Hans-Joachim Jördening

Glycosylation with activated sugars using glycosyltransferases and transglycosidases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a requirement for the general availability of larger amounts of varying carbohydrate structures. Thus the synthesis of oligo-, polysaccharides and glycosylated substances/products represents a major challenge. Activated sugars are key substrates for synthesis and glycosylation. Nucleotide activated sugars are natural tools for highly selective synthesis, providing complex polysaccharides, glycopeptides, glycolipids etc. However their high cost and availability limit their application. Sucrose acts as an activated substrate for a range of sucrase enzymes elaborating natural polysacchrides of the glucan and fructan type, which also serve for the synthesis and technical production of different oligosaccharides. Sucrose analogues have been shown to extend the range of oligosaccharide synthesis making new structures available incorporating further monosaccharides, such as mannose, galactose, xylose, rhamnose and fucose. A short overview of the use of glycosyl phosphates and glycosyl fluorides as substrates is also given.

Comparison of different models of substrate and product inhibition in anaerobic digestion

Abstract In batch-experiments (in lab-scale fluidized bed reactors) the inhibition of acetate- and propionate-degradation by propionate (substrate inhibition) and the inhibition of propionate degradation by acetate (product inhibition) was studied. Various models were compared by fitting them to the experimental data. The importance of independent variation of the acid concentrations and the pH for the experimental design is discussed. The substrate inhibition was best described by a model of inhibition by undissociated acid with an additional independent pH influence. The inhibition by propionic acid was only slight in the practically relevant range of concentrations. However, the propionate degrading bacteria were sensitive against low pH. The product inhibition was best described with the model of competitive inhibition. The inhibition already occurred from an acetate/propionate-ratio of 1 upwards. A complete standstill of the propionate degradation at high acetate/propionate-ratios, as expected because of the proximity to the thermodynamic equilibrium, was not observed.

Kinetics of the dextransucrase acceptor reaction with maltose—experimental results and modeling

The acceptor reaction of the dextransucrase from L. mesenteroides NRRL B-512 F is of technical interest for oligosaccharide synthesis. In this work the acceptor reaction of maltose, the strongest acceptor known so far, has been investigated at different experimental conditions. The data obtained were used for modeling of dextransucrase catalysis with a kinetic model that had been developed earlier.

Co-immobilization of dextransucrase and dextranase for the facilitated synthesis of isomalto-oligosaccharides: Preparation, characterization and modeling

Co‐Immobilization of dextransucrase (DS) and dextranase (DN) into calcium alginate includes the co‐entrapment of soluble DS and adsorbed DN. DS converts sucrose into dextran, which is the substrate for DN, so that isomalto‐oligosaccharides (IMOs) are follow‐up products of dextran hydrolysis. The boundary conditions for the successful preparation are investigated with respect to choice of DN adsorbate, surface modifications using blotting agents and optimal enzyme activity ratios. Further, repetitive batch experiments suggest the selection of medium activity ratios for continuous use (0.3 UDNU−1DS, e.g.). Product formation at various cosubstrate:substrate concentrations as well as at different DN:DS ratios are discussed. Moreover, the complexity of the bi‐enzymatic system can be reduced considering the molar ratios of cosubstrate:substrate (glucose:sucrose). Based on these factors, a mechanistic kinetic model is developed, which distinguishes the corresponding contributions of the two enzymes upon overall product formation. In general, at low glucose:sucrose ratios isomaltose synthesis is featured primarily by DN action. Yet with increasing amounts of glucose both the quantity and quality of DN substrate changes, so that its contribution to product formation decreases in an exponential manner; still the overall product yield continuously increases due to enhanced DS contribution. Biotechnol. Bioeng. 2008;100: 673–683.

Modelling of oligosaccharide synthesis by dextransucrase

Dextransucrase catalyses the formation of dextran, but also of numerous oligosaccharides from sucrose and different acceptors, if appropriate conditions are chosen. Much experimental work has been carried out and a scheme of reactions and a mathematical model have been developed to describe the complex kinetic behaviour of the enzyme. A computer program was used to calculate the parameters of the model from a broad range of experimental data, investigating a large number of kinetic tests with the acceptors maltose and fructose. The results lead to design considerations for a continuous reactor system with immobilized dextransucrase to produce leucrose, a disaccharide of industrial interest.

