Tom Desmet

Enzymes for the biocatalytic production of rare sugars

Carbohydrates are much more than just a source of energy as they also mediate a variety of recognition processes that are central to human health. As such, saccharides can be applied in the food and pharmaceutical industries to stimulate our immune system (e.g., prebiotics), to control diabetes (e.g., low-calorie sweeteners), or as building blocks for anticancer and antiviral drugs (e.g., l-nucleosides). Unfortunately, only a small number of all possible monosaccharides are found in nature in sufficient amounts to allow their commercial exploitation. Consequently, so-called rare sugars have to be produced by (bio)chemical processes starting from cheap and widely available substrates. Three enzyme classes that can be used for rare sugar production are keto–aldol isomerases, epimerases, and oxidoreductases. In this review, the recent developments in rare sugar production with these biocatalysts are discussed.

Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase

Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.

Enzymatic glycosyl transfer: mechanisms and applications

Abstract Glycosylated compounds have numerous applications in the food, pharmaceutical and personal care industries. Their synthesis is, however, far from trivial. Because chemical glycosylation reactions suffer from low yields and lack of selectivity, biocatalytic routes have received increasing attention as efficient and ‘green’ alternatives. Several types of biocatalysts can be used for this purpose, each with its own advantages and disadvantages. The applications of glycoside hydrolases, glycoside phosphorylases, transglycosidases and glycosyl transferases are discussed in light of the most recent insights into the mechanistic features of the enzymes. A thorough understanding of their structure–function relationships should also allow a more efficient engineering of the activity and specificity of these enzymes. Several strategies to enhance the glycosylation potential of natural biocatalysts are presented.

Evaluation of (4-aminobutyloxy)quinolines as a novel class of antifungal agents

Antifungal assessment of eighteen 5-, 6- and 8-(4-aminobutyloxy)quinolines revealed a significant susceptibility of the tested fungi and yeast strains (Candida albicans, Rhodotorula bogoriensis, Aspergillus flavus and Fusarium solani) toward different halo-substituted 8-(4-aminobutyloxy)quinolines. The six most potent compounds displayed antifungal activities similar to those of established antifungal agents such as Amphotericin B, Fluconazole and Itraconazole, and one representative also showed a promising broad-spectrum antifungal profile. The introduction of an aminoalkoxy side chain at the 8-position of a halo-substituted quinoline core might thus provide a new class of lead structures in the search for novel antifungal agents.

Development and application of a screening assay for glycoside phosphorylases

Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzymes substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl beta-glucosides and phenyl beta-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.

Ionic liquids as cosolvents for glycosylation by sucrose phosphorylase: balancing acceptor solubility and enzyme stability

Over the past decade, disaccharide phosphorylases have received increasing attention as promising biocatalysts for glycoside synthesis. Unfortunately, these enzymes typically have a very low affinity for non-carbohydrate acceptors, which urges the addition of cosolvents to increase the dissolved concentration of these acceptors. However, commonly applied solvents such as methanol and dimethyl sulfoxide (DMSO) are not compatible with many intended applications of carbohydrate-derived products. In this work, the solubility of a wide range of relevant acceptors was assessed in the presence of ionic liquids (ILs) as alternative and ‘green’ solvents. The IL AMMOENG 101 was found to be the most effective cosolvent for compounds as diverse as medium- and long-chain alcohols, flavonoids, alkaloids, phenolics and terpenes. Moreover, this IL was shown to be less deleterious to the stability and activity of sucrose phosphorylase than the commonly used dimethyl sulfoxide. To demonstrate the usefulness of this solvent system, a process for the resveratrol glycosylation was established in a buffer containing 20% AMMOENG 101, 1 M sucrose and saturated amounts of the acceptor. A single regioisomer 3-O-α-D-glucopyranosyl-(E)-resveratrol was obtained as proven by NMR spectroscopy.

Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development

Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) long known from plants and more recently discovered in bacteria. The enzyme catalyzes the reversible transfer of a glucosyl moiety between fructose and a nucleoside diphosphate (NDP) (sucrose+NDP↔NDP-glucose+fructose). The equilibrium for sucrose conversion is pH dependent, and pH values between 5.5 and 7.5 promote NDP-glucose formation. The conversion of a bulk chemical to high-priced NDP-glucose in a one-step reaction provides the key aspect for industrial interest. NDP-sugars are important as such and as key intermediates for glycosylation reactions by highly selective Leloir GTs. SuSy has gained renewed interest as industrially attractive biocatalyst, due to substantial scientific progresses achieved in the last few years. These include biochemical characterization of bacterial SuSys, overproduction of recombinant SuSys, structural information useful for design of tailor-made catalysts, and development of one-pot SuSy-GT cascade reactions for production of several relevant glycosides. These advances could pave the way for the application of Leloir GTs to be used in cost-effective processes. This review provides a framework for application requirements, focusing on catalytic properties, heterologous enzyme production and reaction engineering. The potential of SuSy biocatalysis will be presented based on various biotechnological applications: NDP-sugar synthesis; sucrose analog synthesis; glycoside synthesis by SuSy-GT cascade reactions.

Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into fructose and nucleotide (NDP)-glucose. To date, only SuSy’s from plants and cyanobacteria, both photosynthetic organisms, have been characterized. Here, four prokaryotic SuSy enzymes from the nonphotosynthetic organisms Nitrosomonas Europaea (SuSyNe), Acidithiobacillus caldus (SuSyAc), Denitrovibrio acetiphilus (SusyDa), and Melioribacter roseus (SuSyMr) were recombinantly expressed in Escherichia coli and thoroughly characterized. The purified enzymes were found to display high-temperature optima (up to 80 °C), high activities (up to 125 U/mg), and high thermostability (up to 15 min at 60 °C). Furthermore, SuSyAc, SuSyNe, and SuSyDa showed a clear preference for ADP as nucleotide, as opposed to plant SuSy’s which prefer UDP. A structural and mutational analysis was performed to elucidate the difference in NDP preference between eukaryotic and prokaryotic SuSy’s. Finally, the physiological relevance of this enzyme specificity is discussed in the context of metabolic pathways and genomic organization.

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides.

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several β‐glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site‐saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl β‐glucosides, methyl α‐glucoside and cellobiose. In comparison with the wild‐type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413–420.

Creating Space for Large Acceptors: Rational Biocatalyst Design for Resveratrol Glycosylation in an Aqueous System

Polyphenols display a number of interesting properties but their low solubility limits practical applications. In that respect, glycosylation offers a solution for which sucrose phosphorylase has been proposed as a cost-effective biocatalyst. However, its activity on alternative acceptor substrates is too low for synthetic purposes and typically requires the addition of organic (co-)solvents. Here, we describe the engineering of the enzyme from Thermoanaerobacterium thermosaccharolyticum to enable glycosylation of resveratrol as test case. Based on docking and modeling studies, an active-site loop was predicted to hinder binding. Indeed, the unbolted loop variant R134A showed useful affinity for resveratrol (K(m)=185 mM) and could be used for the quantitative production of resveratrol 3-α-glucoside in an aqueous system. Improved activity was also shown for other acceptors, introducing variant R134A as promising new biocatalyst for glycosylation reactions on bulky phenolic acceptors.