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.     

Synthesis of Branched Polyglucans by the Tandem Action of Potato Phosphorylase and Deinococcus geothermalis Glycogen Branching Enzyme

An enzymatic tandem reaction is described in which the enzymes phosphorylase and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) catalyze the synthesis of branched polyglucans from glucose‐1‐phosphate (G‐1‐P). Phosphorylase consumes G‐1‐P and polymerizes linear amylose while Dg GBE introduces branching points on the α‐(1 → 6) positions by reshuffling short oligosaccharides. The resulting branched polyglucans have an unusually high degree of branching of 11%.

     

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.     

Quantification of intermediates of the methionine and polyamine metabolism by liquid chromatography–tandem mass spectrometry in cultured tumor cells and liver biopsies

By means of liquid chromatography–tandem mass spectrometry we showed recently, that the chromosomal deletion or inactivation of the methylthioadenosine phosphorylase (MTAP) gene led to the accumulation of 5′-deoxy-5′-(methylthio)adenosine (MTA) in cancer cells. Here, we expanded the method to other key intermediates of the methionine and polyamine pathways to further elucidate the molecular consequences of a lack of MTAP activity. Employing multiple-reaction monitoring, limits of detection and lower limits of quantification in the range of 2.5–100 and 5.0–500 nM, respectively, were achieved according to the guidelines of the FDA, thus enabling the direct measurement of the metabolites in biological samples without prior enrichment and derivatization with an analytical repeatability of 1–3%. Relative standards deviations for quadruplicate 80% methanol extractions of metabolites from cultured tumor cells ranged from 1.1 to 25.5%, while the combined methodological and biological variability in metabolite concentrations in 10 liver biopsies was 11.8–51.4%. The method enabled the demonstration of changes in the concentration of intermediates of the methionine and polyamine metabolism other than MTA in hepatocellular carcinoma specimens lacking the enzyme MTAP compared to normal liver tissue.     

Use of Pd-catalyzed Suzuki-Miyaura coupling reaction in the rapid synthesis of 5-aryl-6-(phosphonomethoxy)uracils and evaluation of their inhibitory effect towards human thymidine phosphorylase

A number of new 5-aryl substituted pyrimidine acyclic nucleoside phosphonates were synthesized and tested for their ability to inhibit human TP. Their rapid synthesis using Pd-catalyzed Suzuki-Miyaura coupling reactions of various arylboronic acids with 5-bromo-4-(phosphonomethoxy)-2,6-dibutoxypyrimidine was successfully applied. For a series of 5-aryl-6-phosphonomethoxyuracils, an increased inhibitory effect was determined. This effect is supported by the results found for 4-fluorophenyl (K_i^dThd=4.89±0.62) and 3-nitrophenyl (K_i^dThd=3.98±0.46) substituents.     

Preparation of Glycogen‐Based Polysaccharide Materials by Phosphorylase‐Catalyzed Chain Elongation of Glycogen

Facile preparation of glycogen‐based polysaccharide gel materials was carried out by phosphorylase‐catalyzed chain elongation of glycogen using glucose 1‐phosphate (Glc‐1‐P). The resulting solution after the enzymatic reaction gradually turned into a hydrogel form, which was probably caused by formation of junction zones based on the double helix structure of the elongated amylose chains among the glycogen molecules. Furthermore, lyophilization of the hydrogel resulted in formation of a glycogen‐based xerogel. The mechanical properties of the hydrogels and xerogels were affected by the amount of glycogen and the Glc‐1‐P/glycogen ratio in the feed for the enzymatic reaction. The xerogel was also subjected to film formation and re‐hydrogelation with appropriate techniques.

     

QM/MM distortion energies in di‐ and oligosaccharides complexed with proteins*

To investigate whether linkages between monosaccharide residues are unusually distorted by their interactions with proteins, ? and ψ values for fragments of cellulose and starch were taken from the Protein Data Bank. These experimental conformations were then plotted on energy surfaces that were calculated with a hybrid of HF/6‐31G* and MM3(96) energies. Energy values corresponding to each crystallographic conformation were then pooled. Nearly 70% of the 210 structures had energies of 1 kcal mol^?1 or less. A cumulative frequency analysis showed that most points fell on a curve that had an exponential decrease in the number of observed structures as the energy increased. This is analogous to a Boltzmann distribution but at higher temperature. This analysis showed that more than 90% of the linkages were not unusually distorted, and the distribution was similar to that found for small‐molecule crystals of carbohydrates. However, above 2 kcal mol^?1, the observed points deviated from the curve. Most of these high‐energy observations were from linkages being broken by enzymatic attack, but others were not, and some scissile linkages were not unusually distorted. © 2001 John Wiley & Sons, Inc. Int J Quantum Chem 84: 416–425, 2001 *     

Structural Insight into the Binding of Hypoxanthine with Purine Nucleoside Phosphorylase from Streptococcus mutants

Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase(Smu PNP) has been solved by molecular replacement at 1.80  resolution and refined to R factors of 19.9%/23.7%(Rcryst/Rfree) . Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine(HPA) and sulfate ion was solved at 2.24  resolution and refined to R factors of 21.6%/24.1%(Rcryst/Rfree) . It is interesting to note that the resulting electron density indicated the product,HPA,presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site,which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.