Carolina Aguirre

Optimization of yield in kinetically controlled synthesis of ampicillin with immobilized penicillin acylase in organic media

Immobilized penicillin acylase is a moderately priced versatile enzyme, that is able to catalyze the synthesis of derived penicillins and cephalosporins from the corresponding β-lactam nuclei and proper side-chain precursors. Kinetically controlled synthesis is a better strategy when product yield is a key issue. Yield should increase at reduced water activity by depressing the competing hydrolytic reactions in favor of synthesis; therefore, organic cosolvents can be a suitable reaction media for synthesis. Using response surface methodology and product yield as objective function, temperature and pH were optimized in the kinetically controlled synthesis of ampicillin using previously screened cosolvents and reaction conditions. Optimum pH was 6.0 for ethylene glycol (EG) and glycerol (GL) and 6.6 for 1–2 propanediol (PD); optimum temperature was 30°C for GL and for EG and PD was in the lower extreme of the range studied, optimum lying below 26°C. Maximum molar yields predicted by the model were 58,51, and 46% for EG, GL, and PD, respectively, which were experimentally validated. Highest yield in aqueous buffer was always <40%. Molar yields about 60% compare favorably with values reported for the kinetically and thermodynamically controlled synthesis of ampicillin and other derived penicillins.

Synthesis of Cephalexin in Aqueous Medium with Carrier-bound and Carrier-free Penicillin Acylase Biocatalysts

The use of very high substrate concentrations favors the kinetically controlled synthesis of cephalexin with penicillin acylase (PA) not only by Michaelian considerations, but also because water activity is depressed, so reducing the rates of the competing reactions of product and acyl donor hydrolysis. Commercial PGA-450, glyoxyl agarose immobilized (PAIGA) and carrier-free cross-linked enzyme aggregates of penicillin acylase (PACLEA) were tested in aqueous media at concentrations close to the solubility of nucleophile and at previously determined enzyme to nucleophile and acid donor to nucleophile ratios. The best temperature and pH were determined for each biocatalyst based on an objective function considering conversion yield, productivity, and enzyme stability as evaluation parameters. Stability was higher with PAIGA and specific productivity higher with PACLEA, but best results based on such objective function were obtained with PGA-450. Yields were stoichiometric and productivities higher than those previously reported in organic medium, which implies significant savings in terms of costs and environmental protection. At the optimum conditions for the selected biocatalyst, operational stability was determined in sequential batch reactor operation. The experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium.

Enzymatic treatment of stickwater from fishmeal industry with the protease from Cucurbita ficifolia

SummaryThe protease fromC. ficifolia was applied to stickwater prior to evaporation. The evaporator was operated at reduced viscosities with an increase in solid concentration beyond 50% as compared to the untreated substrate.

Enzymatic pretreatment of kraft pulps from pinus radiata D don with xylanolytic complex of pénicillium canescens (CP1) fungi

After screening 14 strains exhibiting high xylanase activities,Penicillium canescens (CP1) andPenicillium janthinellum Biourge (CP2) strains were selected. The β-xylanases produced had an optimum temperature and pH of 50°C and 4.0, respectively. Using a bleaching sequence of D100EP, D80EP, and XD80EP, the effluent color obtained with XD80EP was lower for CP1 and CP2 than at the D100 stage. The color was slightly higher at the XD80EP stage than with the D80EP sequence. In the final pulp obtained with XD80EP pretreatment, the viscosity increase and the Kappa number was similar to that of D100EP in the CP1 and CP2 strains. Brightness in the final pulp was slightly lower than that of control. The selectivity ratio was better for the CP1 and CP2 strains as compared to control. In the XD80EP stage using xylanase extract from CP1 with a pulp consistency of 8 to 15%, the Kappa number was not changed, but the viscosity, brightness, and selectivity ratio were improved proportional to the rise in consistency and delignification. Breaking length, burst and tear index, porosity, and elongation, in the final paper did not change after enzymatic treatment. AOX decreased (26%) in the D80 stage effluent as compared with D100, whereas in the XD80 stage diminished 42%. The enzymatic treatment with CP1 facilitates the lignin release, decreases the CL02 load by 20%, and reduces the AOX without any negative effects on the physical properties of the pulp and paper.

Comparative study of the enzymatic synthesis of cephalexin at high substrate concentration in aqueous and organic media using statistical model

Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.

Amazonian lignocellulosic materials-V: screening of xylanolytic fungi

A plate-agar technique for fungal screening was applied to evaluate the xylanolytic activities of 18Penicillium janthinellum and 10Aspergillus sydowi species from the Amazon region. In order to compare these genera with those of other regions, oneAspergillus sp., one P.janthinellum, and 12 unknown genera from the southern region of Chile were studied. From these fungi strain,A. sydowi (56 strain) (25.2 IU/mL),P. janthinellum (671 strain) (47.3 IU/mL) from Amazonia,P. janthinellum (X4Z2 strain) (9.5 IU/mL), and anAspergillus sp. (X2M1 strain) (33.3 IU/mL) from the southern region of Chile were identified.

Aspergillus sp. 2M1 Xylanase: Production, Characterization and Application in the Pulp and Paper Industry

Aspergillus sp. 2M1 produces high levels of β -xylanases and β-xylosidases. Different carbon sources as inductors of xylanases were used. Production of xylanases were evaluated in a combined packed-bed/air-lift bioreactor (PBAL) in free and immobilized form and with a stationary tray bioreactor (STB). In the free form, xylanase activity was similar to that in Erlenmeyer flasks, but in the immobilized form higher values were observed Similar xylanases activities with the STB were found In order to understand xylanases stabilities, chemical modifications were done. Among others, 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) affected the xylanase activity. On the basis of this study, it was concluded that one or more carboxyl groups are in the active site of the xylanase from Aspergillus sp. 2M1. Xylanases induced by oatt birch and pinus xylans exhibited their optimum pH at 5.5 and at 55°C. At this temperature the half-lives of the xylanase activities were 11.8, 7.2 and 4.4 min for pinus, birch and oat, respectively. Xylanases induced by birch xylan were applied for a pre-bleaching experiment. Pre-bleaching of pinus Kraf pulp followed by a short oxygen-peroxide sequence, resulted in a very high selectivity as compared with commercial xylanases.

New methodology for fungal screening: Xylanolytic enzymes

The technique for determining extracellular xylanolytic activity consists of growing selected fungi for four days on agar medium containing 1% xylan. A section of the agar is then homogenized in buffer and the filtered solution tested for xylanase activity and other related enzymes.