Mikrobielle und enzymatische Synthese von Biotensiden auf der Basis nachwachsender Rohstoffe

Microbial and Enzymatic Synthesis of Biosurfactants from Renewable Resources Renewable resources such as vegetable oils or carbohydrates are suitable substrates for the biocatalysis to produce glycolipids with good physico-chemical properties and biological activities. Potential biotechnological processes include: Multiple-step-biosynthesis by microorganisms (growth-limited cultures, resting cells) and one-step-reaction with lipases or glycosidases. By aid of the microbial method the specific production amounts to more than 50 % conversion of the renewable resources to high value glycolipids. A disadvantage is the limited variability of the molecular structures. The enzymatic method offers the directed synthesis of glycolipids from sugar and fatty acid/fatty alcohol, but needs partly those additional procedures which are necessary in competing chemical methods.     

Digital screening methodology for the directed evolution of transglycosidases

Engineering of glycosidases with efficient transglycosidasesactivity is an alternative to glycosyltransferases or glycosynthasesfor the synthesis of oligosaccharides and glycoconjugates. However,the engineering of transglycosidases by directed evolution methodologiesis hampered by the lack of efficient screening systems for sugar-transferactivity. We report here the development of digital imaging-basedhigh-throughput screening methodology for the directed evolutionof glycosidases into transgalactosidases. Using this methodology,we detected transglycosidase mutants in intact Escherichia colicells by digital imaging monitoring of the activation of non-or low-hydrolytic mutants by an acceptor substrate. We screenedseveral libraries of mutants of β-glycosidase from Thermusthermophilus using this methodology and found variants withup to a 70-fold overall increase in the transglycosidase/hydrolysisactivity ratio. Using natural disaccharide acceptors, thesetransglycosidase mutants were able to synthesise trisaccharides,as a mixture of two regioisomers, with up to 76% yield.     

Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts

Thioglycoligases are recently introduced variants of retaining glycosidases in which the acid-base catalyst has been mutated, rendering them capable of thioglycoside synthesis. The original acid-base mutant of Agrobacterium sp. beta-glucosidase (E170A) was previously shown to be an effective thioglycoligase carrying out glycosyltransfer from 2,4-dinitrophenyl glycosides to several different thio sugar acceptors. Here we report the generation of a screen for improved thioglycoligases, randomized mutagenesis of the acid-base catalyst E170 and identification of variants superior to E170A. Furthermore we have established a coupled assay allowing kinetic analysis of isolated variants and found that Abg E170Q is 5-fold faster than Abg E170A when 2,4-dinitrophenyl glucoside is used as donor and 100-fold faster when glucosyl azide is used. To demonstrate its utility, different acceptor and donor sugar combinations were employed to produce thio-linked di- or trisaccharides in high yields, showing the considerable versatility of the system for the synthesis of carbohydrate mimetics.     

Review Oligosaccharide Synthesis Using Glycosidases*

A wide range of strategies may be considered for the synthesis of oligosaccharides in vitro using enzymes, all of which present significant challenges to the enzyme technologist. Many simple oligosaccharides may be produced by the hydrolysis of readily-available polysaccharides using specific enzymes. However, to produce the complex branched hetero-oligosaccharides of the types which occur N-linked to glycoproteins is more taxing. Materials of this type may be synthesised using the natural synthetic enzymes which employ sugar nucleotides as substrates. These enzymes are highly specific but they are costly to use due to their instability and to the cost of their substrates. It has been demonstrated that glycosidases are capable of synthesising hetero-oligosaccharides when provided with underivatised sugars in conditions of low water activity but that the specificity of synthetic reactions is apparently not high and that yields of material are low. Approaches to these problems are discussed, including the use of immobilised enzymes in packed-bed reactors to allow the ‘ping’ stage of the synthetic reaction to be separated in time from the ‘pong’ stage, and the application of aqueous two-phase systems which may be ‘tailored’ to separate the enzyme and the substrates from the final product. The ability to synthesise a range of oligosaccharides is dependent on the availability of appropriate glycosidases with differing specificities. There is a clear importance of ‘biodiversity’ in providing knowledge of sources of these.     

