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Carbohydrate bioengineering The diversity of carbohydrate structures is a consequence of the broad array of monomers of which they are composed, and of the different ways in which the monomers are joined and, in many instances, modified. Not surprisingly, carbohydrates can exhibit remarkable biological specificities, both as modifiers of proteins and as ligands. Some polysaccharides act as energy and carbon reserves in cells, and some are structural components of plant cell walls; as such, they offer abundant renewable sources of feedstocks for the chemical and fermentation industries. Polysaccharides have many direct applications, including use as lubricants, gelling agents, blood expanders and fibres for making paper and cloth. Currently, there is widespread interest in the structures, activities, synthesis, degradation, modification and applications of carbohydrates. This interest was displayed at a meeting* organized by Sven Pedersen (Novo Nordisk A/S, Copenhagen, Denmark), Steffen Petersen (SINTEF U N I M E D , M R - C e n t r e , Trondheim, Norway) and Birte Svensson (Carlsberg Laboratory, Copenhagen, Denmark). The 34 presentations and 81 posters together covered: the structure and analysis of carbohydrates and carbohydrateactive enzymes; new enzymes; cellulolytic, starch metabolizing and cell wall degrading enzymes; carbohydrates in medicine; applications of protein engineering; transgenic organisms; oligo- and polysaccharides of industrial interest; and the production of new carbohydratecontaining materials.

Carbohydrate structurefunction Nathan Sharon (Weizmann Institute of Science, Rehovot, Israel) opened the proceedings with a discussion of the glycans ofglycoconjugates as modulatory molecules. The attachment of glycans can have marked effects on the physicochemi* The meeting 'CarbohydrateBioengineering' was held in EMnore, Denmark, 23-26 April 1995, and was sponsored by DG XlI of the European Commission, the European Congress of Biotechnology 5, the Nordic Fund for Technology and IndustrialDevelopment, Novo Nordisk A/S, Nutek and SNF. © 1995, ElsevierScience Ltd 0167- 7799/95/$9.50

cal properties and structures of proteins, alter their lifetimes in the circulation and modulate their biological activities. The signals encoded in the glycans are recognized by specific carbohydrate-binding proteins, such as lectins. A variety of pathogens produce lectins that recognize glycans on the surfaces of animal cells. Free carbohydrates that can block such interactions may make the anti-adhesion treatment of infectious diseases possible. Phagocytic cells may be activated by the interaction of their surface glycans with lectins on the surfaces of pathogens. Lectin-carbohydrate interactions also play a role in leucocyte migration, so inhibitors of such interactions may serve as anti-inflammatory agents. Nathan Sharon emphasized the need for detailed information about the binding sites of lectins, and the need for suitable methods to synthesize oligosaccharides. This will require an in-depth understanding of the structures of oligosaccharides in solution, and of the enzymes for potential use in oligosaccharide synthesis. Herman van Halbeek (University of Georgia, Athens, GA, USA) discussed N M R spectroscopic analysis of oligosaccharides in solution. The molecules are usually flexible, with the preferred conformations being stabilized by intramolecular hydrogen bonds. Binding to a lectin locks a molecule into one conformation. Bo Nilsson (NDR_E, Ume~, Sweden) covered the analysis of protein glycosylation by mass spectrometry, and Friedrich Spener (WestfJ.lische Wilhelms Universit~it, Miinster, Germany) spoke about the development of a novel enzyme-based glucose sensor for monitoring blood glucose. In order to analyze the monosaccharide composition and glycosidic-bond positions in glycoprotein glycans, the glucan must be periodate-oxidized and permethylated or acetylated prior to mass spectrometry. The glucose sensor has glucose oxidase from Aspergillus niger immobilized within a silicon chip of appropriate conformation; it uses quinones rather than hydrogen peroxide as oxidants. The enzyme is immobilized via aldehyde groups that are introduced into the glycans of the enzyme by periodate oxi-

