Invertase determination

To characterize modifications in the cell wall composition of transgenic invertase plants we determined the distribution of neutral saccharides (Fig. 1). Obviously, the relative amounts of fucose and mannose were slightly reduced in leaf 6 of transgenic plants, whereas arabinose was increased. The other saccharides remained unchanged. 

The continuous configurations depicted in Figs 5.12.D1 and 5.12.D2 were designed by Nieman s group for application of this sensor to the determination of sucrose (and glucose) in soft drinks, breakfast cereal and cake mix [36]. The analyte is converted into /3-D-glucose, to which the sensor is responsive, in two reaction steps that are catalysed by invertase (INV) and mutarotase (MUT) ... 

The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external invertase.190 Its molecular weight, as determined by SDS-poly(acrylamide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split191 by endo-/3-N-acetylglucosaminidase H (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of d-GIcNAc-PP-DoI, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.190 In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. 

H. Mohammadi, M. El Razi, A. Amine, A.M.O. Brett and C.M.A. Brett, Determination of mercury (II) by invertase enzyme inhibition coupled with batch injection analysis, Analyst, 27 (2002) 1088-1093. 

Determination of methyl mercury in fish tissue using electrochemical glucose oxidase biosensors based on invertase inhibition... 

Since the invertase enzyme was purchased 2 years ago from the time this experiment is performed, it was necessary to determine its activity. Knowing that an enzymatic unit corresponds to the amount of enzyme causing the transformation of 1.0 pM of substrate per minute. The equivalent of the response of the enzyme has been given in concentration of the substrate from the linear regression equation of the glucose response. For both sucrose and glucose the experiments have been done in the same conditions of pH value and applied potential. After extrapolation of the invertase response we have found that the activity of invertase is equal to 140 U/mg. 

The principle of combination of electrochemical glucose oxidase biosensor with the clean-up method for direct extraction and determination of methyl mercury has been successfully demonstrated. The extraction of methyl mercury from the organic solvent has been based on invertase enzyme inhibition. The combination of very low concentration of invertase enzyme and 10 min of incubation time allows the detection of methyl mercury at 5 ppb level. Our method permits the detection of this inhibitor below the legal limit given by the European Union with good recoveries when fish samples were measured. 

This work demonstrates a simple and rapid screening method for determination of methyl mercury in organic solvent using invertase inhibition. We have shown that this method is free from interferences using the solvent extraction combined with the invertase enzyme. 

De Whalley and his coworkers have made extensive studies on the application of paper chromatography to raffinose. To separate raffinose from large proportions of sucrose, a 1-butanol-benzene-pyridine-water solvent was used.140 81 To avoid pyridine, a 1-propanol-ethyl acetate-water mixture was recommended.141 As a color reagent, a-naph-thol-phosphoric acid was used. In this manner, less than 1 % of raffinose in cane sugar could be separated and determined. Partial hydrolysis of the raffinose, using invertase directly on the filter paper, proved useful in some instances.93 142 Melibiose was then detected with 3,5-dinitrosali-cylate142 or with diethyl-p-phenylenediamine sulfite.93... 

The activity of soluble and immobilized invertase was determined by varying the temperature of the standard test within a range of 30-60°C ( 30, 35, 40, 45, 50, 55, and 60°C). A blank was prepared under the same conditions for each temperature employed. The activation energy (E/ kj/ mol) was determined by the Arrhenius method, and the thermodynamic parameters were calculated by conventional equations (Eqs. 3-5) (12). The stability of both forms of invertase (aqueous invertase solution [diluted 1 5000, v/v] or an aqueous suspension of Dowex/invertase (100 mg powder/mL]) was evaluated by maintaining the enzyme solution or suspension for 12 min in acetate buffer (0.010 M, pH 5.5) at each... 

The effect of pH on the activity and stability of soluble and immobilized invertase was determined always at 37°C by mixing the enzyme with buffer solutions at fixed pH. The buffers were prepared according to Bacilla et al. (13). For soluble invertase, the pH values employed were 3.0,3.5,4.0, 4.5, 5.0, 5.5, 6.0, and 6.5, and for immobilized invertase they were 4.5,5.0, 5.5,6.0, and 6.5. The stability against pH was determined by measuring the residual activity of both forms of invertase after 12 min of enzyme-buffer contact. 

In the range of temperatures selected for evaluation, the soluble invertase presented detectable instability only at 55°C (Table 3 and Fig. 4). Hence, the thermodynamic parameters for the heat inactivation of invertase (AG, AH, A S and Ea ) were not determined, because the correlation log k x T 1 might necessarily be established, as proposed by Owusu and... 

Application and Principle This procedure is used to determine the invertase activity of enzyme preparations from yeast Saccharomyces sp (Kluyveromyces). The assay is based on a 30-min hydrolysis of sucrose at 30° 0.1° and at pH 4.62. The degree of hydrolysis is determined by measuring the optical rotation of the solution with a polarimeter. 

By a series of runs at different pH values, determine the pH at which invertase functions most effectively as a catalyst. Is the reaction rate highly dependent on pH Why should pH have anything to do with the catalytic activity of invertase ... 

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