Synthesis of pentylferulate by a feruloyl esterase from Aspergillus niger using water-in-oil microemulsions

Pentylferulate synthesis was achieved at high yields (50–60%) with Aspergillus niger feruloyl esterase using a water-in-oil microemulsion system. The initial rate of synthesis decreased by 15–20% when the water content of the microemulsion was increased from 1.8 to 2.4% (v/v), although a concomitant decrease in conversion was not observed. The enzyme stability was significantly higher in the microemulsion than in an aqueous solution.     

A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.     

Cholesterol biosensors prepared by layer-by-layer technique

The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet–visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.     

Catalytic resolution of (RS)-HMPC acetate by immobilized cells of Acinetobacter sp. CGMCC 0789 in a medium with organic cosolvent

Kinetic resolution of a chiral alcohol, 4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopentenone (HMPC), a key intermediate for the production of prallethrin insecticides, was successfully carried out by enantioselective hydrolysis of (RS)-HMPC acetate using calcium alginate gel-entrapped cells of a newly isolated esterase-producing bacterium Acinetobacter sp. CGMCC 0789. When the effect of different cosolvents was investigated, it was found that isopropanol could markedly enhance the activity and enantioselectivity of the immobilized cells. The optimum concentration of isopropanol was 10% (v/v) where immobilized cells still showed good operational stability. After 10 cycles of reaction, no significant decrease in the enzyme activity was observed. The catalytic specificity constants (V_max/K_m) for both enantiomers of the substrate were determined with partially purified enzyme, giving 0.0184 and 0.671 h⁻¹ for the (S)- and (R)-ester, respectively.     

Effects of tenuifolin extracted from radix polygalae on learning and memory: A behavioral and biochemical study on aged and amnesic mice

Although normal cognitive changes take place when a person becomes older, aging in humans is generally associated with deterioration of cognitive performance and, in particular, of learning and memory. These cognitive deficits can cause debilitating consequences due to aging. There are a number of herbal medicines which are reported to improve brain function including intelligence.In the present study, improving effects of tenuifolin, extracted from Radix Polygalae (RP), on learning and memory in aged and dysmnesia mice were determined using step-down type passive avoidance test or Y type maze trial. Oral administration of tenuifolin (0.02, 0.04, 0.08 g/kg d⁻¹, for 15 d) evidently improved the latency and number of errors in aged and dysmnesia mice. The levels of cortical acetylcholine esterase (AChE) activity and hippocampal neurotransmitters in aged mice given tenuifolin (0.02, 0.04, 0.08 g/kg d⁻¹, for 15 d) were also investigated, and increased levels of norepinephrine (NE), dopamine (DA), decreased activity of AChE were found. However, serotonin (5-HT) had no significant difference from that of aged mice given distilled water. The evident improvement of learning and memory of aged mice is carried out by the effects of tenuifolin on the three stages of memory process, that is, acquisition, consolidation and retrieval. This may do so by relatively increasing the levels of NE, DA in the hippocampus and by decreasing the activity of AChE in the cortex.