Kinetic modeling of oligosaccharide synthesis catalyzed by leuconostoc mesenteroides NRRL B-1299 dextransucrase

The kinetic behavior of soluble and insoluble forms of dextransucrase from Leuconostoc mesenteroides NRRL B-1299 was investigated with sucrose as substrate and maltose as acceptor. To study the parameters involved, a kinetic model was applied that was previously developed for L. mesenteroides NRRL B-512F dextransucrase. There are significant correlations between the parameters of the soluble form of B-1299 dextransucrase and those calculated for the B-512F enzyme; that is, their properties are comparable and differ from those of the insoluble form of B-1299 dextransucrase. Whereas the calculated parameters for high maltose concentrations describe the kinetic behavior very well, the time curves for low maltose concentrations were not described correctly. Therefore, the parameters were calculated separately for the two ranges. Copyright 1999 John Wiley & Sons, Inc.

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.

Extending synthetic routes for oligosaccharides by enzyme, substrate and reaction engineering.

The integration of all relevant tools for bioreaction engineering has been a recent challenge. This approach should notably favor the production of oligo- and polysaccharides, which is highly complex due to the requirements of regio- and stereoselectivity. Oligosaccharides (OS) and polysaccharides (PS) have found many interests in the fields of food, pharmaceuticals, and cosmetics due to different specific properties. Food, sweeteners, and food ingredients represent important sectors where OS are used in major amounts. Increasing attention has been devoted to the sophisticated roles of OS and glycosylated compounds, at cell or membrane surfaces, and their function, e.g., in infection and cancer proliferation. The challenge for synthesis is obvious, and convenient approaches using cheap and readily available substrates and enzymes will be discussed. We report on new routes for the synthesis of oligosaccharides (OS), with emphasis on enzymatic reactions, since they offer unique properties, proceeding highly regio- and stereoselective in water solution, and providing for high yields in general.

Development of a multiphase reaction system for integrated synthesis of isomaltose with a new glucosyltransferase variant

Abstract A new genetically derived variant of the glucosyltransferase from Streptococcus oralis has been characterized physicochemically and kinetically. Compared with the industrially used glucosyltransferase from Leuconostoc mesenteroides, the enzyme variant GTF-R S628D possesses 25 times higher affinity for the specific glucosylation of glucose. For a concept of integrated reaction and product isolation, a fluidized bed reactor with in situ product removal was applied. The technical feasibility and the applicability of the kinetic models for reaction and adsorption could be demonstrated. The immobilized enzyme was stable (20% activity loss after 192 h) and product could be obtained with 90% purity. A bioprocess model was generated which allowed the integral assessment of the enzymatic synthesis and in situ product adsorption. The model is a powerful tool which assists with the localization of optimal process parameters. It was applied for the process evaluation of other glucosyltransferases and demonstrated key characteristics of each enzymatic system.

Improved laminaribiose phosphorylase production by Euglena gracilis in a bioreactor: A comparative study of different cultivation methods

Laminaribiose phosphorylase (EC 2.4.1.31) catalyzes a reversible phosphorolysis reaction in which laminaribiose, a very high value sugar is produced. This enzyme is not being produced commercially therefore, to realize the most effective method for producing laminaribiose phosphorylase and obtaining as much activity units as possible per liter of culture, different cultivation methods of Euglena gracilis were compared. Heterotrophic and mixotrophic cultivations of Euglena gracilis in two different pHs, in flask and bioreactor were performed. The reverse phosphorolysis activity of laminaribiose phosphorylase produced under different cultivation methods was measured. The heterotrophic approach showed to be the more effective cultivation method as 47.6 IU/L was obtained compared to 27 IU/L in the mixotrophic one. The heterotrophic cultivation then was further investigated under two different pH values of the culture media. The culture at pH 6.8 resulted in 7.94 IU/L/day whereas only 4.06 was obtained for the culture at pH 4. Cultivation in a bioreactor resulted in a distinctive amount of 191.5 IU/L and an activity yield of 9.7 IU/g glucose compared to 5.4 in flask cultivation. Heterotrophic cultivation of Euglena gracilis in a bioreactor containing a culture media at pH 6.8 and controlled operation conditions showed enhanced laminaribiose phosphorylase activity production per liter and day of cultivation.