dation. The sensor has been incorporated into a hand-held device, the tip of which is a microdialysis needle that is in contact with the analyte in the subcutaneous tissue. The sessions on carbohydrates in medicine covered a wide range of topics. Strain-dependent variation of the neuraminidase of the influenza virus appears not to include activesite residues. Peter Colman (Biomolecular Research Institute, Parkville, Australia) reported that probes for these residues are used to define potential binding sites for inhibitors, leading to the design of new inhibitors that block virus replication in tissue culture and that have antiviral properties in animal models. Glycogen phosphorylase, a key enzyme in the regulation of glycogen metabolism, is inhibited by glucose. Naturally occurring and synthetic glucose analogues, which are more potent inhibitors of the enzyme than glucose, enhance glycogen deposition and stimulate glucose uptake by isolated hepatocytes. Such analogues are potential agents for the alleviation ofhypoglycaemia in Type II diabetes (Louise Johnson; Laboratory of Molecular Biophysics, University of Oxford, UK). The Escherichia coli maltodextrin phosphorylase is analogous to m a m malian glycogen phosphorylase and, although the structure of glycogen phosphorylase is known in some detail, its carbohydrate-binding site is not defined. Site-directed mutation of maltodextrin phosphorylase is being used to identify residues involved in carbohydrate binding at the active site. The residues chosen for mutation are those that are conserved in all known phosphorylases, and which are located close to a putative channel in phosphorylase b (R.einhard Schinzel; TheodorBoveri-Institut der Universidit Wiirzburg, Germany).

Meeting report

Medical carbohydrates The selectin family of adhesins is involved in the trafficking and recruitment of neutrophils, and trafticking other leucocytes to sites of inflammation. Selectins recognize the glycans (typically the sialyl Lewis X glycans) of the surface glycoproteins of the leucocytes. Chemical and enzymatic methods are being used for the large-scale production of sialyl Lewis X for clinical testing as a moderator of acute and chronic inflammatory diseases, and for the prevention of reperfusion injury TIBTECHNOVEMBER1995 (VOL13)

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forum (James Paulson; Cytel, San Diego, CA, USA). A T-cell response to some glycoprotein antigens on cell surfaces may follow the recognition of the glycans by M H C class II molecules. Glycopeptides bind to the murine E k molecule, but usually less readily than do the corresponding glycan-free peptides; owing to their bulk, the glycans may interfere with binding to the cleft on the M H C molecule. The sugars appear to remain attached to the peptides during antigen processing (Luisa Galli Stampino, M&E A/S, Copenhagen, Denmark).

Enzymes old and new The session on new enzymes presented a lot of new information on well-known enzymes. Apart from its therlnostability, a 13-glycosidase (S-13-gly) from the extremophile Sulfolobus solfataricus is a typical family 1 enzyme. It is a tetramer of identical subunits, with an optimum activity temperature of 87°C. S-13gly hydrolyzes cellooligosaccharides exoglycolytically, and is particularly active on cellotetraose. It transglycosylates, and can be used to synthesize various glycosides in a two-phase system containing 90% octanol. S-13-gly is the first family 1 enzyme to be crystallized in a form suitable for determining its structure (Most P, ossi; Istituto di Biochimica delle Proteine ed Enzimologia del C N R , Napoli, Italy). The lack of specificity of many dehydrogenases and oxidoreductases that act on monosaccharides allows them to be used for the conversion ofmono- and disaccharides to a variety of products, including polyhydroxyalcohols, D- and L-aldonic acids, D- and L-ketosugars, and aromatic alchohols; many of the products can be used as food ingredients. The products are synthesized continuously in membrane reactors, which incorporate secondary enzymes for coenzyme regeneration; this makes coenzymes a minor cost factor (Klaus Kulbe; University of Agriculture, Vienna, Austria). Identifying the residues that determine the substrate and cleavage specificities of the Bacillus licheniformis 1,3-1,4-13-Dglucan-4-glucanohydrolase is a prerequisite for redesigning the properties of the enzyme. The active-site cleft comprises subsites I-IV. An analysis of the competitive inhibition of hydrolysis of a methylumbelliferyl 13-glucan tetrasaccharide substrate by a variety of oligosaccharides TIBTECHNOVEMBER1995 (VOL13)

