Chromatography of mono- and disaccharides on granulated pellets of hydrophobic zeolites.

The chromatographic response of sugars at granulated zeolite pellets in preparative scale liquid chromatography is analyzed with respect to the distribution equilibrium and mass transfer. In contrast to hydrophilic FAU type zeolites their hydrophobic dealuminated counterpart, used here, can separate disaccharides the retention of which can strongly exceed those of the monosugars. The retention is correlated with data of batch adsorption studies from the literature. Whereas the retention decreases with increasing temperature, the peak sharpness shows the opposite trend. The effective mass resistance is calculated for a series of mono- and disaccharides. It increases with the capacity factor. The diffusion coefficient of the trehalulose disaccharide is restricted by a factor of about 2 in the macropores and by a factor of more than 104 in the micropores.

A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase.

Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.

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.

The roots--a short history of industrial microbiology and biotechnology.

Early biotechnology (BT) had its roots in fascinating discoveries, such as yeast as living matter being responsible for the fermentation of beer and wine. Serious controversies arose between vitalists and chemists, resulting in the reversal of theories and paradigms, but prompting continuing research and progress. Pasteur's work led to the establishment of the science of microbiology by developing pure monoculture in sterile medium, and together with the work of Robert Koch to the recognition that a single pathogenic organism is the causative agent for a particular disease. Pasteur also achieved innovations for industrial processes of high economic relevance, including beer, wine and alcohol. Several decades later Buchner, disproved the hypothesis that processes in living cells required a metaphysical 'vis vitalis' in addition to pure chemical laws. Enzymes were shown to be the chemical basis of bioconversions. Studies on the formation of products in microbial fermentations, resulted in the manufacture of citric acid, and chemical components required for explosives particularly in war time, acetone and butanol, and further products through fermentation. The requirements for penicillin during the Second World War lead to the industrial manufacture of penicillin, and to the era of antibiotics with further antibiotics, like streptomycin, becoming available. This was followed by a new class of high value-added products, mainly secondary metabolites, e.g. steroids obtained by biotransformation. By the mid-twentieth century, biotechnology was becoming an accepted specialty with courses being established in the life sciences departments of several universities. Starting in the 1970s and 1980s, BT gained the attention of governmental agencies in Germany, the UK, Japan, the USA, and others as a field of innovative potential and economic growth, leading to expansion of the field. Basic research in Biochemistry and Molecular Biology dramatically widened the field of life sciences and at the same time unified them considerably by the study of genes and their relatedness throughout the evolutionary process. The scope of accessible products and services expanded significantly. Economic input accelerated research and development, by encouraging and financing the development of new methods, tools, machines and the foundation of new companies. The discipline of 'New Biotechnology' became one of the lead sciences. Although biotechnology has historical roots, it continues to influence diverse industrial fields of activity, including food, feed and other commodities, for example polymer manufacture, biofuels and energy production, providing services such as environmental protection, and the development and production of many of the most effective drugs. The understanding of biology down to the molecular level opens the way to create novel products and efficient environmentally acceptable methods for their production.

Tools in oligosaccharide synthesis current research and application.

Oligosaccharides and polysaccharides have found manifold interests in the fields of food, pharmaceuticals, and cosmetics as a result of their various specific properties. Food, sweeteners, and food ingredients constitute important sectors where oligosaccharides are used in substantial amounts. Large amounts of sucrose isomers and derivatives, as well as major amounts of fructo-oligosaccharides are commercialized in Europe and worldwide as sweeteners, prebiotics, and other uses. Increasing attention has been devoted to the sophisticated roles of oligosaccharides and glycosylated compounds at cell or membrane surfaces, and their function, as in infection and cancer proliferation. The challenge for synthetic access is obvious, and convenient approaches using cheap and readily available substrates and enzymes are discussed here. Important examples of commercialized products and recent promising developments are presented in this chapter.

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.

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.

Science--or not? The status and dynamics of biotechnology.

This review traces the emergence of biotechnology as a new scientific discipline since the 1980s, when it became a major economic force. Significant changes in theoretical perception, research strategies, aims, and experimental methods, mainly in genetic engineering techniques, occurred during this period. The article is based on an analysis of its scientific status over four decades: the 60s and 70s when work in the field proceeded in different disciplines with a low level of coherence and little integration, then a significant change during the 80s and 90s when common approaches and the merging of molecular biology and biochemical engineering created a new discipline. The analysis covers scientific highlights and outstanding technical progress, presenting two studies undertaken by scientific and governmental agencies in Germany and the USA, as well as results of interviews and a questionnaire dealing with the scientific status of biotechnology. Answers to the questionnaire were obtained from internationally known scientists and from young scientists with biotechnology degrees. The results collected trace the transition of biotechnology from heterogeneous specialties and approaches towards a scientific discipline of its own. A hypothesis is put forward suggesting a new common paradigm allowing for a coherent perception the of phenomena observed.

Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium.

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His(6)- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His(6)-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates D-Gal-Fru, D-Xyl-Fru, D-Man-Fru, and D-Fuc-Fru.

Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase).

In the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination of d-glycopyranoside residues in (4)C(1) conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the beta-(1-->2)-fructosyl transfer to 2-OH of L-sugars (L-glucose, L-rhamnose, L-galactose, L-fucose, L-xylose) presumably in a (1)C(4) conformation. In those conformations, the L-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis.

A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium.

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.

New regioselective derivatives of sucrose with amino acid and acrylic groups.

We report here a range of new sucrose derivatives obtained from '3-ketosucrose' in aqueous medium with few reaction steps. As an intermediate, 3-amino-3-deoxy-alpha-D-allopyranosyl beta-D-fructofuranoside (1) was obtained via the classical route of reductive amination with much improved yield and high stereoselectivity. Building blocks for polymerization were synthesized by introduction of acrylic-type side chains, for example, with methacrylic anhydride. Corresponding polymers were synthesized. Aminoacyl and peptide conjugates were obtained through conventional peptide synthesis with activated and protected amino acids. Deprotection yielded new glycoderivatives having an unconventional substitution pattern, namely 3-(aminoacylamino) allosaccharides. Both mono- and di-peptide conjugates of allosucrose have been synthesized.

Synthesis and co-polymerization of an unsaturated 1,5-anhydro-D-fructose derivative.

An unsaturated derivative, 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-enopyranos-2-ulose (3), was obtained via regioselective elimination and acetylation of monohydrated 1,5-anhydro-D-fructose (1) in a single step reaction. High yield (80%) was achieved without any dimeric by-products. Its co-polymerization to saccharide polymers was investigated with different commercial vinyl co-monomers. Co-polymers were obtained and characterized.

Oligosaccharides and derivatives--integrating biocatalyst selectivity and chemical diversity.

Several biocatalytic pathways are available to synthesize rare sugars, oligosaccharides and derivatives. As examples selective hydrolysis, transglycosylation and regioselective oxidation are presented illustrating their potential. From inulin, difructose anhydrides and inulobiose are accessible via specific hydrolases with transglycosylation activity. Glycosyltransferases of the sucrase type utilise sucrose as a versatile substrate for the selective transfer of either glucosyl- or fructosyl units to a broad range of acceptors, sugars as well as derivatives, to yield oligosaccharides of different type. Kinetic analysis and reaction engineering can provide high product yields and concentration, as will be shown for glucose, yielding isomaltooligosaccharides as an example. Several new acceptor saccharides, including sugar alcohols and acids, have been shown to give new oligosaccharides. Regioselective oxidation of disaccharides like sucrose, lactose and others with resting cells of Agrobacterium tumefaciens yield 3-keto-disaccharides. These can further be functionalized in aquous systems without protecting groups via established chemical routes, such as catalytic hydration. As an example allosucrose is thus obtained from 3-keto-sucrose with high yield and stereochemical selectivity, which in turn can easily be hydrolysed and separated from fructose to give allose. Amino acid and peptide conjugates are accessible via reductive amination and acylation, as are building blocks for polymerisation.

Oligosaccharide synthesis by dextransucrase: new unconventional acceptors.

The acceptor reactions of dextransucrase offer the potential for a targeted synthesis of a wide range of di-, tri- and higher oligosaccharides by the transfer of a glucosyl group from sucrose to the acceptor. We here report on results which show that the synthetic potential of this enzyme is not restricted to 'normal' saccharides. Additionally functionalized saccharides, such as alditols, aldosuloses, sugar acids, alkyl saccharides, and glycals, and rather unconventional saccharides, such as fructose dianhydride, may also act as acceptors. Some of these acceptors even turned out to be relatively efficient: alpha-D-glucopyranosyl-(1-->5)-D-arabinonic acid, alpha-D-glucopyranosyl-(1-->4)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-mannitol, alpha-D-fructofuranosyl-beta-D-fructofuranosyl-(1,2':2,3')-dianhydride, 1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol ('D-glucal'), and may therefore be of interest for future applications of the dextransucrase acceptor reaction.