Effect of enzyme dehydration on alcalase-catalyzed dipeptide synthesis in near-anhydrous organic media.

The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N-protected phenylalanine in near-anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0-80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration-induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration-induced inactivation that takes place during the first activity measurement, (2) a phase of first-order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well.

Assessment of intraparticle biocatalytic distributions as a tool in rational formulation.

Research has shown that the intraparticle biocatalytic distribution has extensive effects on the properties of various (industrial) biocatalytic particles and their performance in (bio-) chemical reactions. In recent years, advances in molecular chemistry have led to the development of many different specific (immuno-) labeling and light-microscopic detection techniques. Furthermore, high-quality image-digitizing devices and enhanced computing power have made image analysis readily accessible. These technologies may lead to the assessment and improvement of the internal biocatalyst profile as an integral part of biocatalytic particle optimization.

Membrane-facilitated bioproduction of 3-methylcatechol in an octanol/water two-phase system.

Bioproduction of 3-methylcatechol from toluene by Pseudomonas putida MC2 was studied in the presence of an additional 1-octanol phase. This solvent was used to supply the substrate and extract the product, in order to keep the aqueous concentrations low. A hollow-fibre membrane kept the octanol and aqueous phase separated to prevent phase toxicity towards the bacterium. Volumetric production rates increased approximately 40% as compared to two-phase 3-methylcatechol production with direct phase contact. Preliminary investigations on downstream processing of 3-methylcatechol showed that 1 M of sodium hydroxide selectively extracted the disodium salt of 3-methylcatechol into an aqueous phase.