Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei

Summary The new enzyme d-2-hydroxyisocaproate dehydrogenase (NAD⁺-dependent) was detected in strains of the genus Lactobacillus and related genera. Straight and branched chain aliphatic as well as aromatic 2-ketocarboxylic acids are stereospecifically reduced to the corresponding d-2-hydroxycarboxylic acids according to the following equation:R-CO-COOH + NADH + H⁺ R-CHOH-COOH + NAD⁺ The enzyme is called d-hydroxyisocaproate dehydrogenase by us because 2-ketoisocaproate is the substrate with the lowest K_M-value. NAD(H) as a cofactor cannot be replaced by NADP(H). Because of its broad substrate specificity we chose the strain Lactobacillus casei ssp. pseudoplantarum (DSM 20 008) for enzyme production and characterization. d-2-hydroxyisocaproate dehydrogenase could be purified 180-fold starting with 500 g of wet cells.The purification procedure involved liquid-liquid extraction with aqueous two-phase systems and ion-exchange chromatography. At this stage the enzyme has a specific activity of 25 U/mg and can be used for technical applications. Further purification up to a homogeneous protein with a specific activity of 110 U/mg can be achieved by chromatography on Amberlite CG 50 at pH 3.5. Properties important for technical application of the d-HicDH were investigated, especially the substrate specificity and the optimum pH- and temperature ranges for activity and stability of the catalist.     

Localization of the enzyme 3-deoxy-d-manno-2-octulosonic acid aldolase

Summary Several strains of Gram-negative microorganisms were screened for maximum 3-deoxy-d-manno-2-octulosonic acid (KDO) aldolase (EC 4.1.2.23) activity. Although this enzyme has been noted to be inducible on special medium, no induction was found. By centrifugation studies the KDO aldolase was found to be localized in the cell wall or membrane fraction. The enzyme activity was very susceptible to small amounts of detergent in solution. Offprint requests to: M.-R. Kula     

An approach to integrated antibody production: Coupling of fluidized bed cultivation and fluidized bed adsorption

Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.The help of H. Schmitz, A. Bader, J. Gätgens and M. Halfar during the experiments is gratefully acknowledged. This work was partially funded by the ministry of science and research of the Federal Republic of Germany within the project Stoffumwandlung mit Biokatalysatoren.     

Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption

To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose-based matrix (Stream-line-SP), and a self-derivatized material based on controlled-pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose-based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. (c) 1995 John Wiley & Sons, Inc.     

Studies of the interaction between aminoacyl-tRNA synthetase and transfer ribonucleic acid by equilibrium partition.

The partition behavior of isoleucyl-tRNA synthetase, leucyl-tRNA synthetase and tRNA in aqueous two-phase systems composed of the polymers poly(ethyleneglycol) and dextran was investigated. From the results of this investigation a two-phase system could be derived which can be employed for the study of the interactions between synthetases and their cognate tRNAs by equilibrium partition. These measurements show that in each case one molecule of cognate tRNA is bound per molecule of enzyme. The binding constants were in the range 1-5micronM-1. It could be demonstrated that equilibrium partition is a useful method for the study of interactions between macromolecules.     

Studies on immobilized α-glucosidase from brewers yeastSaccharomyces carlsbergensis

Summary -Glucosidase isolated from brewer's yeast (Saccharomyces carlsbergensis) was immobilized using hydroxymethacrylate activated by cyanogen bromide as a carrier. Up to a hundred-fold increase in the stability of the enzyme was observed after immobilization. The yield in activity (bound/applied) was up to 30%. Before developing the process of enzymatic cleavage of maltose further we evaluated the kinetic properties of the enzyme catalyst, as we had observed earlier that the soluble enzyme is strongly inhibited by the product glucose. This is even more pronounced with the immobilized -glucosidase leading in this case to a linear relation between initial rate and substrate concentration, so K_M (approx.) values can no longer be defined due to the dominating influence of the product inhibition.     

Evaluation of crude dextran as phase-forming polymer for the extraction of enzymes in aqueous two-phase systems in large scale

A detailed study of the influence of crude dextran on enzyme extractions in aqueous phase systems is presented in this article. The physical parameters of crude dextran, a purified T-500 fraction from Pharmacia, and a hydrolyzed crude dextran are compared and their influence on the phase system parameters investigated. Initially there is a drastic increase in the viscosity of the lower dextran-rich phase and a significant shift in the macroscopic structure of these phases, observed as the "gel-forming" properties of the dextran phases. The latter can be important for the partition of any enzyme by influencing the effect of phosphate concentration on the partition of proteins, although these experiments show that the partition coefficient of several enzymes is not much altered. The partition parameters allow the substitution of Dextran T-500 fractions by crude dextran or unfractionated, slightly hydrolyzed fractions. Using crude dextrans the performance and technical realization of enzyme extraction processes are demonstrated for pullulanase from Klebsiella pneumoniae and formate dehydrogenase from Candida boidinii.Both enzymes were recovered in comparable high yields. The equipment performance was quite good, as indicated by the high throughput values of the separators employed. Especially when using nozzle separators for phase separation there is a better performance in comparison to the Dextran T-500 fraction. No serious technical problems were encountered when replacing the expensive fractionated dextran with a crude dextran. In this way aqueous two-phase systems containing dextran become more feasible for enzyme purification from an economic point of view. The price of about 1.30 German marks (DM) per liter for a useful phase system already appears acceptable for the production of valuable intracellular enzymes.     

A two-step enzymatic synthesis of dipeptides

A simple system is introduced to produce dipeptides continuously by enzyme catalyzed condensation of amino acid esters and amino acid amides. Synthesis of N-terminal free dipeptide-amides is achieved by means of carboxypeptidase Y. The peptide-amide is deamidated utilizing a newly isolated peptide-amide is deamidated utilizing a newly isolated peptide-amidase. Separation of substrates and products is accomplished by anion-exchange chromatography. Modeling of the reactions shows that the two reactions have to be carried out in a cascade of two reactors in order to prevent hydrolysis of the peptide by the carboxypeptidase. Continuous production of Kyotorphin (H-TyrArg-OH) with a space-time yield of 257 g/L . d shows the feasibility of this concept.