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The last step of the production of four phthalimide-derived acids, designed to act as antiasthma drugs, was performed by enzymatic hydrolysis of the respective methyl or ethyl esters. The esters 4-ethyl-[2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic methyl ester (PHT-MET), 4-ethyl-[2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic ethyl ester, 4-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester, and 2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester were hydrolyzed by immobilized lipase. The enzymatic reaction could be used only to produce the desired 4-substituted compounds. The best result that was found to hydrolysis of PHT-MET, and, therefore, that ester was selected for optimization experiments in a three-phase system. Reactions were performed with solid biocatalyst (Lipozyme® RM IM), organic solvent phase (ethyl acetate), and aqueous phase (saturated NaCO solution). To optimize the reaction conditions, an experimental design optimization procedure was used. The variables studied were the amount of enzyme, the temperature, and the volume of the aqueous solution. Time course experiments were then performed for different initial enzyme concentrations (0.5, 0.9, and 1.4 U/mL of solvent). The optimized reaction conditions found were 20 mg of Lipozyme (0.9 U/mL) and 5.0 mL of NaCO at 40°C for 6 h.

When Tyr245 in endocellulase Cel5A from was changed to Gly (Y245G) by designed mutation, the value of for inhibition of the enzyme by the product cellobiose was increased more than 1480%. This reduction in product inhibition enabled the mutant enzyme (used in conjunction with cellobiohydrolase-I) to release soluble sugars from biomass cellulose at a rate as much as 40% greater than that achieved by the wild-type (WT) enzyme. The mutant was designed on the basis of the previously published crystal structure of the WT enzyme/substrate complex (at a resolution of 2.4 Å), which provided insights into the enzyme mechanism at the atomic level and identified Tyr245 as a key residue interacting with a leaving group. To determine the origin of the change in activity, the crystal structure of Y245G was solved at 2.4-Å resolution to an -factor of 0.19 (-free = 0.25). To obtain additional information on the enzyme-product interactions, density functional calculations were performed on representative fragments of the WT Cel5A and Y245G. The combined results indicate that the loss of the platform (Y245G) and of a hydrogen bond (from a conformational change in Gln247) reduces the binding energy between product and enzyme by several kilo calories per mole. Both kinetic and structural analyses thus relate the increased enzymatic activity to reduced product inhibition.

Glucose oxidase (GO) (EC 1.1.3.4) was used as catalyst for oxidizing glucose into gluconic acid utilizing a 10-mL Bioengineering Enzyme Membrane Reactor® or a 400-mL Millipore Stirred Ultrafiltration Cell (MSUC) coupled with a Millipore UF membrane (cutoff of 100 kDa) and operated for 12 h under an agitation of 100 rpm, pH 5.5, and 30°C. The effect of feeding rate (0.10, 0.15, or 0.20 min), glucose (2.5 or 5.0 m), and GO (1.0 or 2.0 mg/mL) concentrations on the catalysis were studied. A yield of about 75% was attained when the MSUC filled with 1.0 mg/mL of GO was fed with 2.5 m glucose solution at a rate of 0.15 min.

Economic barriers preventing commercialization of lignocellulose-toethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.

Organosolv (ethanol/water and acetosolv) pulps were treated with xylanase for 4,8, and 12 h and compared with commercial Cartazyme HS xylanase-treated pulps. Treatment of ethanol/water pulps with xylanase increased viscosity by 40% in 8 h of treatment compared with pulps treated without enzyme. However, acetosolv pulps treated with xylanase lost viscosity. Ethanol/water pulps treated with Cartazyme had a viscosity of 18.5 cP in 4 h of treatment. In the acetosolv pulps treated with commercial enzyme, the loss of viscosity was 20% compared with pulps treated without enzyme. Ethanol/water pulps treated with and Cartazyme had similar effects: a 44% reduction in kappa number for pulps treated with enzyme followed by alkaline extraction compared with pulps treated with alkaline extraction. In acetosolv pulps treated with , the kappa number was from 12 to 18, compared with pulps treated without enzyme, which had a 40% reduction in 4 and 12 h and a 60% reduction in 8 h. Cartazyme-treated acetosolv pulps had a kappa number of 14 in 4 and 8 h of treatment. For 12 h of treatment, the kappa number was 8. Fourier transform infrared spectra of the pulps showed that enzyme-treated pulps had changes in the 1000 cm absorption owing to a C-O bond present in esters. Using principal component analysis, it is possible to differentiate the unbleached pulps and enzyme-treated pulps.