Enzymatic Thioxyloside Synthesis: Characterization of Thioglycoligase Variants Identified from A Site‐Saturation Mutagenesis Library of Bacillus Circulans Xylanase

Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site‐saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.     

Developing promiscuous glycosidases for glycoside synthesis: residues W433 and E432 in Sulfolobus solfataricus beta-glycosidase are important glucoside- and galactoside-specificity determinants

Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification. The E432C and W433C mutations removed key interactions with the OH-4 and OH-3 of the sugar substrates, thus reducing the discrimination of glucose, galactose and fucose with respect to other glycosides. This resulted in two glycosidases with greatly broadened substrate specificities. Observed changes include a 24-fold increase in Man:Gal activity and an 18-fold increase in GalA:Gal activity. This promiscuous substrate tolerance was further illustrated by the parallel synthesis of a beta-glycoside library of glucose, galactose, xylose and mannose in one pot at 50 degrees C, in organic solvent. The synthetic potential of the catalysts was further evaluated through alkyl glycoside transglycosylation yields, including the first examples of synthesis of beta-mannosides and beta-xylosides with SsbetaG.     

Towards Tuneable Retaining Glycosidase‐Inhibiting Peptides by Mimicry of a Plant Flavonol Warhead

Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in‐depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α‐amylase (HPA). Notably, incorporation of l ‐DOPA into this peptide affords an inhibitor of HPA with potency that is tenfold higher (K_i=480 pm ) than that of the previously found consensus sequence. This represents a first successful step in converting a recently discovered natural‐product‐derived motif, already specific for the catalytic side‐chain arrangement conserved in the active sites of retaining glycosidases, into a tuneable retaining glycosidase inhibition warhead.     

Production of combi-CLEAs of glycosidases utilized for aroma enhancement in wine

Glycosidases are frequently used in winemaking to liberate glycosidically bound aroma compounds. Since most of the glycosidases used for diglycoside hydrolysis act sequentially, their co-immobilization is an attractive alternative from a technical and economical perspective. The enzymes α-l-arabinosidase (ARA) and β-d-glucosidase (βG) from the preparation Rapidase^®AR2000 were co-immobilized in CLEAs (combi-CLEAs), evaluating the effect of bovine serum albumin (BSA) addition and the concentration of glutaraldehyde (Glu) on enzyme immobilization yield and expressed activity. Combi-CLEAs prepared with a Glu to Rapidase protein mass ratio of 0.053 and BSA to Rapidase protein mass ratios of 1, 0.33, and 0.2 were selected, evaluating their stability at simulated winemaking conditions: 25 °C, pH 3.5, and 10% (v/v) of ethanol. All combi-CLEAs were more stable than the soluble enzymes, the best result being obtained at a BSA to Rapidase protein mass ratio of 0.33. Half-lives of βG and ARA in combi-CLEAs were 43.9 and 54.9 days, respectively, whereas in the case of the soluble enzymes were only 1.3 and 6.2 days, respectively. Immobilization yields were 79.1 and 47.1% in terms of βG and ARA activity, respectively. Combi-CLEAs of glycosidases are technologically relevant robust biocatalysts for their application as aroma enhancers in winemaking.     

Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates.     

Engineering Glyco-Enzymes for Substrate Identification and Targeting

Glycoconjugates are assembled by the coordinate actions of glycosyltransferases, which add sugars, and glycosidases, which remove sugars. These glyco-enzymes comprise families of enzymes that catalyze the same reaction, making it difficult to identify the direct substrates of each isozyme. To solve this challenge, mutagenesis of glyco-enzymes has been used to enable incorporation of unnatural sugar analogs that can be employed to tag and isolate the protein substrates of an individual glycosyltransferase. A second challenge arises in efforts to determine which substrates mediate biological effects. Engineering a glycosyltransferase to target its activity toward select acceptor substrates allows deconvolution of the roles of specific glycosylation events. Similarly, glycosidases can be engineered to target specific substrates, with basic science and translational applications. This review describes recent efforts at engineering glyco-enzymes to identify and target their distinct substrates.     

Chemoenzymatic Synthesis and Inhibitory Activities of Hyacinthacines A1 and A2 Stereoisomers

A novel straightforward chemoenzymatic procedure for the synthesis of hyacinthacine stereoisomers based on the aldol addition of dihydroxyacetone phosphate (DHAP) to N‐Cbz‐prolinal under catalysis by L ‐rhamnulose 1‐phosphate aldolase from E. coli is presented. The synthesis is complemented by a simple and effective purification protocol consisting of ion‐exchange chromatography on CM‐sepharose. As examples, (−)‐hyacinthacine A₂ [the enantiomer of (+)‐hyacinthacine A₂], 7‐deoxy‐2‐epialexine (the enantiomer of 3‐epihyacinthacine A₂), ent‐7‐deoxyalexine (the enantiomer of 7‐deoxyalexine) and 2‐epihyacinthacine A₂ were synthesized by these procedures and characterized for the first time. These new isomers were assayed as inhibitors of glycosidases. As a result, (−)‐hyacinthacine A₂ demonstrated to be a good inhibitor of α‐D ‐glucosidase from rice whereas the natural enantiomer, hyacinthacine A₂, was not. Moreover, a new family of inhibitors of α‐L ‐rhamnosidase was uncovered.     

Characterization of a Galactosynthase Derived from Bacillus circulans β‐Galactosidase: Facile Synthesis of D‐Lacto‐ and D‐Galacto‐N‐bioside

Glycosynthases—retaining glycosidases mutated at their catalytic nucleophile—catalyze the formation of glycosidic bonds from glycosyl fluorides as donor sugars and various glycosides as acceptor sugars. Here the first glycosynthase derived from a family 35 β‐galactosidase is described. The Glu→Gly mutant of BgaC from Bacillus circulans (BgaC‐E233G) catalyzed regioselective galactosylation at the 3‐position of the sugar acceptors with α‐galactosyl fluoride as the donor. Transfer to 4‐nitophenyl α‐D ‐N‐acetyl‐glucosaminide and α‐D ‐N‐acetylgalactosaminide yielded 4‐nitophenyl α‐lacto‐N‐biose and α‐galacto‐N‐biose, respectively, in high yields (up to 98 %). Kinetic analysis revealed that the high affinity of the acceptors contributed mostly to the BgaC‐E233G‐catalyzed transglycosylation. BgaC‐E233G showed no activity with β‐(1,3)‐linked disaccharides as acceptors, thus suggesting that this enzyme can be used in “one‐pot synthesis” of LNB‐ or GNB‐containing glycans.     

Glycosidase mechanisms: anatomy of a finely tuned catalyst

In order to accelerate the hydrolysis of glycosidic bonds by factors approaching 10(17)-fold, glycosidases have evolved finely tuned active sites optimally configured for transition-state stabilization. Structural analyses of various enzyme complexes representing stable intermediates along the reaction coordinate, in conjunction with detailed mechanistic studies on wild-type and mutant enzymes, have delineated the contributions of nucleophilic and general acid/base catalysis, as well as the roles of noncovalent interactions, to these impressive rate enhancements.     

A High‐Throughput Mass‐Spectrometry‐Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

The most commonly employed glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position in potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a qualitative mass spectrometry–based glycosidase assay amenable to high‐throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high‐throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved through the observation of an appropriate decrease in mass between a potential sugar substrate and its corresponding product by electrospray ionization mass spectrometry (ESI‐MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.     

N-Glycosidase-carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation

A carbohydrate-binding module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM-EndoF1 and CBM-PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after production by Escherichia coli. CBM-EndoF1 is as effective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM-PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for at least 1 month at 4 degrees C. The hexahistidine tag can be removed with thrombin, leaving the CBM as the only affinity tag. The CBM can be removed with factor Xa if required.     

Alkaloidal Glycosidase Inhibitors and Digestive Glycosidase Inhibition in Specialist and Generalist Herbivores of Omphalea diandra

