Melanin Standard Method: Particle Description

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin—K⁺ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K⁺ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.     

Improved design of peptides exhibiting angiogenic activities for clinical applications

Vascularization is one of the key steps for engraftment in regenerative medicine. Previously one of the authors had discovered peptides exhibiting significant angiogenic activities designated AGP and elucidated the active core. For neovascularization basic fibroblast growth factor is used although permeation can be envisaged. The original AGPs did not suffer from this although their half-life times are short because of decomposition by endogenous enzymes. Several new AGP-libraries have been constructed and their enzymatic resistance has been investigated by the use of human umbilical vein endothelial cells to find candidates for clinical applications.     

Enzymatic and Non-Enzymatic Esterification of long Polyunsaturated Fatty Acids and Lysophosphatidylcholine in Isooctane

Phosphatidylcholine containing large amounts of long polyunsaturated fatty acid, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), was synthesized in isooctane. Immobilized phospholipase A₂ was used as a catalyst. A parallel non-enzymatic esterification reaction was investigated in separate experiments.

The concentrations of lyso-phosphatidylcholine, polyunsaturated fatty acids, water and the enzyme were varied over wide ranges as were the temperature and the reaction time. The type of enzyme, carrier and immobilization procedure were held constant.

The yield of phosphatidylcholine was relatively high (about 21%) when the concentration of polyunsaturated fatty acids was high (300 mg/g of reaction mixture) and the water content was low (below 30% of the dry immobilized enzyme). The highest yield of phosphatidylcholine was found at 80 hours and 75°C. However, at this temperature an extensive non-enzymatic reaction between polyunsaturated fatty acids and lyso-phosphatidylcholine occurred. At 80°C the polyunsaturated fatty acids were partly oxidized. Therefore, a temperature of 45°C to 65°C is probably the optimum temperature for the reaction.     

Enzymatic Esterification of Long Polyunsaturated Fatty Acids and Lyso-Phosphatidylcholine in Isooctane and Ethanol

Phosphatidylcholine (PC) was synthesized from lyso-PC and long poly-unsaturated (n-3) fatty acids (PUFA) using immobilized phospholipase A₂. The esterification was performed using the fatty acids as the main solvent and isooctane or ethanol (99.5%) at low concentrations (7–45%) as additional solvents. The temperature was kept constant at 45d`C and the water concentrations were carefully controlled.

The best yield of PC (22%) was found in the isooctane system at a low water content (22% of the dry immobilized enzyme). In the ethanol system, the yield of PC was only half. The best yields were attained when the concentrations of both isooctane and ethanol were below 7% and the reaction time was very long (9 days). Improved contact between the enzyme and the substrates would probably increase the reaction rate.     

A Theory for the Displacement of Proteins and Viruses with Polyethylene Glycol

The displacement action of polyethylene glycol of different molecular weights may be linked to the ability of the polymers to form coiled particles in solution. From conclusions drawn from their sedimentating properties in centrifugal fields the polyethylene glycols of low molecular weights, as expected, are les randomly coiled than those of higher molecular weight. It is suggested that protein molecules have the ability to diffuse into the coils of the polyethylene glycol from which they are excluded when the random coiling increases with increasing polymer concentration. From considerations based on the interaction of the polymer filament with the displaced particle the distribution of the substance between the coils and the intermolecular spaces may be predicted semi-quantitatively.     

Chemo-enzymatic Synthesis of 3-Deoxy-β-D-ribofuranosyl Purines and Study of Their Biological Properties

Abstract

9-(3-Deoxy-β-d-erythro-pentofuranosyl)-2,6-diaminopurine (2) was synthesized by an enzymatic transglycosylation of 2,6-diaminopurine using 3′-deoxycytidine (1) as a donor of the sugar moiety. Nucleoside 2 was transformed to 3′-deoxy guanosine (3), 9-(3-deoxy-β-d-erythro-pentofuranosyl)-2-amino-6-oxopurine (3′-deoxyisoguanosine; 4), and 9-(3-deoxy-β-d-erythro-pentofuranosyl)-2-fluoroadenine (5). Compounds 2–5 were evaluated for their anti-HIV activity.     

Extended Parker–Sochacki method for Michaelis–Menten enzymatic reaction model

In this article, a new approach—namely, the extended Parker–Sochacki method (EPSM)—is presented for solving the Michaelis–Menten nonlinear enzymatic reaction model. The Parker–Sochacki method (PSM) is combined with a new resummation method called the Sumudu–Padé resummation method to obtain approximate analytical solutions for the model. The obtained solutions by the proposed approach are compared with the solutions of PSM and the Runge–Kutta numerical method (RKM). The comparison proves the practicality, efficiency, and correctness of the presented approach. It serves as a basis for solving other nonlinear biochemical reaction models in the future.     

一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 *

羟基化氨基酸是一种新型氨基酸衍生物,可广泛用作化工材料的前体物及医药合成的中间体。将来源于Nostoc minutum的新型L-亮氨酸5-羟化酶 (NmLEH) 通过重组质粒在大肠杆菌中异源表达。结果表明,在BL21(DE3) 宿主细胞中,诱导温度为25℃,IPTG诱导浓度为0.5mmol/L,诱导10h时,蛋白质表达量最高 (0.45mg/ml);通过Ni-亲和层析和凝胶过滤层析两步分离纯化获得了高度纯化的重组NmLEH蛋白;对NmLEH的酶学性质进行了表征,该酶的最适反应温度为25℃,最适pH 为7.5,在pH 7.0~9.0较为稳定,最适底物为亮氨酸和甲硫氨酸;同源序列分析表明NmLEH属于亚铁和α-酮戊二酸依赖性双加氧酶家族[Fe(II)/αKG-Dos],并预测了该酶的保守催化活性位点(H150、D152、H236);通过同源建模得到了该蛋白质的模拟结构,分析了该蛋白质催化活性中心的形成机制。