Manuel G. Roig

Studies on the occurrence of non-enzymatic browning during storage of citrus juice

Abstract In freshly produced commercial citrus juice, aseptically filled in TetraBrik cartons, it has been demonstrated that nonenzymatic browning was mainly due to carbonyl compounds formed from l -ascorbic acid degradation. Contribution from sugar–amine reactions is negligible as evident from the constant total sugar content of degraded samples. The presence of amino acids and possibly other amino compounds, however, enhance browning. Although formation of 5-hydroxymethyl-furaldehyde (5-HMF) has been detected in degraded juice samples, its presence could not be used as an index of browning. 5-HMF has been found to be unreactive in the browning process in citrus juices and its contribution to browning in this type of products is insignificant, if not negligible. Increasing the l -ascorbic acid concentration extends the nutritional value of the product but also increases the severity of browning. Hence where l -ascorbic acid is added as an antioxidant in foods, care should be taken so that the amount added is just proportional to the level of oxygen present or to the amount of oxidizable substances.

Bioremediation of uranium-bearing wastewater: Biochemical and chemical factors influencing bioprocess application

A biotechnological process for the removal of heavy metals from aqueous solution utilizes enzymatically liberated phosphate ligand which precipitates with heavy metals (M) as cell-bound MHPO(4). The enzyme, a phosphatase, obeys Michaelis-Menten kinetics in resting and immobilized cells; an integrated form of the Michaelis-Menten equation was used to calculate the apparent K(m) (K(m app.)) as operating in immobilized cells in flow-through columns by a ratio method based on the use of two enzyme loadings (E(o1), E(o2)) or two input substrate concentrations (S(o1), S(o2)). The calculated K(m app.) (4.08 mM) was substituted into an equation to describe the removal of metals by immobilized cells. In operation the activity of the bioreactor was in accordance with that predicted mathematically, within 10%. The initial tests were done at neutral pH, whereas the pH of industrial wastewaters is often low; an increase in the K(m app.) at low pH was found in previous studies. Immobilized cells were challenged with acidic mine drainage wastewaters, where the limiting factors were chemical and not biochemical. Bioreactors initially lost activity in this water, but recovered to remove uranyl ion with more than 70% efficiency under steady-state conditions in the presence of competing cations and anions. Possible reasons for the bioreactor recovery are chemical crystallization factors. (c) 1997 John Wiley & Sons, Inc.

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.

Biochemical process for the removal of uranium from acid mine drainages

Abstract A biochemical process has been assessed with a view to removing heavy metals from acid mine drainages in which the metal cation removed is accumulated in situ as insoluble metal phosphate on the surface of Citrobacter N14 cells (Roig et al. , 1995). The localized presence of inorganic phosphate (P i ) is brought about via the hydrolysis of a “donor” organic phosphate added to the solution of metals with precipitation as MHPO 4 bound to the cells. The present work explores the potential of immobilized Citrobacter biomass for the recovery of uranium from the acid drainage waters of the “Faith” mine exploited by ENUSA (Ciudad Rodrigo, Salamanca). A physicochemical characterization of the acid waste-water from ENUSA was carried out and flow injection analysis methods for the determination of uranium and P i in such water were developed and improved. The efficiencies of chemical precipitation (by the addition of P i to the acid water) with regard to bioinsolubilization (supplementing the water with an organic phosphate that is (later) hydrolysed to P i ) were investigated and compared. Additionally, the efficiency of chemical and biochemical precipitation as phosphates of uranium present in ENUSA acid drainage water were assessed. Furthermore, the relative importance of chemical precipitation (by the addition of P i plus alcohol) was established. To do so, a series of mass balances for chemical precipitation and for bioinsolubilization of the metal phosphate was performed. Once the efficiency of the bioprocess as regards the removal of uranium when glycerol-2-phosphate is used as a substrate had been determined, a major question was forthcoming: the search for an efficient and much more economical substrate for the process. In this sense, sodium tripolyphosphate, one of the main components of many formulations of commercial detergents, proved to be a poor substrate, such that the quest for an appropriate substrate (cheap, by-product or waste) remains open.

Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability.

The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization.