with 13-1,3 and 13-1,4 linkages has led to a proposed model for substrate binding to the cleft, with a 3-O-glucopyranose being bound preferentially at subsite I (Antoni Planas, Universitat Ramon Hull, Barcelona, Spain). E n z y m e structure-function The session on structure and function of carbohydrate-active enzymes reviewed the variety of approaches for analyzing the catalytic mechanisms of the enzymes, identifying catalytically important residues and modifying enzyme activity. Hydrolysis of glycosides by glycosidases occurs with either retention or inversion of configuration at the anomeric centre. Retaining enzymes use a double-displacement mechanism that involves a glycosylenzyme covalent intermediate at the catalytic nucleophile. The nucleophile is identified by trapping the covalent intermediate with a mechanism-based inhibitor, hydrolyzing it with an appropriate protease, and detecting and sequencing the glycosyl-peptide by electrospray tandem mass spectrometry. However, the acid-base catalyst in a retaining enzyme is harder to identify, but it has been identified in several enzymes by detailed kinetic analysis of mutants in which highly conserved Asp or Glu residues are replaced with Ala (Stephen Withers; University of British Columbia, Vancouver, Canada). Glycosides that have the interosidic bond formed with a sulfur atom instead of an oxygen atom are resistant to hydrolysis by glycosidases, making them powerful substrate analogues for analysis of the enzymes. Oligosaccharides that have one or more sulfur linkages in appropriate positions for analysis of exo- or endoglucosidases can be synthesized (Hugnes Drignez; CN1KS, Grenoble, France). Glucoamylase from Aspergillus is an inverting exo-ci-glucanase with a modular structure, comprising an (odo06 catalytic domain, a Ser/Thrrich linker, and a starch-binding domain. X-ray crystallography, sitedirected mutation and kinetic and thermodynamic analyses have enabled substrate recognition and the mechanism of catalysis to be understood in considerable detail. In contrast to glucoamylase, the catalytic domains ofo~-amylases, are generally (cx/13)8 barrels. The structure-function relationship in barley a-amylase has been examined by isozyme

hybrid formation, random mutation of a binding sequence, and sitedirected mutation (Birte Svensson; Carlsberg Laboratory). Chemical modification ofxylanase A, a retaining enzyme from SchizophyUum commune, has identified Glu87 as the nucleophile; in addition, Tyr97 is essential for catalytic activity. The substrate protects Tyr97 from modification by tetranitromethane. Glu87 and Tyr97 are conserved in all of the family 11 xylanases (Anthony Clarke; University of Guelph, Guelph, Ontario, Canada).

Protein engineering Protein engineering can be used to modify enzymes in a variety of ways to achieve specific goals. The cyclodextrin glycosyltransferase from Bacillus circulans strain 251 forms a mixture of(x, [3 and y cyclodextrins that can be separated by selective precipitation with organic solvents; however, this is an expensive procedure. The enzyme is modular, with an (cx/13)8 barrel catalytic domain, a starch-binding domain and three other domains. Protein engineering is being used in an attempt to obtain an enzyme(s) that produces predominantly one type of cyclodextrin (Lubbert Dijkhuizen; University of Groningen, Groningen, The Netherlands). TermamyFM is an amylase from B. licheniformis that has superior performance at high pH and high temperature, making it potentially useful for laundry and dishwashing detergents. Unfortunately, it is oxidation labile, with Met197 being the major cause of lability; substitution of Met197 with Thr, Ser, Gly, Ala or Asn increases thermoactivation (Torben Borchert; Novo-Nordisk A/S, Bagsvaerd, Denmark). The electrostatic fields that surround proteins are thought to influence substrate recognition, protein aggregation, protein stability and pH optima. Iterative computational tools for pH-dependent electrostatic descriptions of proteins have been developed for application to proteins of known, inferred, or modelled structure. The information that is obtained helps when engineering a protein (Steffen Petersen; SINTEFUNIMED). Glycosylation of a protein can change its properties significantly. The chemical removal of the glucans from horseradish peroxidase C does not change its thermal stability, but the unfolding rate constant for irreversible unfolding in

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EDTA-guanidinium hydrochloride is increased threefold, showing the importance of distinguishing between thermodynamic and kinetic protein stability; deglycosylation decreases the solubility in salt solutions by two orders of magnitude. The properties of peroxidase derivatives with different levels of glycosylation from Coprinus show that the magnitude of such effects is proportional to the extent of glycosylation (Karen Welinder; University of Copenhagen, Copenhagen, Denmark).

