of salts and, therefore, find applications in the food industry. Therefore, this review summarizes the structural and biochemical features of marine bacterial alginate lyases along with their applications. This comprehensive information can aid in the expansion of future prospects of alginate lyases.     

Two‐Dimensional Tin Selenide (SnSe) Nanosheets Capable of Mimicking Key Dehydrogenases in Cellular Metabolism

While dehydrogenases play crucial roles in tricarboxylic acid (TCA) cycle of cell metabolism, which are extensively explored for biomedical and chemical engineering uses, it is a big challenge to overcome the shortcomings (low stability and high costs) of recombinant dehydrogenases. Herein, it is shown that two‐dimensional (2D) SnSe is capable of mimicking native dehydrogenases to efficiently catalyze hydrogen transfer from 1‐(R)‐2‐(R′)‐ethanol groups. In contrary to susceptible native dehydrogenases, lactic dehydrogenase (LDH) for instance, SnSe is extremely tolerant to reaction condition changes (pH, temperature, and organic solvents) and displays extraordinary reusable capability. Structure–activity analysis indicates that the single‐atom structure, Sn vacancy, and hydrogen binding affinity of SnSe may be responsible for their catalytic activity. Overall, this is the first report of a 2D SnSe nanozyme to mimic key dehydrogenases in cell metabolism.     

Lactucin,a Bitter Sesquiterpene from Cichorium intybus,Inhibits Cancer Cell Proliferation by Downregulating the MAPK and Central Carbon Metabolism Pathway

Lung cancer, especially adenocarcinoma, is the second most occurring and highest fatality-causing cancer worldwide. Many natural anticancer compounds, such as sesquiterpene lactones (SLs), show promising anticancer properties. Herein, we examined Lactucin, an SL from the plant Cichorium intybus, for its cytotoxicity, apoptotic-inducing, cell cycle inhibiting capacity, and associated protein expression. We also constructed a biotinylated Lactucin probe to isolate interacting proteins and identified them. We found that Lactucin stops the proliferation of A549 and H2347 lung adenocarcinoma cell lines while not affecting normal lung cell MRC5. It also significantly inhibits the cell cycle at G₀/G₁ stage and induces apoptosis. The western blot analysis shows that Lactucin downregulates the MAPK pathway, cyclin, and cyclin-dependent kinases, inhibiting DNA repair while upregulating p53, p21, Bax, PTEN, and downregulation of Bcl-2. An increased p53 in response to DNA damage upregulates p21, Bax, and PTEN. In an activity-based protein profiling (ABPP) analysis of A549 cell’s protein lysate using a biotinylated Lactucin probe, we found that Lactucin binds PGM, PKM, and LDHA PDH, four critical enzymes in central carbon metabolism in cancer cells, limiting cancer cells in its growth; thus, Lactucin inhibits cancer cell proliferation by downregulating the MAPK and the Central Carbon Metabolism pathway.     

Alcohol Dehydrogenases with anti-Prelog Stereopreference in Synthesis of Enantiopure Alcohols

Biocatalytic production of both enantiomers of optically active alcohols with high enantiopurities is of great interest in industry. Alcohol dehydrogenases (ADHs) represent an important class of enzymes that could be used as catalysts to produce optically active alcohols from their corresponding prochiral ketones. This review covers examples of the synthesis of optically active alcohols using ADHs that exhibit anti-Prelog stereopreference. Both wild-type and engineered ADHs that exhibit anti-Prelog stereopreference are highlighted.     

