A Modular Approach for Interlocking Enzymes in Whatman Paper

A potentially universal approach is presented for enzyme attachment to cellulose that significantly enhances enzyme stability while retaining high activity, and involves no chemical functionalization of cellulose. Bovine serum albumin (BSA) was interlocked in cellulose to form a protein‐friendly surface (named BSA‐Paper), while also providing COOH and NH₂ groups for subsequent attachment of enzymes. The desired enzyme is then mixed with additional BSA and interlocked on BSA‐Paper. The second BSA layer dilutes and crosslinks the enzyme for improved stability. Laccase was tested as a model enzyme for interlocking on BSA‐Paper, and was found to retain over 100 % activity and was 240 times more stable at 25 °C (half life=180 d) than laccase. This new approach was also tested with a few other enzymes with encouraging results, thus providing a potentially universal method for stabilization of enzymes on cellulose with retention of high activities.     

An Enzyme‐Responsive “Turn‐on” Fluorescence Polymeric Superamphiphile as a Potential Visualizable Phosphate Prodrug Delivery Vehicle

The development of inexpensive and highly efficient enzyme‐responsive polymers has significantly contributed to targeted drug delivery systems. Here, a superamphiphile with a capability of fluorescent dissociation sensing is designed. It is constructed with negatively charged adenosine 5′‐triphosphate (ATP) and negatively charged fluorescein diphosphate (FDP), which are used as fluorescence detection, and a cationic diblock copolymer methoxy‐poly(ethylene glycol)₁₁₃‐b‐poly(2‐dimethyl‐aminoethyl methacrylate)₇₀. Upon addition of calf intestinal alkaline phosphatase, the superamphiphile disintegrates, presumably due to the enzymatic hydrolysis of ATP. This process is accompanied by an increase in the fluorescence emission intensity of fluorescein owing to the hydrolysis of FDP. The in vitro application of the superamphiphile is also proven. Thus, the “turn‐on” fluorescence of the superamphiphile serves as a real‐time module for detection of the disintegration of superamphiphile.     

Antioxidative sulphated polygalactans from marine macroalgae as angiotensin-I converting enzyme inhibitors

Antioxidant and antihypertensive potential of the sulphated polygalactans isolated from the marine macroalgae Kappaphycus alvarezii and Gracilaria opuntia were assessed by utilising different in vitro systems. The galactans isolated from K. alvarezii possessed significantly greater antioxidative properties as determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH IC₉₀ 0.97 mg/mL) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS^.+ IC₉₀ 0.72 mg/mL) scavenging activities than those isolated from G. opuntia (DPPH IC₉₀ 1.2 mg/mL and ABTS 0.86 mg/mL). The sulphated polygalactan →4)-4-O-sulphonato-(2-O-methyl)-β-D-galactopyranosyl-(1→4)-3,6-anhydro-(2-O-methyl)-α-D-galactopyranan from K. alvarezii showed greater angiotensin-I-converting enzyme (ACE) inhibitory activity (IC₅₀ 0.02 μg/mL) than →3)-4-O-sulphonato-(6-O-acetyl)-β-D-galactopyranosyl-(1→4)-3,6-anhydro-(2-O-sulphonato)-α-D-galactopyranosyl-(1→3)-4-O-sulphonato-(6-O-acetyl)-β-D-xylosyl-(1→3)-4-O-sulphonato-(6-O-acetyl)-β-D-galactopyranosyl-(1→4)-3,6-anhydro-(2-O-sulphonato)-α-D-galactopyranan motif extracted from G. opuntia (IC₅₀ 0.70 μg/mL). Structure activity correlation studies displayed that the ACE inhibitory properties of titled polygalactans were directly proportional to their electronic properties and inversely with the steric and hydrophobic characteristics. Putative ACE inhibitory mechanism of action of sulphated galactans from marine macroalgae corroborated the structure bioactivity correlation analysis.     

聚乙烯亚胺改性聚丙烯腈中空膜固定脂肪酶

聚丙烯腈是富腈基的高分子聚合物,易修饰改性,广泛应用于膜分离应用.我们以聚丙烯腈中空膜为载体,采用化学法交联聚乙烯亚胺并固定脂肪酶,固定过程中引入海藻酸钠,用CaCl_2进行后处理,得到固定化脂肪酶PAN-PEI-SA/E-CaCl_2载酶量为31.70(mg enzyme)/(g support),酶活为50.20 U/(g support),15次重复使用可保留58.77%的酶活,与游离酶相比耐酸性和耐温性有所提高,相同条件下与Nov 435相比,酶活更高,这表明最终得到的固定化脂肪酶有良好的工业应用前景.     