The aim of this experimental study was to isolate and partially purify protease enzyme from and . Protease enzyme is obtained by inducing spore genesis of bacteria from species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.

Organosolv (ethanol/water and acetosolv) pulps were treated with var. and compared with Cartazyme HS xylanase-treated pulp. The ethanol/water pulps treated with had the same viscosity as unbleached pulps (8 cP). Ethanol/water pulps treated with Cartazyme had higher viscosity than H. grisea-treated pulps (12 cP). Acetosolv pulps treated with and Cartazyme presented a reduction in viscosity; however, the pulps treated with had a lower reduction in viscosity than Cartazyme-treated pulps. Ethanol/water pulps treated with had a 23% reduction in kappa number in 4 and 8 h of treatment, compared with the unbleached pulps. Cartazyme-treated pulps had a kappa number similar to that of the control pulps for 4 h of treatment. Extending the treatment time to 12 h resulted in a reduction of 33%. The acetosolv pulp treated with had a kappa number reduced to 23% in 4 h. Cartazyme treatment resulted in a reduction of 55 and 44% in kappa number for 4 and 8 h of treatment, respectively, when compared with control pulp. Extending the treatment time to 12 h decreased the kappa number 72%. Fourier transform infrared spectra and principal component analysis showed differences among unbleached, H. grisea-treated, and Cartazyme-treated pulps.

In previous work, purification procedures and zymogram analysis conducted with supernatants of crude extracts from aerobic mycelium of the YR-1 strain of isolated from petroleum-contaminated soils indicated the existence of only one soluble alcohol oxidase (sAO) activity. In the present work enzymatic activity of alcohol oxidase (AO) was also detected in the mixed membrane fraction (MMF) of a high-speed centrifugation procedure after drastic ballistic cellular homogenization to break the mycelium from strain YR-1. When mycelial cells were gently broken by freezing the mycelium with liquid nitrogen, smashing in a mortar, and submitting the samples to an isopyenic sucrose gradients (10–60% sucrose), AO activity was detected in particular and discrete fractions of the gradient, showing specific density values quite different from the density of peroxisomes. The results suggest that there could be a different intracellular pattern of distribution of the microsomal fraction in aerobically grown mycelium depending on the carbon source used in the culture media, including alcohols and hydrocarbons, but not in glucose. In working with particulate fractions, we found two AO activities: a new membrane alcohol oxidase (mAO) activity and the sAO. Both activities appear to be located in the inner of the cells in specific compartments different from the peroxisomes, so mAO could be in the membrane of these compartments and sAO in the lumen of the vesicles. We also assayed other enzymatic activities involved in hydrocarbon biodegradation to establish its intracellular location and other enzymatic activities such as peroxidase to use them as intracellular markers of different organelles. In the case of monooxygenase, the first enzymatic step in the hydrocarbon biodegradation pathway, its location was in the same fractions where AOs were located, suggesting the existance of a specific organelle that contains the enzymatic activities involved in hydrocarbon biodegradation.

Softwood residues are the most abundant feedstock available for bioconversion in many northern countries. However, the high costs for delignification and enzymatic hydrolysis currently deter commercialization of softwood bioconversion processes. This study evaluates the abilities of two novel fungal preparations (MSUBC1 and MSUBC2) and two commercial cellulase preparations (TR1 and TR2) to hydrolyze cellulose in Douglas-fir pretreated by steam explosion or ethanol organosolv process. MSUBC1 showed significantly better performance than the other preparations on both lignocellulosic substrates. In particular, MSUBC1 achieved >76% cellulose conversion for hydrolysis of steam-exploded Douglas-fir (∼44% lignin) after 72 h at low enzyme loading (10 filter paper units/g of cellulose) and without β-glucosidase supplementation.

This work investigated the production of fatty acid ethyl esters (FAEEs) from soybean oil using -hexane as solvent and two commercial lipases as catalysts, Novozym 435 and Lipozyme IM. A Taguchi experimental design was adopted considering the variables temperature (35–65°C), addition of water (0–10 wt/wt%), enzyme (5–20 wt/wt%) concentration, and oil-to-ethanol molar ratio (1:3–1:10). It is shown that complete conversion in FAEE is achieved for some experimental conditions. The effects of process variables on reaction conversion and kinetics of the enzymatic reactions are presented for all experimental conditions investigated in the factorial design.