Volker Kasche

Immobilized enzymes: crystals or carriers?

The advantages of immobilized over soluble enzymes arise from their enhanced stability and ease of separation from the reaction media, leading to significant savings in enzyme consumption. Immobilization methods range from binding to prefabricated carrier materials to packaging in enzyme crystals or powders. During their use, mass-transfer effects can produce substrate or pH gradients, which reduce the reaction rates and product yields. The costs of immobilized enzymes must be minimized in order to increase their competitiveness for technical applications.

Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of β-lactam antibiotics, peptides and other condensation products

Abstract Hydrolases can be used to catalyse the synthesis of condensation products such as β-lactam antibiotics, peptides, oligosaccharides and glycerides. In biotechnological processes, synthesis to achieve maximum yields may be carried out either as an equilibrium controlled or kinetically controlled reaction. Only in the later case is the yield of condensation product influenced by the properties of the enzyme that act as a transferase in this reaction. With the same amount of enzyme the maximum yield is also obtained much more rapidly than in the equilibrium controlled process. Hydrolases with high ratios of transferase to hydrolase activity are favourable for use. Recent results on the mechanisms of enzyme catalysed condensations allow a rational analysis of how yield controlling factors (pH, temperature, ionic strength, enzyme and substrate properties) may be changed to obtain optimum yields. This can be used to evaluate whether these biotechnological processes can compete with the chemical methods currently used for the synthesis of these products. It can also be used to plan rational protein engineering of the enzymes that in kinetically controlled synthesis of the condensation products may give yields that can compete favourably with the existing chemical processes to produce these compounds.

TRANSIENT SPECIES IN THE PHOTOCHEMISTRY OF EOSIN

Abstract— Photochemical reactions of eosin in aqueous solution were studied using the flash photolysis technique. In deaerated solution the dye was converted quantitatively to the triplet state during flashing. The triplet dye decayed by first and second order reactions which partly regenerated the dye in the ground state and partly produced semioxidized and semireduced eosin. These radical species were formed in an electron dismutation reaction between two triplet molecules and also in a reaction between one triplet and one unexcited molecule. The radicals recombine rapidly to give the dye in the ground state.

Purification of monoclonal antibodies by simulated moving-bed chromatography

A simulated moving bed (SMB) system has been developed for the biospecific purification of monoclonal antibodies. Adsorption and desorption of the desired product is performed under different conditions. To increase the purity and yield of the antibodies, two purge steps have to be introduced. The steady-state performance of the SMB system was modelled by solving the governing differential equations using a linear driving force approximation. The model parameters were determined independently in batch experiments. They were used to determine the operating conditions of the SMB system for the purification of monoclonal antibodies from cell culture supernatant. The antibodies could be isolated with a yield of > or = 90. SDS gel electrophoresis of the feed and product stream showed that more than 99% of the contaminating proteins were removed in a single step by SMB chromatography.

Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli.

The penicillin amidase (PA) from Escherichia coli belongs to a group of proteolytically processed bacterial enzymes. The mechanism of the maturation of the single polypeptide proenzyme has been studied for the PA from E. coli using a slowly processing mutant proenzyme. The mutant proenzyme was constructed by replacing Thr with Gly in the Thr(263)-Ser(264) bond that must be hydrolysed in active PA. The mutant proenzyme was purified by biospecific affinity chromatography using an immobilized monoclonal antibody against PA. The maturation of the free and covalently immobilized purified proenzyme was studied in vitro. For the free proenzyme the same products with PA activity as observed in homogenates of wild-type PA-producing E. coli cells were found to be formed during this process. A kinetic analysis of the possible inter- and intramolecular processes involved in the maturation demonstrated that unambiguous evidence for the existence of intramolecular processes can only be obtained in systems where intermolecular processes are excluded. The Gly(263)-Ser(264) bond was found to be hydrolysed first in the free and immobilized mutant proenzyme, based on determinations of mass spectra, N-terminal sequences and active site concentrations. In the system with immobilized proenzyme intermolecular processes are excluded, demonstrating that this bond is hydrolysed by intramolecular autoproteolysis. Based on the known three-dimensional structure of the PA from E. coli the same maturation mechanism should apply for the wild-type proenzyme.

