Fermentative production of isobutene

Isobutene (2-methylpropene) is one of those chemicals for which bio-based production might replace the petrochemical production in the future. Currently, more than 10 million metric tons of isobutene are produced on a yearly basis. Even though bio-based production might also be achieved through chemocatalytic or thermochemical methods, this review focuses on fermentative routes from sugars. Although biological isobutene formation is known since the 1970s, extensive metabolic engineering is required to achieve economically viable yields and productivities. Two recent metabolic engineering developments may enable anaerobic production close to the theoretical stoichiometry of 1isobutene + 2CO2 + 2H2O per mol of glucose. One relies on the conversion of 3-hydroxyisovalerate to isobutene as a side activity of mevalonate diphosphate decarboxylase and the other on isobutanol dehydration as a side activity of engineered oleate hydratase. The latter resembles the fermentative production of isobutanol followed by isobutanol recovery and chemocatalytic dehydration. The advantage of a completely biological route is that not isobutanol, but instead gaseous isobutene is recovered from the fermenter together with CO2. The low aqueous solubility of isobutene might also minimize product toxicity to the microorganisms. Although developments are at their infancy, the potential of a large scale fermentative isobutene production process is assessed. The production costs estimate is 0.9 € kg−1, which is reasonably competitive. About 70% of the production costs will be due to the costs of lignocellulose hydrolysate, which seems to be a preferred feedstock

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs

An overview of the recent developments on fructooligosaccharide production and applications

Over the past years, many researchers have suggested that deficiencies in the diet can lead to disease states and that some diseases can be avoided through an adequate intake of relevant dietary components. Recently, a great interest in dietary modulation of the human gut has been registered. Prebiotics, such as fructooligosaccharides (FOS), play a key role in the improvement of gut microbiota balance and in individual health. FOS are generally used as components of functional foods, are generally regarded as safe (generally recognized as safe status—from the Food and Drug Administration, USA), and worth about 150€ per kilogram. Due to their nutrition- and health-relevant properties, such as moderate sweetness, low carcinogenicity, low calorimetric value, and low glycemic index, FOS have been increasingly used by the food industry. Conventionally, FOS are produced through a two-stage process that requires an enzyme production and purification step in order to proceed with the chemical reaction itself. Several studies have been conducted on the production of FOS, aiming its optimization toward the development of more efficient production processes and their potential as food ingredients. The improvement of FOS yield and productivity can be achieved by the use of different fermentative methods and different microbial sources of FOS producing enzymes and the optimization of nutritional and culture parameter; therefore, this review focuses on the latest progresses in FOS research such as its production, functional properties, and market data.Agencia de Inovacao (AdI)-Project BIOLIFE reference PRIME 03/347. Ana Dominguez acknowledges Fundacao para a Ciencia e a Tecnologia, Portugal, for her PhD grant reference SFRH/BD/23083/2005

Activity and stability of immobilized penicillin amidase at low pH values

Penicillin amidase is being applied widely in the production of semi-synthetic beta-lactam antibiotics. Usually the processes are at pH 7-8, but for many new applications the range of pH 3-6 is of interest too. Therefore, we studied the activity of penicillin amidase at 25 degreesC in potassium phosphate buffer of pH 3.7-9, as well as its stability in potassium phosphate buffer of pH 3-6. At each pH, the enzyme was stable during at least 32 days. On the other hand, immobilized penicillin amidase incubated in butyl acetate lost its stability, showing after 32 days a decrease of 52% in relation to its initial enzymatic activity value. In phosphate buffer, the enzyme showed the highest activity at pH 8-9. A gradual decrease to about 20% of this activity occurred when the pH was decreased to 3.7. At even lower pH, the enzyme activity could not be determined with the assay that was used due to a very low stability of penicillin G (PenG). The course of penicillin G conversion and 6-aminopenicillanic acid (APA) production, during enzymatic hydrolysis at pH 4, could be quantitatively described by a simple model when the thermodynamic equilibrium of the hydrolysis was taken into account. (C) 2003 Elsevier B.V. All rights reserved.271293

Solution crystallization kinetics of 6-aminopenicillanic acid

Semisynthetic penicillins, derived from 6-aminopenicillanic acid (APA), constitute an important group of antibiotics. APA is produced by enzymatic hydrolysis of penicillin G (PenG). This study concerns the determination of the crystallization kinetics of APA in the presence of other components that may be present after the PenG hydrolysis, i.e., remaining PenG and the byproduct phenylacetic acid. This evaluation is based on the analyses of growth rate and crystal size distribution, at pH 4 and 5. The results show that the impurities have no significant influence on the APA crystallization, within the range of pH and impurity concentrations evaluated. A mathematical model, based on the population balance, gives a good prediction of the crystallization rate and crystal size distribution with a single set of parameters.45206740674

Liquid-Liquid Extraction of Fermentation Inhibiting Compounds in Lignocellulose Hydrolysate

Several compounds that are formed or released during hydrolysis of lignocellulosic biomass inhibit the fermentation of the hydrolysate. The use of a liquid extractive agent IS Suggested as a method for removal of these fermentation inhibitors. The method can be applied before or during the fermentation. For a series of alkanes and alcohols, partition coefficients were measured at low concentrations of the inhibiting compounds furfural, hydroxymethyl furfural, vanillin, syringaldehyde, coniferyl aldehyde, acetic acid, as well as for ethanol as the fermentation product. Carbon dioxide production was measured during fermentation in the presence of each organic solvent to indicate its biocompatibility. The feasibility of extractive fermentation of hydrolysate was investigated by ethanolic glucose fermentation in synthetic medium containing several concentrations of furfural and vanillin and in the presence of decanol, oleyl alcohol and oleic acid. Volumetric ethanol productivity with 6 g/L vanillin in the medium increased twofold with 30% volume oleyl alcohol. Decanol showed interesting extractive properties for most fermentation inhibiting compounds, but it is not Suitable for in situ application due to its poor biocompatibility.102513541360Netherlands Ministry of Economic AffairsB-Basic partner organization