Generalist herbivores of the neotropical liana Omphalea diandra (Euphorbiaceae) were compared to the specialist herbivore, larvae of the uraniid moth Urania fulgens, with respect to their ability to accumulate the alkaloidal glycosidase inhibitors (AGIs) produced by the plant and the resistance of their digestive glycosidases to inhibition by these AGIs. The generalist herbivores did not accumulate the AGI aglycones 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) and 2,6-dideoxy-2,6-imino-D-glycero-L-gulo-heptitol (HNJ) to the levels detected in larvae of U. fulgens, which contained 0.05–0.11% dry weight DMDP and 0.17–0.35% HNJ. Glucosides of DMDP and HNJ that were synthesized by O. diandra were either absent from both the generalist and the specialist herbivores or present at low levels (less than 0.01%), even though HNJ-glucoside was often the most abundant AGI in the foliage. Analyses of the herbivores' feces indicated that failure to accumulate AGIs was due to the compounds being metabolized rather than excreted. The digestive glycosidases of U. fulgens larvae were more resistant to inhibition by AGI aglycones than those of the generalist herbivores. Similarly, sucrose and maltose hydrolysis in two of the generalist lepidopteran herbivores, larvae of Panthiades ballus and Theope virgilius, was more resistant to inhibition by DMDP than in larvae of Spodoptera littoralis, a lepidopteran which does not encounter O. diandra in nature. There was little difference in the susceptibility to AGIs of glycosidases from the generalist coleopteran Rhabdopterus fulvipes, which naturally feeds on O. diandra, compared with the coleopteran Dermestes maculatus, which does not. The glucoside of HNJ was found to be a very potent inhibitor of trehalase activity in all the insects examined. AGIs are considered to reduce the nutritional value of O. diandra to nonadapted herbivores rather than be acutely toxic. Nevertheless, U. fulgens does appear to be unique among Omphalea-feeding insects in its ability to accumulate AGIs, suggesting that it gains some advantage from storing these compounds.     

糖苷酶在茶叶增香及香气形成中的应用研究进展

茶香气物质在鲜叶中主要以糖苷类前体物质的形式存在,糖苷酶作为一种茶叶的内源酶在茶叶加工中对香气物质释放起到重要作用。就糖苷酶的种类和特性,以及其在茶叶加工、茶叶深加工过程中的增香应用等方面内容进行综述,为糖苷酶酶解技术在茶叶和茶深加工领域的进一步应用提供理论指导。     

N‐Alkyl‐, 1‐C‐Alkyl‐, and 5‐C‐Alkyl‐1,5‐dideoxy‐1,5‐imino‐(l)‐ribitols as Galactosidase Inhibitors

A series of 1,5‐dideoxy‐1,5‐imino‐(l )‐ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4‐epi‐isofagomine (4‐epi‐IFG) mimics and were expected to behave as selective inhibitors of β‐galactosidases. All compounds were indeed found to be highly selective for β‐galactosidases versus α‐glycosidases, as they generally did not inhibit coffee bean α‐galactosidase or other α‐glycosidases. Some compounds were also found to be inhibitors of almond β‐glucosidase. The N‐alkyl DIR derivatives were only modest inhibitors of bovine β‐galactosidase, with IC₅₀ values in the 30–700 μm range. Likewise, imino‐l ‐ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced β‐galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the ‘pseudo‐anomeric’ configuration in this series does not appear to play a role. Human lysosomal β‐galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC₅₀ values in the 100 μm range), while 4‐epi‐IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1‐gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7‐fold.     

Mechanistic Insights into the Activities of Major Families of Enzymes in Bacterial Peptidoglycan Assembly and Breakdown

Serving as an exoskeletal scaffold, peptidoglycan is a polymeric macromolecule that is essential and conserved across all bacteria, yet is absent in mammalian cells; this has made bacterial peptidoglycan a well-established excellent antibiotic target. In addition, soluble peptidoglycan fragments derived from bacteria are increasingly recognised as key signalling molecules in mediating diverse intra- and inter-species communication in nature, including in gut microbiota-host crosstalk. Each bacterial species encodes multiple redundant enzymes for key enzymatic activities involved in peptidoglycan assembly and breakdown. In this review, we discuss recent findings on the biochemical activities of major peptidoglycan enzymes, including peptidoglycan glycosyltransferases (PGT) and transpeptidases (TPs) in the final stage of peptidoglycan assembly, as well as peptidoglycan glycosidases, lytic transglycosylase (LTs), amidases, endopeptidases (EPs) and carboxypeptidases (CPs) in peptidoglycan turnover and metabolism. Biochemical characterisation of these enzymes provides valuable insights into their substrate specificity, regulation mechanisms and potential modes of inhibition.