JoséM. Moreno

Immobilization of lipase from Candida cylindracea on inorganic supports

Abstract Lipase from Candida cylindracea has been covalently immobilized on trichlorotriazine activated supports (alumina, silica and two types of controlled pore glass). The optimum conditions of the activation process have been determined (pretreatment, solvent, gram of activating agent/gram of support ratio and reaction time). The influence of the enzyme concentration and of the temperature on the immobilization process has been evaluated. The immobilized derivatives on silica and alumina exhibited greater residual activity and were more resistant to inactivation by temperature (50°C) than their immobilized counterpart on controlled pore glasses. The derivatives obtained on alumina and silica have been used in the hydrolysis of ( R,S ) ethyl 2-phenylpropionate only yielding the S (+) acid. The influence of Na I and Ca II on the lipase activity is discussed. The immobilized derivative on silica, stored at 50°C, was 37 times more stable than the native enzyme and displayed 80% residual activity after 336 h of operating time

Sensitivity of fire occurrence to meteorological variables in Mediterranean and Atlantic areas of Spain

Abstract Wildland fires are common in most areas of Spain, even in areas with a mesic climate. Weather plays a crucial role in fire, but the relationship between fire incidence and meteorological variables is not usually strong. Assessing the relationship between meteorological variables and fire from the recent past can eventually aid in evaluating fire risk in future climate scenarios. In this paper we report on the relationships between the number of fires and the surface burned per year and several yearly temperature and precipitation variables for three areas of Spain: North, Central and the Levant. The three areas differ in their climate as well as in the ignition sources. We used the fire records from 1974 to 1988. The relationships between meteorological and fire variables was tested by least square simple regression models. Our results show that some of the meteorological variables used are good predictors of either number of fires or surface burned in all three areas. The northern area was better related to meteorological variables than the Levant. Fire occurence in the northern and central areas was mainly related to temperature variables whereas in the Levant it was related to precipitation variables. Fires caused by pasture burning or by lightning had less relationship to meteorological variables than those caused accidentally. Fires caused by arsonists were very highly related to meteorological variables in all three areas. Fires caused by unknown sources had a similar pattern of relationships to that found for arsonist-caused fires. Our study indicates that the degree of relationship between meteorological variables and fire occurence may be related to the current climate and weather patterns of a given area.

A systematic analysis of the variables that control a highly stereoselective resolution of racemic non-steroidal antiinflammatory drugs using immobilized lipase from Candida cylindracea

An enzymatic method for the production of S( + ) 2-arylpropionic acids has been developed. The process consists of the stereoselective hydrolysis of the racemic ethyl esters catalyzed by covalent immobilized Candida cylindracea lipase. The performance and yield of the reaction were evaluated as a function of the critical reaction parameters such as temperature, substrate concentration, pH, ionic strength and stirring speed. An increase in the stirring speed, a diminution of the substrate concentration and pH = 7.0 favors the hydrolysis of esters. The influence of the alkyl chain and the alcoholic residue of the ester have been studied. The ethyl and butyl esters are the most interesting esters for carrying out hydrolysis. High enantioselective hydrolysis of the racemates (yielding S( + ) isomer; ee ≥ 95%) can be achieved using the immobilized derivatives. The different yields of S( + ) 2-arylpropionic acids produced with the several immobilized derivatives are related to structural parameters of the substrate, which were studied by molecular mechanics, and to the relation between the hydrophilicity of the supports and the lipophilicity of the substrates. A model of the active site is proposed in relation to substrates used and reaction yields. The immobilized derivatives were re-used in three cycles and showed an 80% residual activity after 336 h of operating time.

Covalent immobilization of pure lipases A and B from Candida rugosa

Abstract Covalent immobilization on agarose and SiO2 of pure lipase A (LIP.A) and B (LIP.B) from C. rugosa is described. The results obtained are compared with the data obtained in the covalent binding of commercial lipase (CL) to the same supports. The immobilization of LIP.A and LIP.B on agarose affords more stable biocatalysts than on SiO2. Kinetic studies of all these lipases and derivatives in the hydrolysis of (R)-(+) and (S)-(−) methyl 2-chloropropionates were performed and their enantiomeric ratios ( E = ( V max K m ) fast ( V max K m ) slow ) calculated. The results show that (i) purification of the commercial lipase increased the E value 2.5-fold; (ii) both isoenzymes have similar E values, and (iii) in general, the E value increases with the immobilization process.

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.

Organic reactions catalyzed by immobilized lipases. Part I. Hydrolysis of 2-aryl propionic and 2-aryl butyric esters with immobilized Candida cylindracea lipase

Abstract The alteration of the selectivity of enzymes due to the immobilization methodology is discussed. The hydrolysis of esters of ( R,S ) 2-phenyl propionic and 2-phenyl butyric acids has been used as a reaction test. Lipases from Candida cylindracea immobilized on agarose and alumina have been used as enzymatic derivatives. The stirring speed, [Substrate]/[Enzyme] ratio and pH are the main variables that control the process. An increase in the stirring speed, a diminution of the [Substrate]/[Enzyme] ratio and pH = 7.0 favours the hydrolysis of esters. The effect of the support on the enzymatic activity is discussed. Inorganic supports such as Al 2 O 3 or SiO 2 stabilize the oil/water interface acting in the same way as Na(I) or Ca(II) in the case of native enzyme. Enzymatic derivative on Al 2 O 3 is the most interesting biocatalyst. The effect of the alkyl chain of the ester is not related to the steric hindrance but to the stability of the microemulsion. The butyl ester is the most interesting ester for carrying out the hydrolysis. A model of the ester-active site interaction is proposed to explain the increase observed in the stereoselectivity of the hydrolysis of ( R,S )-ester. High enantioselective hydrolysis of the racemates (yielding S (+) isomer; ee ≤ 98%) can be achieved using the immobilized derivatives.

Contribution to the study of the enzymatic activity of benzonase

Abstract The hydrolytic activity of benzonase has been studied at different values of Mg(II) concentration, pH, temperature and percentages of water-miscrible organic solvents (DMSO, THF, ACN and DMF). The action of these parameters on the UV spectra of benzonase has been analyzed. The best experimental conditions (pH = 8.0, T = 37 °C, [Mg(II)] = 2 mM) lead to a well-defined conformation. This conformation is active vs. DNA and RNA. Changes in these parameters give conformational alterations which can be monitored by changes in the UV spectra. Organic solvents deactivate the enzyme by hydrophobic interaction of the lipophilic solvent molecules with the aliphatic residues of the protein. DMF, the most hydrophilic solvent tested by us, gives slight deactivation of the enzyme. Benzonase hydrolyzes native DNA, heat-denatured DNA and RNA. The active site seems to be the same in all cases. Benzonase has been immobilized for the first time, retaining high enzymatic activity.

Hydrolysis of nucleic acids in single-cell protein concentrates using immobilized benzonase

Hydrolysis of nucleic acids for single-cell protein concentrates has been carried out in one step using immobilized benzonase on corn cob. The immobilization is carried out by tosylation of primary alcohols of cellulose of corn cob. The immobilized benzonase is more stable vs pH changes than native benzonase, but the same optimum values of [Mg(II)] and temperature are obtained. The DNase activity is greater than the RNase activity. The percentage of DNA is reduced to 3-6% and that of RNA to 50%. The protein loss is negligible (1%). The enzymatic activity per weight unit of enzyme is greater in the case of benzonase that in reported data for other nucleases insolubilized on corn cob by the same procedure.