Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered β-hexosaminidase immobilized on solid support

The D746E variant of Bifidobacterium bifidum β-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective β-1,3-glycosylation of lactose from a d -glucosamine 1,2-oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145–200 mM) at quantitative yield from the donor substrate. The wild-type β-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d -glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80–100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.     

Donor substrate promiscuity of bacterial β1–3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1–4-galactosyltransferases

β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN₃ and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.     

Enzymatic synthesis of oligosaccharides by two glycosyl hydrolases of Sulfolobus solfataricus

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an alpha-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.     

New Approaches to Enzymatic Oligosaccharide Synthesis: Glycosynthases and Thioglycoligases

Abstract

An overview of the applications of engineered glycosynthases and thioglycoligases for the enzymatic synthesis of O- and S-glycosidic linkages in oligosaccharides is presented. Glycosynthases lack the catalytic nucleophile of retaining glycosidases and use glycosyl fluorides with inverted anomeric stereochemistry as glycosyl donors. To date, nine enzymes from seven different glycosyl hydrolase families have been engineered to perform the glycosynthase reaction. Thioglycoligases lack the catalytic acid/base residue of retaining glycosidases and use dinitrophenyl glycosides as donors and deoxy-thiosugars as acceptors. The regioselectivity of the transglycosylation reaction is entirely controlled by the position of the thiol in the acceptor. To date, two retaining exo glycosidases and one endo glycanase, all from different glycosyl hydrolase families, have been engineered in this fashion.     

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

Rice BGlu1 β-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. The X-ray crystal structures of the E386G glycosynthase with and without α-glucosyl fluoride were solved and the α-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites +2, +3, and +4. Mutants in which these residues were replaced in BGlu1 β-glucosidase hydrolyzed cellotetraose and cellopentaose with k(cat) /K(m) values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNP-cellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.     

Development of a microbioreactor for glycoconjugate synthesis

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl-l-asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.     

A universal approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A universal approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with amino group in aglycone moiety can be conjugated with PBC carboxy derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a condensing agent. Both N-and O-glycosides differing in the conformation mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides isolated from natural sources can be transformed into N-glycosides with a functional group in aglycone. The approach was tested by conjugation of the carboxy derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand of lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

A uniform approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A uniform approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with an aglycone moiety amino group can be coupled with PBC carboxyl derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a coupling agent. Both N- and O-glycosides differing in the conformational mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides. For example, not only oligosaccharides obtained by chemical synthesis but also reducing oligosaccharides isolated from natural sources can be transformed into N-glycosides. The approach was tested by the example of conjugation of the carboxyl derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand for lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Enzymatic hydrolysis of 1,3-1,4-beta-glucosyl oligosaccharides by 1,3-1,4-beta-glucanase from Synechocystis PCC6803: a comparison with assays using polymer and chromophoric oligosaccharide substrates

The specificity of 1,3-1,4-β-glucanase from Synechocystis PCC6803 (SsGlc) was investigated using novel substrates 1,3-1,4-β-glucosyl oligosaccharides, in which 1,3- and 1,4-linkages are located in various arrangements. After the enzymatic reaction, the reaction products were separated and determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As a result, SsGlc was found to hydrolyze the pentasaccharides, which possess three contiguous 1,4-β-glycosidic linkages (cellotetraose sequence) adjacent to 1,3-β-linkage, but none of the other oligosaccharides were hydrolyzed. To further analyze the specificity, kinetic measurements were performed using polymeric substrates and 4-methylumbelliferyl derivatives of laminaribiose and cellobiose (1,3-β-(Glc)₂-MU and 1,4-β-(Glc)₂-MU). The k_cat/K_m value obtained for barley β-glucan was considerably larger than that for lichenan, indicating that SsGlc prefers 1,3-1,4-β-glucan possessing a larger amount of cellotetraose sequence. This is consistent with the data obtained for 1,3-1,4-β-glucosyl oligosaccharides. However, the k_cat/K_m value obtained for 1,4-β-(Glc)₂-MU was considerably lower than that for 1,3-β-(Glc)₂-MU, suggesting inconsistency with the data obtained from the other natural substrates. It is likely that the kinetic data obtained from such chromophoric substrates do not always reflect the true enzymatic properties.     

Synthesis of building blocks of human milk oligosaccharides. Fucosylated derivatives of the lacto- and neolacto-series

The synthesis of protected fucosylated derivatives of a Galβ(1→3)GlcNAc and of lactosamine Galβ(1→4)GlcNAc building blocks contained in human milk oligosaccharides is described. Both chemical and enzymatic methods have been exploited for selective protection of the disaccharide. Fucosylation of the appropriate derivatives allowed an easy and relatively short access to different products from common precursors.     

Affinity chromatography of carbohydrate-specific immunoglobulins: coupling of oligosaccharides to sepharose.

A simple method has been developed for the coupling of oligosaccharides to Sepharose. The sugars are reacted with β-(p-aminophenyl)-ethylamine to form N-alkylglycosides which are then reduced with sodium borohydride to stable secondary amines. The derivatives are then coupled to cyanogen bromide-activated Sepharose through their arylamino groups. Yields are essentially quantitative based on starting oligosaccharides. An affinity column containing lacto-N-difucohexaose I coupled to Sepharose by this method was used for the purification of an antibody directed against this oligosaccharide. The antibody is absorbed by the gel and is specifically eluted by the free sugar.     

Carbohydrate-binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for the synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a “non-catalytic” lectin-like domain or carbohydrate-binding modules (CBMs) in the catalytic step for glycosidic bond synthesis using β-cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. This study provides evidence for the direct participation of CBMs in the catalytic reaction step for β-glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intradomain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM-based CAZyme chimeras for the synthesis of bespoke oligosaccharides using simple activated sugar monomers.     

New derivatives of reducing oligosaccharides and their use in enzymatic reactions: efficient synthesis of sialyl Lewis a and sialyl dimeric Lewis x glycoconjugates

The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbonylaminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyaldi tol oligosaccharide derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars. Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene treatment, and protein coupling was carried out.     

Enzymatic Peptide Synthesis in Organic Media. Synthesis of CCK-8 Dipeptide Fragments

The enzymatic synthesis of the seven consecutive dipeptide fragments of the cholecysto kinin C-terminal octapeptide (CCK-8) in organic media is reported. The influence of the reaction medium composition, the protease, and the structure of N-α and C-α protecting groups of both carboxyl and amino components was evaluated. α-Chymotrypsin, papain and thermolysin adsorbed on Celite were used as catalysts, under thermodynamic and kinetic control. The carboxamidomethyl, methyl and allyl ester derivatives of acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl and fluoren-9-ylmethoxycarbonyl amino acids, were assayed as carboxy components. Amino acid amide and ester derivatives were employed as nucleophiles with a preference for the latter, since the dipeptide product obtained could be used directly, without any further chemical modification, as acyl-donor in subsequent coupling steps. All dipeptides selected were successfully synthesized, using the optimal combination of protecting groups, reaction media and enzyme different for each coupling reaction. The information gained with this study should be instrumental in designing an optimal strategy for the total enzymatic synthesis of cholecystokinin C-terminal octapeptide (CCK-8).     

Enzymatic synthesis of novel oligosaccharides by use of transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of novel oligosaccharides. When (Man)6(GlcNAc)2Asn was used as a substrate for the transglycosylation, (Man)6GlcNAc-Glc, (Man)6GlcNAc-Man, (Man)6GlcNAc-chitobiose, and (Man)6GlcNAc-gentiobiose were synthesized. Their structures were identified by HPLC, ion spray mass spectrometry, and digestion with glycosidases. Endo-beta-N-acetylglucosaminidases hydrolyzed the pyridylamino derivatives of these oligosaccharides.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

Studies on the synthesis of Lewis-y oligosaccharides

Lewis-y histo-blood group oligosaccharides are tumour-associated antigens prevalent in several different types of cancer, and they may also be secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. The key step in the synthesis of these sterically congested oligosaccharides involves difucosylation of partially protected lactosamine derivatives. Existing methods require either prolonged reaction times or elaborate glycosyl donors to ensure high stereoselectivity. Herein we report an optimised procedure for using a simple thioglycoside donor that leads to the desired products in high yield and excellent stereoselectivity. It is found that initial glycosylation of the 3′-hydroxy group of lactosamine derivatives in dichloromethane solution can inhibit subsequent glycosylation at the 2-position; however, reaction in toluene solution leads to Lewis-y oligosaccharides in high yield.     

One-step immobilization and purification of genetic engineering CBD fusion EndoS on cellulose for antibodies Fc-glycan remodeling

The endoglycosidase (EndoS and its glycosynthase mutants D233A, D233Q) gene was fused with cellulose binding domain (CBD) using pET-35b vector and the fusion enzymes were successfully expressed in Escherichia coli. Then a simplified approach for one-step immobilization and purification of EndoS enzymes using cellulose as matrices were developed and excellent loading efficiency (81–90%) was achieved in optimal condition. The cellulose immobilized CBD-EndoS and the glycosynthase mutants presented high catalytic activity and were successfully applied in a two-step antibody Fc N-glycan remodeling, generating a therapeutic antibody with homogeneous glycoform in high efficiency. The cellulose immobilized CBD-EndoS and its mutants (D233A and D233Q) displayed excellent storage stability when stored at 4 degrees for one month. Reusability studies demonstrated that the cellulose immobilized CBD-EndoS and its mutants could be recycled for five times without obvious activity loss.     

Impact of donor binding on polymerization catalyzed by KfoC by regulating the affinity of enzyme for acceptor

BackgroundCurrently marketed chondroitin sulfate isolated from animal sources and structurally quite heterogeneous. Synthesis of structurally defined chondroitin sulfate is highly desired. The capsular polysaccharide from Escherichia coli strain K4 is similar to chondroitin, and its biosynthesis requires a chondroitin polymerase (KfoC). The essential step toward de novo enzymatic synthesis of chondroitin sulfate, synthesis of chondroitin, could be achieved by employing this enzyme.MethodsStructurally defined acceptors and donor-sugars were prepared by chemoenzymatic approaches. In addition, surface plasmon resonance was employed to determine the binding affinities of individual substrates and donor–acceptor pairs for KfoC.ResultsKfoC has broad donor substrate specificity and acceptor promiscuity, making it an attractive tool enzyme for use in structurally-defined chimeric glycosaminoglycan oligosaccharide synthesis in vitro. In addition, the binding of donor substrate molecules regulated the affinity of KfoC for acceptors, then influenced the glycosyl transferase reaction catalyzed by this chondroitin polymerase.Conclusion and general significanceThese results assist in the development of enzymatic synthesis approaches toward chimeric glycosaminoglycan oligosaccharides and designing future strategies for directed evolution of KfoC in order to create mutants toward user-defined goals.