Kinetic and enantioselective behavior of the lipase from Candida cylindracea: A comparative study between the soluble enzyme and the enzyme immobilized on agarose and silica gels

Candida cylindracea lipase has previously been covalently immobilized on agarose by the tosylation method and on silica by the trichlorotiazine method. We describe a simultaneous study of the soluble enzyme and its counterpart immobilized derivatives and quantify the differences between the kinetic behavior of the three forms. The dependence of lipase activity on Na(I) and Ca(II) concentrations has been examined for the three forms of the enzyme, and results interpreted. Kinetic studies with p-nitrophenyl acetate (esterase activity) and tributyrine (lipase activity) as substrates revealed that the immobilized derivatives have lower kcatapp and Kmapp values than the soluble enzyme. With both substrates, the parallel decrease in kcatapp and Kmapp produces the favorable effect of maintaining the kcatappKmapp ratios of the supported enzymes at values comparable to those of the soluble enzyme. Kinetic studies with methyl (R)-(+) and methyl (S)-(−)-2-chloro-propionates (enantioselective activity) point to certain deviations in the Michaelis-Menten behavior. Accordingly, the v versus [S] curves were fitted by the cubic splines method and interpreted using the areas under the curves. A new type of average enantiomeric excess calculated from these areas varied from 6.4% for the soluble enzyme to 24 and 42% when immobilized on agarose and silica, respectively. This is interpreted in terms of a rigidification of the enzyme produced by the covalent immobilization.

Enzymically-enhanced extraction of uranium from biologically leached solutions

Abstract Many wastes contain heavy metals which are toxic and refractory: further problems arise in the production and discharge of waste radionuclides which have additional radiotoxic effects on the biosphere. Currently the problem may be tackled in four ways: (i) direct chemical methods; (ii) electrochemical treatments; (iii) ion exchange and biosorption methods; (iv) intracellular sequestration by growing microbial cells. A hybrid approach exploits the advantages of processes (iii) and (iv) with the disadvantages of neither. In this context, a biotechnological process for removing and recovering heavy metals from aqueous solutions has been evaluated at low pH. Metal uptake relies upon the in situ cumulative deposition of insoluble metal phosphate tightly bound to the cell surface of Citrobacter N14. Localized high loading of phosphate is contributed via a phosphatase-catalysed hydrolysis of an organic phosphate ‘donor’ molecule added to the metal solution with precipitation of metals (M) as cell-bound MHPO 4 . The present work reports on the potential of this immobilized microbial biomass for uranium recovery from the dilute uranium acid drainage of ENUSA mine in the Ciudad Rodrigo district of Spain. A range of supports (organic and inorganic) and immobilization methods for Citrobacter cells have been screened. Finally, biofilm and entrapped cells on polyurethane foam and cells covalently immobilized on silanized and glutaraldehyde coated Al 2 O 3 Raschig rings were chosen, characterized and evaluated for stability and suitability for large scale use. The physico-chemical monitoring and chemical composition of naturally bioleached waters from ENUSA mine were evaluated and improved methods for flow injection analysis (FIA) of uranium and inorganic phosphate were developed. Optimization studies of the operational conditions and performance of a plug flow bioreactor of immobilized Citrobacter for uranium removal at 30°C gave the following conclusions: (1) a working pH of 4·5 in the absence of any metal complexing agent (citrate) has been established, (2) a [glycerol 2-phosphate]/[ UO 2 2+ ] ratio of 39 is necessary for an optimum uranium removal; (3) at a flow rate of 50 ml/h (residence time of 1·4 h) the efficiency of removal was 50%.

Perspectives for Chemical Modifications of Enzymes

AbstractThe contents of this review include current information on advances regarding the chemistry and chemical modifications (surface and internal) of enzymes that affect their kinetic behavior. As an illustrative example of identifying catalytically essential amino acid residues at the active site(s), the case of liver alcohol dehydrogenase is shown.

Peroxidase stability related to its calcium and glycans

Peroxidases are known to be very stable enzymes. The reasons for such have not yet been fully investigated. Cationic peroxidase from cultured peanut peroxidase can be obtained in substantial amounts and can easily be purified. It is thus an ideal enzyme for study. Through immunological assays its site in the cell has been found and a function determined. With crystals and X-ray diffraction thereof, a 3-D structure of the protein is available. The sites of the heme as well as the 2 calcium ions have been located. With the cDNA it was possible to determine the sites for three glycan chains on the protein. Good progress is being made on the elucidation of the structure of these glycan chains. While both calcium and glycans influence the stability of the protein, the search for how the glycans control the folding pattern is harder than to define the role of calcium. Site-directed mutagenesis has been carried out in each of the three binding sites in turn to determine the role of each glycan. Further work with Mass Spectroscopy. using Electron Spin Ionization tandem Mass Spectroscopy (ESI MS/MS) is underway.

Thermal stability of peroxidase from Chamaerops excelsa palm tree at pH 3

The structural stability of a peroxidase, a dimeric protein from palm tree Chamaerops excelsa leaves (CEP), has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism and steady-state tryptophan fluorescence at pH 3. The thermally induced denaturation of CEP at this pH value is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, leading to the conclusion that in solution CEP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of CEP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate.