Microwave and Ultrasound Irradiation‐Assisted Synthesis of Novel Disaccharide‐Derived Arylsulfonyl Thiosemicarbazides

The synthesis of 1‐arylsulfonyl‐4‐(1′‐N‐hepta‐O‐acetyl‐β‐lactosyl)thiosemicarbazides by reaction of hepta‐O‐acetyl‐α‐D‐lactosyl isothiocyanate with substituted phenylsulfonyl hydrazines has been shown to occur in less than 1 min under microwave activation and 8 min under ultrasound irradiation at room temperature. It is noteworthy that when ultrasound and microwaves (MW) were utilized, a cleaner reaction accompanied with higher yields was observed.     

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.     

Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage

Purification tags are robust tools that can be used to purify a variety of target proteins. However, tag removal remains an expensive and significant issue that must be resolved. Based on the affinity and the trans-splicing activity between the two domains of Ssp DnaB split-intein, a novel approach for tag affinity purification of recombinant proteins with controllable tag removal by inducible auto-cleavage has been developed. This system provides a new affinity method and avoids premature splicing of the intein fused proteins expressed in host cells. The affinity matrix can be reused. In addition, this method is compatible with his-tag affinity purification technique. Our methods provide the insights for establishing a novel recombinant protein preparation system.     

A novel synthesis method for cyclodextrins from maltose in water-organic solvent systems

A novel enzymatic synthesis method of cyclodextrin (CD) from low-mol-wt maltose using cyclomaltodextrin glucanotransferase (CGTase) fromBacillus macerans has been developed in various water-organic solvent systems. A Β-CD was synthesized in a two-phase system consisting of water and cyclohexane. However, no CDs could be synthesized in an aqueous buffer solution. A maximal yield of Β- CD has been obtained at a cyclohexane content volume of 44%. This synthesis has been obtained only at low temperatures, i.e., 7‡C, and did not take place at 50‡C. In addition, various organic solvents have been used for the enzymatic synthesis of CD from maltose. Consequently, Β-CD could be synthesized in various water-organic solvent systems, e.g., cyclohexane, benzene, xylene, and chloroform, but no enzymatic reaction occurred using aliphaticn-hydrocarbon solvents such as hexane, dodecane, and hexadecane. Furthermore, α- and Β- CD could be synthesized in water mixture solutions using organic solvents having an alcoholic group (e.g., ethanol, propanol, butanol, and pentanol) in a wide range of the reaction temperatures, typically 7–50‡C. In this temperature range, α- and Β-CD were also formed and the maximal yield from maltose to Β-CD of approx 13% was reached in 60 h.     

麦芽糖作选择剂西酞普兰手性毛细管电泳分析及拆分机理研究

研究了以麦芽糖为选择剂的毛细管电泳手性拆分方法。以抗抑郁药物西酞普兰对映体的分离和定量测定为实例,考察了分离条件,在40%(m/m)麦芽糖、8.0×10-2mol/L磷酸盐运行缓冲液(pH5.0)中,分离电压20kV时,西酞普兰对映体分离度达2.3。测定S-( )-西酞普兰中R-(-)异构体的含量,在0.05~4.00g/L浓度范围内线性关系良好。R-(-)-西酞普兰与S-( )-西酞普兰的检出限分别为0.0453g/L和0·0473g/L,线性相关系数均>0.9978。以荧光光谱法对西酞普兰与麦芽糖的相互作用进行了考察,并较系统地对拆分机理进行了研究。证明麦芽糖的手性识别能力与其浓度有关,当麦芽糖达到一定浓度后将形成聚集体,而麦芽糖的拆分作用就主要体现在其聚集体疏水空腔的立体作用上。     

HF/6‐31G* energy surfaces for disaccharide analogs

The HF/6‐31G* level of theory was used to calculate relaxed potential energy surfaces for 12 analogs of disaccharides. The analogs were made by replacing glucose with tetrahydropyran and fructose with 2‐methyltetrahydrofuran. Molecules had zero, one or two anomeric carbon atoms, and di‐axial, axial‐equatorial, and di‐equatorial linkages. Despite the absence of hydroxyl groups, the surfaces account well for conformations that are observed in crystals of the parent disaccharides. Thus, torsional energy and the simple bulk of ring structures are major factors in determining disaccharide conformation. The contour shapes around the global minima depend on the number of anomeric carbons involved in the linkage, while the presence of alternative minima that have relative energies less than 4 kcal/mol mostly requires equatorial bonds. However, molecules with two adjacent anomeric centers gave exceptions to these rules. Flexibility values related to a partition function show that the di‐axial trehalose analog is the most rigid. The di‐equatorial pseudodisaccharide analog with no anomeric centers is most flexible. Reproduction of these surfaces is proposed as a simple test of force fields for modeling carbohydrates. Also, these surfaces can be used in a simple hybrid method for calculating disaccharide energy surfaces. © 2000 John Wiley & Sons, Inc. * J Comput Chem 22: 65–78, 2001     

Determination of Damaged Starch in Wheat Flour Using an Electrochemical Bienzyme Maltose Probe

Abstract

The development of an electrochemical biosensor based on a bienzyme maltose probe and a third enzyme α-amylase in solution is reported for the rapid and inexpensive determination of damaged starch. Analytical parameters, such as probe stability, pH, temperature and response time, were optimised. Damaged starch was measured in the range of 5 × 10^?6 - 5 × 10^?4 mol/L as maltose produced by the enzymatic reaction and the detection limit was calculated according with the free maltose and/or glucose in the sample. The damaged starch was determined in different wheat flours, and the data significantly correlated with those obtained using a reference procedure (r² = 0.994; P ≤ 0.0001). In addition the results showed a comparable precision (CV < 5%). This method is rapid, inexpensive and friendly for unskilled operators.     

Activation volumes of hydrolyses of maltose and maltotriose over an immobilized glucoamylase catalyst

Abstract

Initial rates of hydrolysis of maltose and maltotriose over an immobilized glucoamylase have been measured up to 127 MPa at 25±0.1°C. The observed rates have been analyzed showing the reaction pathways of both hydrolyses to be E+S?ES*?ES7ast;E+P, where E, S, P, ES*, and ES denote the enzyme, the substrate, the product, a substrate-subsite complex, and a substrate-active site complex, respectively. The apparent maximum rate r^mand the apparent Michaelis constant K^m as well as their respective pressure dependences in terms of the apparent activation volume Δ V_app ^# and the apparent volume of reaction Δ V_app have been evaluated. Small absolute values of Δ V_app ^num; and Δ V_app for both reactions have been discussed on the basis of the reaction mechanism.     

A HPLC method for specific determination ofα-amylase and glucoamylase in complex enzymatic preparations

Summary In the hydrolysis of soluble starch by mixtures of α-amylase and glucoamylase, the ratios maltose/glucose and maltoriose/glucose linearly depend, over a wide range, on the relation between both enzymes and are independent on the activity level of the enzymatic preparation. HPLC determination of hydrolysis products (glucose, maltose and maltotriose) of soluble starch by mixtures of these enzymes, after incubation under controlled conditions, is a rapid method for the evaluation of the relative levels of each enzyme in the mixtures. The method, first developed using pure commercial amylases, is applied, with consistent results, to cell free media ofAspergillus niger cultures on a glycogen-rich effluent.