Synthesis of Cysteine-Functionalized Silver Nanoparticles Using Green Tea Extract with Application for Lipase Immobilization

The application of cysteine-capped silver nanoparticles synthesized using green tea as the reducing agent to immobilize lipase has been reported in the present work. The reducing property of green tea is due to the presence of polyphenolic compounds in its extract which are not oxidized at ambient atmospheric conditions and hence is a suitable reducing agent for green synthesis of nanoparticles. Cysteine-capped silver nanoparticles were synthesized under alkaline conditions by reducing the silver salt by green tea extract in the presence of cystine. Various parameters such as the cystine concentration, pH, temperature, and amount of reducing agent were standardized and their effect on the synthesis process has been initially evaluated by surface plasmon resonance peak analysis. Furthermore, the synthesized nanoparticles were also characterized using X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy. The particle size analysis revealed the average size of the particles to be around 20?nm. The glutaraldehyde-deactivated amino group on cysteine-capped nanoparticles was used to immobilize lipase on its surface. Both crude and immobilized lipases were checked for activity and protein content under standard assay conditions and their activity was found to be 37.7 and 24.9?U?mL^?1, respectively. The lipase nanoparticle bioconjugates exhibited a good shelf life of 60 days with a marginal decrease in activity. The bioconjugates showed 15% loss in its initial activity at the end of five reusability cycles. This immobilized reusable system has the potential to be utilized for various applications pertaining to the exploitation of lipase in various industries.     

Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing

The article reviews the use of electrochemical biosensors for detecting lactate, a key metabolite of the anaerobic glycolytic pathway. This compound plays an important role in (sports) medicine, in the nutritional sector, in food quality control and touches environmental concerns. Amperometric biosensors offer a sensitive and selective means to monitor organic analytes like lactate. A detailed study on different aspects of amperometric lactate biosensor preparation is described: the main configuration aspects are compiled regarding electrode materials, biorecognition elements, immobilization methods, mediators and cofactors as well as fields of application. Comparative studies are conducted correlating different configuration aspects and performance of the resulting biosensors. This review contains 214 references from the years 1974 to 2007. Correspondence: Beate Strehlitz, UFZ, Helmholtz Centre for Environmental Research – UFZ, Environmental and Biotechnology Centre (UBZ), Permoserstrasse 15, D-04318 Leipzig, Germany     

四硫富瓦烯为电子媒介体的葡萄糖生物传感器的研究

本采用四硫富瓦烯（ＴＴＦ）作为葡萄糖氧化酶与玻碳电极之间的电子媒介体，把葡萄糖氧化酶因定在Ｎａｆｉｏｎ－ＴＴＦ修饰电极上，制成葡萄糖生物传感器。用这种传感器测定人体血清中葡萄糖的含量，其线性范围在４．０×１０＾－５～１０＾－３ｍｏｌ／Ｌ之间，响应时间为２０ｓ。该传感器具有选择性、重现性好，灵敏度高等优点。     

Electrochemical enzyme immunoassay using immobilized antibody on gold film with monitoring of surface plasmon resonance signal

Sandwich immunoassay was conducted on a thin gold film set in a surface plasmon resonance (SPR) cell. Monochronal antibody (anti-IgG) was immobilized onto the gold film via 4,4′-dithiodibutyric acid (DDA) and avidin-biotin bonding. Next, IgG sample and alkaline phosphatase-conjugated anti-IgG (ALP anti-IgG) were introduced into the cell successively. Finally, p-aminophenyl phosphate (PAPP) was injected as an enzyme substrate, and the produced p-aminophenol (PAP) was electrochemically measured. Flow did not need to be stopped for incubation for the enzyme reaction, because of the thinness of the cell. In these processes, all the antigen-antibody reactions took place on the gold film. Therefore, the immobilization was performed quickly, and each process could be confirmed by SPR signal. This system had the advantage that the middle of the complicated process could be monitored. For example, the amount of antibody immobilized, which affected on the final electrochemical signal, could be confirmed in the course of immobilization. It was also convenient to investigate process conditions, such as removal of used antigens and labeled antibodies. Good correlation was obtained between the electrochemical current and the SPR signals due to the adsorption of IgG and ALP anti-IgG, and the sensitivity of the electrochemical measurement was much higher than the SPR’s.     

Kinetic Analysis of Multi Enzyme Systems: A Case Study of the Closed System of Creatine Kinase, Hexokinase and Glucose 6-Phosphate Dehydrogenase

This paper describes a general methodology to handle closed multi enzyme systems using mixture of symbolic (which depends on the Gröbner Basis technique) and numerical computation methods. The applicability of the proposed method has been examined for the closed three-enzyme system of rabbit heart creatine kinase (EC 2.7.3.2), yeast hexokinase (EC 2.7.1.1) and human erythrocyte glucose 6-phosphate dehydrogenase (EC 1.1.1.49) using experimental data.