Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris Mut(S) strain.

Pichia pastoris is currently one of the most preferred microorganisms for recombinant enzyme production due to its efficient expression system. The advantages include the production of high amounts of recombinant proteins containing the appropriate posttranslational modifications and easy cultivation conditions. α-L-Rhamnosidase is a biotechnologically important enzyme in food and pharmaceutical industry, used for example in debittering of citrus fruit juices, rhamnose pruning from naringin, or enhancement of wine aromas, creating a demand for the production of an active and stable enzyme. The production of recombinant α-L-rhamnosidase cloned in the Mut(S) strain of P. pastoris KM71H was optimized. The encoding gene is located under the control of the AOX promoter, which is induced by methanol whose concentration is instrumental for these strain types. Fermentation was upscaled in bioreactors employing various media and several methanol-feeding strategies. It was found that fed batch with BSM media was more effective compared to BMMH (Buffered Methanol-complex Medium) media due to lower cost and improved biomass formation. In BSM (Basal Salt Medium) medium, the dry cell weight reached approximately 60 g/L, while in BMMH it was only 8.3 g/L, without additional glycerol, which positively influenced the amount of enzyme produced. New methanol feeding strategy, based on the level of dissolved oxygen was developed in this study. This protocol that is entirely independent on methanol monitoring was up scaled to a 19.5-L fermenter with 10-L working volume with the productivity of 13.34 mgprot/L/h and specific activity of α-L-rhamnosidase of 82 U/mg. The simplified fermentation protocol was developed for easy and effective fermentation of P. pastoris Mut(S) based on dissolved oxygen monitoring in the induction phase of an enzyme production.

Protein engineering study of ß-mannosidase to set up a potential chemically efficient biocatalyst.

This study is focused on the analysis and mutagenesis of ß-mannosidase from Bacteroides thetaiotaomicron with the aim of broadening its substrate specificity to 2-acetamido-2-deoxy-ß-d-mannopyranosyl (ß-ManNAc) derivatives. Various conformations ((4)C1, (4)H5 and (1)S5) of native and modified ligands were docked to the binding site of the protein to determine the most suitable conformation of sugars for further hydrolysis. Key amino acid residues were mutated in silico focusing on stabilizing the acetamido group of ß-ManNAc as well as forming the oxazoline intermediate needed for hydrolysis. The results of large set of 5 ns molecular dynamic simulations showed that the majority of the active site residues are involved in substrate interaction and do not exhibit a higher flexibility except for Asn178. Mutations of Asn178 to alanine and Asp199 to serine could lead to a stabilization of the acetamido group in the binding site. So far, in vitro mutagenesis and the screen of a large variety of biological sources were unable to extend ß-mannosidase's activity to include ß-ManNAc derivatives.

Re-evaluation of binding properties of recombinant lymphocyte receptors NKR-P1A and CD69 to chemically synthesized glycans and peptides.

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

Chemoenzymatic synthesis of α-L-rhamnosides using recombinant α-L-rhamnosidase from Aspergillus terreus.

This study describes an efficient, large scale fermentation of a recombinant α-L-rhamnosidase originating from Aspergillus terreus. High-cell-density Pichia pastoris fermentation resulted in yields up to 627 U/L/h. The recombinant enzyme was used for the reverse rhamnosylation of various small organic compounds. A full factorial experimental design setup was applied to identify the importance of temperature, substrate concentrations, solvent type and concentration as well as the acidity of the reaction mixture. Careful optimization of these parameters allowed the synthesis of a range of α-L-rhamnosides among which cyclohexyl α-L-rhamnopyranoside, anisyl α-L-rhamnopyranoside and 2-phenylethyl α-L-rhamnopyranoside. In addition, α-L-rhamnosylation of phenolic hydroxyls in phenols such as hydroquinone, resorcinol, catechol and phenol was observed, which is a rather unique reaction catalyzed by glycosidases.

Crystallization and preliminary X-ray crystallographic analysis of recombinant ß-mannosidase from Aspergillus niger.

ß-Mannosidase (EC 3.2.1.25) is an important exoglycosidase specific for the hydrolysis of terminal ß-linked mannoside in various oligomeric saccharide structures. ß-Mannosidase from Aspergillus niger was expressed in Pichia pastoris and purified to clear homogeneity. ß-Mannosidase was crystallized in the presence of D-mannose and the crystal diffracted to 2.41 Šresolution. The crystal belonged to space group P1, with unit-cell parameters a=62.37, b=69.73, c=69.90 Å, α=108.20, ß=101.51, γ=103.20°. The parameters derived from the data collection indicate the presence of one molecule in the asymmetric unit.

Carbohydrate synthesis and biosynthesis technologies for cracking of the glycan code: recent advances.

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.

Production of Aspergillus niger ß-mannosidase in Pichia pastoris.

ß-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal ß-linked mannoside in various sugar chains. cDNA corresponding to the ß-mannosidase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Pichia pastoris. The ß-mannosidase gene contains an open reading frame which encodes the protein with 933 amino acid residues. The wild type and recombinant proteins were purified to apparent homogeneity and biochemically characterized (K(M) 0.28 and 0.44 mmol/l for p-nitrophenyl ß-d-mannopyranoside, pI 4.2 and 4.0, and their pH optima were at pH 4.5 and 5.5 and 65°C, respectively).

Facile synthesis of nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranosides from ManNAc-oxazoline.

The synthetic procedures for a large-scale preparation of o- and p-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside are described. The synthetic pathway employs the glycosylation of phenol with ManNAc oxazoline, followed by nitration of the aromatic moiety yielding a separable mixture of the o- and p-nitrophenyl derivative in a 2:3 ratio.

Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast.

α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.

Preparatory production of quercetin-3-ß-D-glucopyranoside using alkali-tolerant thermostable α-L-rhamnosidase from Aspergillus terreus.

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 100-300 g/L, thus enabling very high volumetric productivity of the reaction product quercetin-3-ß-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 10 kg 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.

Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors.

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 ß-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental ß1,4-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 ß1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.

The α-galactosidase type A gene aglA from Aspergillus niger encodes a fully functional α-N-acetylgalactosaminidase.

Two genes in the genome of Aspergillus niger, aglA and aglB, have been assigned to encode for α-d-galactosidases variant A and B. However, analyses of primary and 3D structures based on structural models of these two enzymes revealed significant differences in their active centers suggesting important differences in their specificity for the hydrolyzed carbohydrates. To test this unexpected finding, a large screening of libraries from 42 strains of filamentous fungi succeeded in identifying an enzyme from A. niger CCIM K2 that exhibited both α-galactosidase and α-N-acetylgalactosaminidase activities, with the latter activity predominating. The enzyme protein was sequenced, and its amino acid sequence could be unequivocally assigned to the enzyme encoded the aglA gene. Enzyme activity measurements and substrate docking clearly demonstrated the preference of the identified enzyme for α-N-acetyl-d-galactosaminide over α-d-galactoside. Thus, we provide evidence that the α-galactosidase type A gene aglA from A. niger in fact encodes a fully functional α-N-acetylgalactosaminidase using a retaining mechanism.

Enzymatic processing of bioactive glycosides from natural sources.

A number of biologically active natural products are glycosides. Often, the glycosidic residue is crucial for their activity. In other cases, glycosylation only improves their pharmacokinetic parameters. Enzymatic modification of these glycosides - both extension of the glycoside moiety and its selective trimming - is advantageous due to their selectivity and mildness of the reaction conditions in the presence of reactive and sensitive complex aglycones. Enzymatic reactions enable the resulting products to be used as "natural products", e.g., in nutraceuticals. This chapter concentrates on naturally occurring glycosides used in medicine but also in the food and flavor industry (e.g., sweeteners). Both "classical" and modern methods will be discussed.

Large propeptides of fungal beta-N-acetylhexosaminidases are novel enzyme regulators that must be intracellularly processed to control activity, dimerization, and secretion into the extracellular environment.

Filamentous fungi produce and secrete beta-N-acetylhexosaminidases, Hex, as important components of the binary chitinolytic systems involved in the formation of septa and hyphenation. Enzyme reconstitution experiments published previously indicate that Hex can occur in the form of two molecular species containing either one or two molecules of the propeptide noncovalently associated with the enzyme dimer. Here, we describe a novel mechanism for the regulation of the activity of Hex based on the association of their catalytic subunits with the large N-terminal propeptides in vivo. We show that the enzyme precursor is processed early in the biosynthesis, shortly after the addition of N-glycans through the action of a dibasic peptidase, cleaving both before and after the dibasic sequence. The processing site for this unique dibasic peptidase, different from that of kexins, is conserved among the beta-N-acetylhexosaminidases from filamentous fungi, and inhibition of the dibasic peptidase abrogates enzyme folding and activation. Binding of the released propeptide to the catalytic subunit of Hex is essential for its activation. An examination of the kinetics of Hex activation and dimerization in vitro allowed us to understand the unusually high efficiency of the assembly of this enzyme. We also report that the fungus is able to actively regulate the concentration of the processed propeptide in endoplasmic reticulum and thus the specific activity of the produced Hex. This novel regulatory mechanism enables the control of the catalytic activity and architecture of the secreted enzyme according to the needs of the producing cell at various stages of its growth cycle.

Fungal beta-N-acetylhexosaminidases with high beta-N-acetylgalactosaminidase activity and their use for synthesis of beta-GalNAc-containing oligosaccharides.

About 60 fungal strains were tested for production of extracellular beta-N-acetylhexosaminidases. A unique beta-N-acetylhexosaminidase with the beta-GalNAc-ase/beta-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) MgSO(4) increased the beta-GalNAc-ase/beta-GlcNAc-ase ratio to the value of 3.35. Cultivation conditions influence this ratio as well. beta-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 beta-D-GalpNAc-(1-->4)-D-GlcpNAc, beta-D-GalpNAc-(1-->6)-D-GlcpNAc, beta-D-GalpNAc-(1-->6)-D-GalpNAc, beta-D-GalpNAc-(1-->4)-alpha-D-GlcpNAcOAll and beta-D-GalpNAc-(1-->6)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAcOAll to demonstrate the application of these new enzymes.

Clustered ergot alkaloids modulate cell-mediated cytotoxicity.

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.

Exploitation of a library of alpha-galactosidases for the synthesis of building blocks for glycopolymers.

After screening a library of fungal alpha-galactosidases for the synthesis of functionalized alkyl alpha-D-galactopyranosides, four enzymes (isolated from Aspergillus terreus CCM55, Aspergillus commune CCM 2969, Penicillium vinaceum CCM 2384, or Penicillium brasilianum 2155) proved to be suitable for these biotransformations. The effect of different concentrations of alcohol on activity and stability of these enzymes was investigated. After optimization of the reaction conditions, three galactose derivatives (allyl, 2-nitroethyl and 2-(2',2',2'-trifluoroacetamido)-ethyl alpha-D-galactopyranoside, 1a, 3a, and 4a, respectively), suitable for subsequent chemical polymerization, were synthesized using either the "reverse hydrolysis" or the "transglycosylation" protocols.