Cellulose-degrading enzymes Cellulolytic enzymes are of widespread interest because of their potential industrial applications. However, relatively little is known about their three-dimensional structures and catalytic mechanisms. The most striking aspects of the threedimensional structures of the catalytic domains ofcellobiohydrolases I and II from Trichoderma reeseiare the tunnel-shaped active sites into which the ends of cellulose molecules can thread; these account for the exoglycolytic activities of the enzymes. Catalytic and substrate-binding residues are identified by structure analysis and site-directed mutations (Tuula Teeri; VTT, Espoo, Finland). Endoglucanase I from Humicola insolens is structurally similar to CbhI from T. reesei, but it has an open active-site cleft with seven potential sugar-binding subsites. Endoglucanase V from H. insolens also has an open active-site cleft, but a loop moves to close the cleft when substrate is bound, bringing Asp 144 into the active site and a hydrophobic residue over the substrate (Gideon Davies; University of York, York, UK). The catalytic domain of endoglucanase A, a retaining enzyme from Clostridium cellulolyticum, is an (ci/[3)8 barrel. Examining the structure and mutating Glu170 and Glu307 has revealed that Glu170 is the acid-base catalyst, and that Glu307 is the nucleophile. The structure is virtually superimposable on that of the catalytic domain of Cex, a family 11 retaining exo-glycosidase from Celtulomonasfimi(Valerie Ducros; LCCMB, C N R S - U R A , Marseille, France). Most of the cellulases from Clostridium thermocellum are found in a multi-enzyme complex - the cellulosome. Scaffoldin is a multifunctional non-catalytic protein that has multiple attachment sites (cohesins) for binding specific dock-

ing domains (dockerins) on cellulases. Scaffoldin also has a cellulosebinding domain that adsorbs the cellulosome to cellulose. The discrete domains within the proteins of the cellulosome can be exploited for a variety of applications by using gene fusions or chemical cross-linking. Dockerin fusions bind to scaffoldin and, hence, to cellulose. Constructing fusions or cross-linking to the cellulase-binding domain also allows binding to cellulose (Edward Bayer; The Weizmann Institute of Science, Rehovot, Israel). The eight [3-1,4glucanases from C. fimi that have been characterized to date are modular proteins. They interact with substrate in two ways: the catalytic domains hydrolyze cellulose molecules, but bind only transiently to cellulose during the hydrolytic event; or, the cellulose-binding domains adsorb strongly to cellulose but have no hydrolytic activity. The isolated cellulose-binding domain from endoglucanase A has a disruptive effect on cotton fibres, despite lacking hydrolytic activity; it interacts synergistically with the catalytic domain of CenA in the release of reducing sugars from cotton fibres (Tony Warren; University of British Columbia, Vancouver, Canada).

Transgenic organisms There were two fascinating presentations on the application of transgenic organisms to carbohydrate engineering. Martin Quanz (Institut f'tir Genbiologische Forschung, Berlin, Germany) discussed how the structure and formation of starch, and the starch content of tubers, can be modified in transgenic potato plants. The expression of glycogen-biosynthesis genes in potato plants, coupled with the antisense-RNA control of starch biosynthesis genes, results in the synthesis of amylose-free starch. Such modified starches have commercial potential. Simi Ali (University of Newcastle, Newcastle, UK) proposed how nutrition of nonruminant livestock could be improved dramatically by the presence of cellulase activity in their small intestines. The expression of a gene fusion, encoding endoglucanase E from C. thermocellumwith a glycosylphosphatidylinositol anchor, in the exocrine pancreas oftransgenic mice leads to the appearance of low levels of non-glycosylated, protease-resistance endoglucanase in the small intestine.