Enzyme denaturation in supercritical CO2: Stabilizing effect of S-S bonds during the depressurization step

SummaryThe stability of the monomeric enzymes α-chymotrypsin and trypsin, and the oligomeric enzyme penicillin amidase in supercritical CO2 has been studied. They were found to be partly denatured during the depressurization step. The degree of denaturation was larger in humid CO2 than in dry CO2. Enzymes with S-S bridges (α-chymotrypsin; trypsin) were denatured to a lesser degree than the enzyme without cysteine (penicillin amidase). These results and electrophoretic and spectroscopic analysis indicated that the denaturation was caused by partial unfolding during the depressurization step.

Nitrate removal of drinking water by means of catalytically active membranes

The reduction of nitrate to nitrogen in aqueous solutions by means of catalytically active membranes has been investigated. A heterogeneous catalyst (Pd/Cu) has been incorporated in a microporous polyetherimide membrane. After saturation with hydrogen nitrate containing water was filtered through these membranes. The nitrate reduction was studied as a function of pH, volume flow and temperature. It could be demonstrated that the catalyst remained active in the polymer matrix. The calculated activation energy for nitrate reduction is indicating that the reaction is dominated by mass transfer and diffusion.

Correlation of experimental and theoretical data for artificial and natural systems with immobilized biocatalysts

Abstract External and internal mass transfer limitations that influence the rate of substrate conversion by immobilized biocatalysts in artificial (biotechnical) and natural systems can be expressed by the following system properties: (i) dimensionless numbers that are ratios between the maximum rate of substrate conversion and the maximum rate of substrate transfer (Damkohler or Thiele modulus); (ii) stationary and operational effectiveness factors. Their magnitude must be estimated in applied and fundamental studies on immobilized biocatalysts in artificial and natural (cells) systems where observed rates should be directly proportional to the biocatalyst content, or to optimize technical systems by minimizing external and internal mass transfer limitations. Different procedures to calculate these properties are described and compared with respect to their potential use to predict experimental data.

A theoretical model describing steady-state catalysis by enzymes immobilized in spherical gel particles. Experimental study of α-chymotrypsin-sepharose

Abstract Analytical expressions describing steady-state kinetics are given for systems with enzymes enclosed or covalently bound in spherical gel particles. In experiments using immobilized CT ∗ the rate of product formation was found to be proportional to the square root of the enzyme content at low substrate concentration, and proportional to the enzyme content at high substrate content. These findings are in agreement with the results predicted from the theoretical model. The determination of the turnover number and Michaelis-Menten constant for immobilized enzymes is outlined. Some possible consequences for the description of enzyme kinetics in vivo are discussed.

Equilibrium and kinetically controlled synthesis with enzymes: semisynthesis of penicillins and peptides.

Publisher Summary This chapter focuses on equilibrium and kinetically controlled synthesis with enzymes. Generally the biosynthesis of condensation products is kinetically controlled. Whether this kinetically controlled process can compete with the equilibrium-controlled process in the biotechnological synthesis of condensation products must be answered by studies on the mechanism and the yield-controlling factors. Some of these factors are analyzed in the chapter in connection with the enzyme- catalyzed semisynthesis of β-lactam antibiotics and peptides. Higher yields are obtained in the kinetically controlled synthesis of condensation products catalyzed by enzymes than in the corresponding equilibrium-controlled synthesis. Contrary to that of the latter process, the yield of the former process depends on the properties of the used enzyme. In addition, large product yields can be obtained at the kinetically controlled maximum even when the (final) yield of the corresponding equilibrium-controlled process is negligible. Thus, the kinetically controlled maximum is a suitable end point in the synthesis of condensation products catalyzed by immobilized hydrolases.