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Abstract: The glycan code of glycoproteins can be conceptually defined at molecular level by the sequence of well characterized glycans attached to evolutionarily predetermined amino acids along the polypeptide chain. Functional consequences of protein glycosylation are numerous, and include a hierarchy of properties from general physicochemical characteristics such as solubility, stability and protection of the polypeptide from the environment up to specific glycan interactions. Definition of the glycan code for glycoproteins has been so far hampered by the lack of chemically defined glycoprotein glycoforms that proved to be extremely difficult to purify from natural sources, and the total chemical synthesis of which has been hitherto possible only for very small molecular species. This review summarizes the recent progress in chemical and chemoenzymatic synthesis of complex glycans and their protein conjugates. Progress in our understanding of the ways in which a particular glycoprotein glycoform gives rise to a unique set of functional properties is now having far reaching implications for the biotechnology of important glycodrugs such as therapeutical monoclonal antibodies, glycoprotein hormones, carbohydrate conjugates used for vaccination and other practically important protein–carbohydrate conjugates. [Copyright &y& Elsevier]

Abstract: Extensive screening for a robust producer of α-l-rhamnosidase activity from well-defined strains of filamentous fungi, including multifactorial optimization (inducers, cultivation conditions) was accomplished. Enzyme production of the optimal producer Aspergillus terreus (non-toxigenic) was scaled up to 50L. α-l-Rhamnosidase, which was fully characterized, proved to be thermo- and alkali-tolerant, thus enabling effective operation at 70°C and pH 8.0. These conditions allow for a very high substrate (rutin) load up to 100–300g/L, thus enabling very high volumetric productivity of the reaction product quercetin-3-β-d-glucopyranoside (isoquercitrin). Here, a novel concept of “immobilised substrate” is used. Isoquercitrin is a highly effective and biocompatible antioxidant with strong anti-inflammatory activities. Rutin biotransformation was optimized and scaled up to ca 10kg production and thus the robustness of the large-scale production was demonstrated. Isoquercitrin can be produced to a very high purity (98%) in multikilogram amounts, without any quercetin and directly applicable in nutraceuticals. [Copyright &y& Elsevier]

The hydrolytic and transglycosylation capabilities of 35 fungal β-N-acetylhexosaminidases with p-nitrophenyl 2-amino-2-deoxy-β-d-glucopyranoside and its four N-acyl derivatives (CH&z.dbnd;O, COCH2OH, COCH2CH3, COCF3) as substrates were tested. The preparation of four novel p-nitrophenyl disaccharides from these unnatural substrates catalysed by enzymes from Aspergillus oryzae, Penicillium oxalicum and Talaromyces flavus represents a considerable extension of the synthetic potential of glycosidases. [Copyright &y& Elsevier]

About 60 fungal strains were tested for production of extracellular β-N-acetylhexosaminidases. A unique β-N-acetylhexosaminidase with the β-GalNAc-ase/β-GlcNAc-ase ratio of 2.3–2.8 was found in the culture filtrates of some strains of Penicillium oxalicum. Addition of 20% (w/v) MgSO4 increased the β-GalNAc-ase/β-GlcNAc-ase ratio to the value of 3.35. Cultivation conditions influence this ratio as well. β-N-Acetylhexosaminidases from P. oxalicum CCF 2430 and Aspergillus oryzae CCF 1066 considerably differing in the GalNAc-ase activity were used for the synthesis of the following structures β-d-GalpNAc-(1→4)-d-GlcpNAc, β-d-GalpNAc-(1→6)-d-GlcpNAc, β-d-GalpNAc-(1→6)-d-GalpNAc, β-d-GalpNAc-(1→4)-α-d-GlcpNAcOAll and β-d-GalpNAc-(1→6)-β-d-Galp-(1→4)-α-d-GlcpNAcOAll to demonstrate the application of these new enzymes. [Copyright &y& Elsevier]

Highlight: [•] α-l-Rhamnosidase was heterologously produced in P. pastoris. [•] Chemoenzymatic rhamnosylation of alcohols was optimized using full factorial setup. [•] Phenolic acceptors can be glycosylated with α-l-rhamnosidase using free rhamnose. [Copyright &y& Elsevier]

Abstract: α-l-Rhamnosidase (EC 3.2.1.40) is a biotechnologically important enzyme used for derhamnosylation of many natural compounds. The extracellular α-l-rhamnosidase was purified from the culture of Aspergillus terreus grown on l-rhamnose-rich medium. This enzyme was found to be thermo- and alkali-tolerant, able to operate at 70°C and pH 8.0. The α-l-rhamnosidase cDNA was cloned from A. terreus, sequenced, and expressed in the yeast Pichia pastoris as a fully functional protein. The recombinant protein was purified to apparent homogeneity and biochemically characterized. Both the native and the recombinant α-l-rhamnosidases catalyzed the conversion of rutin into quercetin-3-glucopyranoside (isoquercitrin), a pharmacologically significant flavonoid usable in nutraceutics. This procedure has high volumetric productivity (up to 300g/L) and yields the product void of unwanted quercetin. The significant advantage of our expression system consists in shorter production times, up to fourfold increase in enzyme yields and the absence of unwanted β-d-glucosidase as compared to the native production system. Thanks to its unique properties, this enzyme is applicable in a selective synthesis/hydrolysis of various rhamnose containing structures. [Copyright &y& Elsevier]

Abstract: This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a β-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental β1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental β1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials. [Copyright &y& Elsevier]