Industrial production of carbohydrates A number of polysaccharides are currently produced commercially using microorganisms. There is a growing interest in applying the enzymes involved to the synthesis of novel oligosaccharides and polysaccharides of controlled molecular weight. The enzymes of greatest commercial interest are those that do not use nucleotide-activated sugar derivatives as donors. Leuconostoc mesenteroides strains produce several different glucansucrases; these synthesize glucans with varying structures from sucrose, via covalent glucosyl or glucanyl-enzyme complexes. Glucosyl units are transferred to the reducing end of the growing glucan chain by a two-site insertion mechanism. Branching of the glucans occurs when a glucan acts as an acceptor and attacks the covalent enzyme--substrate complex. Carbohydrates other than sucrose can act as glucose acceptors, leading to the formation of novel oligosaccharides (John Robyt; Iowa State University, Ames, Iowa, USA). L. mesenteroides and its enzymes can be used to produce carbohydrates and derivatives as diverse as dextran, fructose, mannitol, leucrose (a noncariogenic disaccharide), glucose 1-phosphate (Wim Soetaert; Pfeifer & Langen, Dormagen, Germany). Neisseriaperflava and Neisseria polysaccharea produce amylosucrase, which synthesizes an amylopolysaccharide-like o~-glucan from sucrose. The gene encoding the enzyme from N. polysaccharea is expressed by E. coli. Using traces of glycogen as a primer, the recombinant enzyme converts sucrose to an insoluble glucan with >90% ¢x-1,4 linkages. The enzyme transfers glucose to a variety of acceptor molecules (Magali RemaudSimeon; INSA-CNRS, Toulouse, France). Enzymes from saprophytic filamentous fungi are also used commercially to produce polysaccharides from plant cell wall materials. The organisms generally produce a variety of enzymes for the effective degradation of the walls, but for many commercial applications monocomponent enzymes are desirable. Cloning the gene encoding a desired gene into a heterologous host provides an effective means of obtaining monocomponent enzymes. Enzymes from fungi such as H. insolens and Aspergillus aculeans are obtained by random cloning in yeast, screening TIBTECHNOVEMBER1995 (VOL13)

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forul~l clones for the desired activity, and expressing the gene in Aspergillus (Lene Kofod; N o v o Nordisk A/S). Hybrid compounds of carbohydrates and other molecules also have great commercial potential, bfft they are difficult to synthesize chemically. However, enzymatic synthesis may allow the production of a variety of materials. Sugar-based polymers that are biodegradable and biocompatible are synthesized by combining enzymatic and chemical methods. Subtilisin is used to esterify sugars or oligosaccharides with an acid, such as vinylacrylate, which can then be chemically polymerized to give a polyacrylate backbone with pendant sugars. Disubstituted sugars can be used to produce crosslinked hydrogels that swell to many hundred times their weight in water. Subtilisin can also be used to synthesize an alternating copolymer of sucrose and adipate (Jonathan Dordick; University of Iowa).

Fatty acid esters ofethyl-D-glucoside can be synthesized in mixtures of ethyl-D-glucopyranoside, a melted fatty acid, and an immobilized lipase from Candida antarctica that is specific for esters of primary alcohols; yields of greater than 95% of the 6-O-monoester are obtained. These ethylglucoside esters have potential applications in a variety of soaps and detergents, because they are biocompatible and biodegradable (Otto Andresen; N o v o Nordisk A/S). The conference closed with a discussion by Ten Feizi ( M R C Glycosciences Laboratory, Harrow, UK) of new approaches to harnessing carbohydrate-mediated cell-effector functions. Oligosaccharide-protein interactions are crucial to inflammation and host defence. Natural killer cells are important in innate immunity, and specific carbohydrate structures on some types of cancer cell bind strongly to a protein on natural killer

cells, thereby triggering their cytotoxic activity. If these carbohydrates are embedded in the surfaces of resistant cancer cells by hnking them to lipids (neoglycolipids), the resistant cells become susceptible to natural killer cells. This finding also has ramifications for moderating graftversus-host reactions in organ transplant recipients. In conclusion, the organizers are to be congratulated for successfully organizing such a stimulating conference at which the variety of topics presented almost matched that of the structures of carbohydrates. Unfortunately, space limitations preclude comment on the many excellent posters that complemented and extended the oral presentations.

Tony Warren Department of Microbiology and Immunology, and Protein Engineering Network Centres of Excellence, University of BritiSh Columbia, Vancouver, BC, Canada V6T 1Z3.

TIBTECH Editorial Policy Trends in Biotechnology is a news, reviews and commentary journal designed to keep its international readership up-to-date with the current developments in biotecl~nology. TIB occupiesa niche between primary research journals and conventional review journals - it is not a vehicle for the publication of origina research data or methods.

experts in the field alike. All articles are subject to peer-review and are prepared to strict standards to ensure clarity, scientific accuracy and readability. Commissioning does not guarantee publ cation.

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Letters to the Editor. Biotopics: A column providing a lighter, more-personal t r e ~ e n t of topics ranging from serious science and novel techniques, to finance and ethical questions. patenting, commercial opportunities and regulatory aspects. TIBTECHNOVEMBER1995 (VOL13)