海洋用钢在含硫酸盐还原菌的海泥中的应变电化学响应

海洋用钢在海底泥土区常承受具大的应力 ,而应力将对海洋用钢的腐蚀破坏行为产生影响 .本文选用一种常用的海洋用钢 ,采集含有硫酸盐还原菌 (SRB)的天然海泥 ,研究试样在海泥中的腐蚀行为 ,以及试样在有裂纹和无裂纹存在时的电流响应特征     

血红蛋白在壳聚糖修饰碳纳米管上的电化学特性及对过氧化氢的电催化分析

用壳聚糖对多壁碳纳米管进行修饰,构建了一种用于固定血红蛋白的新型复合材料,并研究了血红蛋白在该碳纳米管上的电化学性质及其对过氧化氢的电催化活性.扫描电镜结果表明,壳聚糖修饰的多壁碳纳米管呈单一的纳米管状,并能均匀分散在玻碳电极表面.紫外光谱分析表明血红蛋白在该复合膜内能很好地保持其原有的二级结构.将该材料固定在玻碳电极上后,血红蛋白能成功地实现其直接电化学.根据峰电位差随着扫描的变化,计算得到血红蛋白在壳聚糖修饰的碳纳米管膜上的电荷转移系数为0.57,表观电子转移速率常数为7.02 s-1.同时,该电极对过氧化氢显示出良好的催化性能,电流响应信号与H2O2浓度在1.0×10-6 ～1.5×10-3 mol/L间呈线性关系,检出限为5.0×10-7 mol/L.修饰电极显示了良好的稳定性.     

基于带强负电性物质作电极界面的电流型转铁蛋白免疫传感器的研究

将角叉胶、溶胶—凝胶与碳粉相结合,构成了一种具有强负电性可更新电极界面,用于静电吸附固定免疫材料,研制成一种安培型转铁蛋白免疫传感器。研究了电极膜组成、工作电位、底物pH、酶标转铁蛋白浓度及培育时间等实验条件对响应电流的影响。响应电流与转铁蛋白浓度在1．5～60mg／L范围呈线性关系：检出限为1．5mg／L。将传感器初步用于人血清中转铁蛋白的测定,结果良好。     

Simplified universal method for determining electrolyte temperatures in a capillary electrophoresis instrument with forced-air cooling

Temperature increase due to resistive electrical heating is an inherent limitation of capillary electrophoresis (CE). Active cooling systems are used to decrease the temperature of the capillary, but their capacity is limited; and in addition, they leave "hot spots" at the detection interface and at the capillary ends. Until recently, the matter was complicated by the lack of a fast and generic method for temperature determination in efficiently and inefficiently cooled regions of the capillary. Our group recently introduced such a method, termed "Universal Method for determining Electrolyte Temperatures" (UMET). UMET is a probe-less approach that requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. Here, we apply UMET to develop a Simplified Universal Method of Temperature Determination (SUMET) for a CE instrument with a forced-air cooling system using an Agilent 7100 CE instrument (Agilent Technologies, Saint Laurent, Quebec, Canada) as an example. We collected a wide set of empirical voltage-current data for a variety of buffers and capillary diameters. We further constructed empirical equations for temperature calculation in efficiently and inefficiently cooled parts of the capillary that require only the data from a single 1-min voltage-current measurement. The equations are specific for the Agilent 7100 CE instrument (Agilent Technologies) but can be applied to all kinds of capillaries and buffers. Similar SUMET approaches can be developed for other CE instruments with forced-air cooling using our approach.     

Designing a 0D/2D S-Scheme Heterojunction over Polymeric Carbon Nitride for Visible-Light Photocatalytic Inactivation of Bacteria

Constructing heterojunctions between two semiconductors with matched band structure is an effective strategy to acquire high-efficiency photocatalysts. The S-scheme heterojunction system has shown great potential in facilitating separation and transfer of photogenerated carriers, as well as acquiring strong photoredox ability. Herein, a 0D/2D S-Scheme heterojunction material involving CeO₂ quantum dots and polymeric carbon nitride (CeO₂/PCN) is designed and constructed by in situ wet chemistry with subsequent heat treatment. This S-scheme heterojunction material shows high-efficiency photocatalytic sterilization rate (88.1 %) towards Staphylococcus aureus (S. aureus) under visible-light irradiation (λ≥420 nm), which is 2.7 and 8.2 times that of pure CeO₂ (32.2 %) and PCN (10.7 %), respectively. Strong evidence of S-scheme charge transfer path is verified by theoretical calculations, in situ irradiated X-ray photoelectron spectroscopy, and electron paramagnetic resonance.