Klaus Buchholz

Industrial carbohydrate biotransformations

Nearly all major industrial processes which involve carbohydrates, include biotechnological transformations. This is due to the complex nature of carbohydrates where stereo- and regioselectivity are highly complex and difficult to control. Enzymes and microorganisms work highly selectively and efficiently in water solution, and provide high yield in general. The article focuses on different types of reactions, including large-scale processes. Topics are hydrolytic reactions, including starch processing, oxidation and reduction transformations including organic acids, such as gluconic and ketogluconic acids and vitamin C synthesis, and isomerization and transfer reactions, which are established on a very large scale to produce glucose/fructose syrups and sucrose isomers. The article will further discuss some mechanistic aspects which are relevant for technology and present selected details of industrial-scale processing. Finally an outlook outlines perspectives of future processes.

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.

Enzymatic Degradation of (Ligno)cellulose

Glycoside-degrading enzymes play a dominant role in the biochemical conversion of cellulosic biomass into low-price biofuels and high-value-added chemicals. New insight into protein functions and substrate structures, the kinetics of recognition, and degradation events has resulted in a substantial improvement of our understanding of cellulose degradation.

Synthesis of novel fructooligosaccharides by substrate and enzyme engineering.

Fructooligosaccharides (FOSs) and polyfructosides (PSs) have received particular attention due to its beneficial effects as prebiotics. Here we report the synthesis of a new class of fructooligosaccharides by substrate and enzyme engineering. Using an engineered levansucrase enzyme (SacB of Bacillus subtilis), and sucrose analogues (alpha-Xyl-1,2-beta-Fru or alpha-Gal-1,2-beta-Fru), the product profile shifted from the fructan (levan) polymer to a range of new higher oligosaccharides (xylooligofructosides), or polysaccharides (galactopolyfructosides), of varying size. Further the enzyme was tailored by random mutagenesis, for the synthesis of short-chain fructooligosaccharides to yield variant A5 (N242H), which is unable to produce polymers. It shifts its product pattern to short-chain oligosaccharides and hydrolysis and enabled in combination with the sucrose analogue Xyl-Fru for the first time the direct synthesis of a 6-kestose analogue (alpha-Xyl-1,2-beta-Fru-2,6-beta-Fru). The different glycopyranosyl-residues (i.e. galactose and xylose) that cap fructooligosaccharides may alter prebiotic and biochemical properties.

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.

Unconventional immobilization of dextransucrase with alginate

Abstract An unconventional immobilization of dextransucrase via entrapment in alginate is described, which provides high activity yield and good operational stability. This method is normally restricted to the immobilization of complete cells or parts of cells. Experiments were designed to provide evidence for the immobilization mechanism. It was shown that a typical globular protein, α-chymotrypsin, is neither immobilized in an alginate matrix nor by formation of an additional network of dextran inside alginate. The results suggest that dextransucrase immobilization in alginate is due to a unique supramolecular structure.

A Bacillus megaterium Plasmid System for the Production, Export, and One-Step Purification of Affinity-Tagged Heterologous Levansucrase from Growth Medium

ABSTRACT A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His6-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.

Regioselective synthesis of new sucrose derivatives via 3-ketosucrose.

3-Ketosucrose (alpha-D-ribo-hexopyranosyl-3-ulose-beta-D-fructofuranoside), obtained from sucrose via microbial oxidation with Agrobacterium tumefaciens, was shown to be an appropriate and versatile synthon for regioselective syntheses. Condensation with hydroxylamine and its derivatives with allyl and benzyl groups leads to the oxime and the corresponding substituted products. By reductive amination 3-amino-3-deoxy-alpha-D-allopyranosyl-beta-D-fructofuranoside is obtained which can readily be submitted to further functionalization to methacryloyl and fatty acid derivatives. After silylation of 3-ketosucrose the 3-allyl and butylene-substituted as well as decyl- and dodecyl-substituted sucrose can be obtained via Grignard reaction, the side chains being C-C linked to the saccharide.

Productivity of immobilized dextransucrase for leucrose formation

Abstract Continuous leucrose formation with dextransucrase entrapped in calcium alginate beads was investigated. A plug-flow packed bed reactor was used as the reaction vessel and the conversion of sucrose as a function of residence time was measured at different initial sucrose concentrations. Leucrose productivity by the immobilized enzyme at different reaction conditions was determined and compared to the productivity of a discontinuous operation process. Productivities of the continuous process were in a range of 0.7–3.6 g U−1 whereas only 0.7 g U−1 could be reached in the discontinuous process; moreover, inactivation constants of the immobilized enzyme in the continuous operation mode were determined. The enzyme was continually inactivated with increasing reaction time. This was due to transport limitations inside the enzyme particles that could be confirmed by investigation of the swelling behavior of the enzyme beads due to the formation of dextran.

Microbial modification of sugars as building blocks for chemicals

SummaryInvestigations on the microbial modification of sucrose to the corresponding 3-keto-derivative were carried out with resting cells of Agrobacterium tumefaciens NCPPB 396. This highly specific oxidation to yield the 3-keto-derivative has been analysed kinetically with varying substrate and cell mass concentrations. The formation of the corresponding 3-keto-derivative depended strongly on the reaction time and the aeration rate, and was maximal at aeration rates up to 11.5 volume air/cultivation volume per minute with resting cells. The product formation increased with increasing substrate concentrations. However, the product yield was maximal at substrate concentrations below 20 g/l. Data pertaining to the production of active cell mass as well as for maximal 3-keto-derivative formation are presented in this paper. Also included are some applications for these derivatives.