Transglycosylation reaction of xylanase B from the hyperthermophilic Thermotoga maritima with the ability of synthesis of tertiary alkyl beta-D-xylobiosides and xylosides

The recombinant xylanase B (XynB) of Thermotoga maritima MSB8 was characterized and was found to cleave p-nitrophenyl beta-D-xyloside via the transglycosylation reaction in the previous study. XynB was activated in the presence of alcohols, and XynB activity was increased by iso-propanol (2M) to 2.1-fold. This type of activation was investigated and was shown to be due to the transglycosylation activity with p-nitrophenyl beta-D-xylobioside being converted to alkyl beta-D-xylobiosides in the presence of XynB and alcohols. Through the transglycosylation reaction, alkyl beta-xylosides and xylobiosides were simultaneously produced in the presence of xylan and alcohols. Primary alcohols were found to be the best acceptors. The highest yields of alkyl beta-xylosides and xylobiosides were 33% and 50% of the total sugar, respectively. XynB showed a great ability to transfer xylose and xylobiose to secondary alcohol acceptors, and was unique for being able to synthesize the tertiary alkyl beta-xylosides and xylobiosides with high yields of 18.2% and 11.6% of the total sugar, respectively. This is the first report of a xylanase with the ability to synthesize tertiary alkyl beta-xylosides and xylobiosides. The specificity of the beta-linkage was confirmed by the proton nuclear magnetic resonance ((1)H NMR). Thus, XynB of T. maritima appears to be an ideal enzyme for the synthesis of useful alkyl beta-xylosides and xylobiosides.     

Glycosidase-catalysed synthesis of alkyl glycosides

Glycosidases catalyse the synthesis of anomerically pure alkyl glycosides in one step. In contrast, chemical synthesis of anomerically pure glycosides is circuitous and expensive. Two methodologies are used in enzymatic glycosylation: thermodynamically controlled reversed hydrolysis and kinetically controlled transglycosylation. The advantages and limitations of both approaches are delineated. Glycosidases exhibit broad specificity with regard to the aglycon: in addition to simple alcohols, hydroxy amino acids, nucleosides, ergot alkaloids and cardiac genins are glycosylated. Non-alcohol acceptors such as oximes and thiols also function as substrates whereas pyranoid glycals act as non-natural donors. Glycosidases exhibit absolute selectivity with regard to the stereochemistry at the anomeric centre and show a high degree of chemoselectivity for different hydroxyl groups, e.g., the order of reactivity is primary>secondary alcohols>phenols; tertiary alcohols are unreactive. Chiral primary alcohols are poorly discriminated, but the enantioselectivity towards a hydroxyl group that is directly attached to a (pro)chiral carbon atom is often high. The synthetic utility of glycosidases would be considerably improved if methods could be found for maintaining their catalytic activity in non-aqueous media.     

Optimization of alkyl beta-D-galactopyranoside synthesis from lactose using commercially available beta-galactosidases

Commercially available lactase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) enzymes produced from Kluyveromyces fragilis and Kluyveromyces lactis were accessed as catalysts for use in the production of beta-galactopyranosides of various alcohols using lactose as galactosyl donor. The yield of galactoside was enhanced by using the highest practical concentrations of both lactose and alcohol acceptor. The concentrations and thus yield, were limited by the solubility of the substrates. The increase in galactoside yield with increasing lactose concentration appeared to be specific to the lactose substrate and not due to water activity alterations, because addition of maltose to a fixed concentration of lactose had no effect. During the course of the reaction, the yield of galactoside peaked after around 70% to 80% of the lactose was consumed, due to hydrolysis of the product by the enzyme. A wide variety of compounds with primary or secondary hydroxyl groups could act as acceptors, the essential requirement being at least some water solubility. Addition of organic cosolvents had little effect on galactoside yield except when it increased the water solubility of sparingly soluble alcohols. Some galactosides were synthesized on a gram scale to determine practical product recoveries and improve purification methods for large-scale synthesis. Initial purification by hydrophobic chromatography (for galactosides of hydrophobic alcohols) or strong anion-exchange chromatography (for galactosides of hydrophilic alcohols) separated galactosides, galactobiosides, and higher oligomers from reducing sugars. A facile separation of the galactoside and galactobioside could then be effected by flash chromatography on silica gel. (c) 1993 John Wiley & Sons, Inc.     

Glycosidases in organic solvents: I. Alkyl-β-glucoside synthesis in a water-organic two-phase system

A low-water organic solvent two-phase system suitable for glycosylation of hydrophobic substrates is described. Almond β-glucosidase adsorbed on polymeric supports has been shown to catalyse alkyl-β-glucoside synthesis via a transferase reaction or through direct condensation of the glucosidic bond. High concentrations of glucosyl donors were present in the aqueous phase, while water-immiscible primary alcohols, which form the organic phase, served as acceptors of glucose. Reaction yield appeared to be thermodynamically controlled. The influence of various support materials, glucosyl donors, and glucosyl acceptors on reaction rate and product yield was investigated.     

Production of alkyl glucoside from cellooligosaccharides using yeast strains displaying Aspergillus aculeatus β-glucosidase 1

For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of β-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl β-d-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond β-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates.     

Unique transglycosylation potential of extracellular α-d-galactosidase from Talaromyces flavus

The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail.     

Alpha- and beta- aspartyl peptide ester formation via aspartimide ring opening.

The undesirable reaction of aspartimide formation has been proved to occur under both acid and base conditions in solid-phase peptide synthesis and is dependent on the beta-carboxyl protecting group, the acid or base used during the synthesis, as well as the peptide sequence. The hydrolysis of aspartimide-containing peptides, especially during HPLC purification, yields a mixture of alpha- and beta-aspartyl peptides that can not be purified easily. A previous study demonstrated that treatment of aspartimide-containing peptides with methanol in the presence of 2% diisopropylethylamine in solution leads to alpha- and beta-aspartyl peptide methyl esters. Taking advantage of these results and aiming at elucidating the optimal conditions for aspartimide ring opening, the effect of different types and concentrations of alcohols (primary and secondary) and bases (diisopropylethylamine, collidine, 4-pyrrolidinopyridine, 1-methyl-2-pyrrolidone, piperidine and KCN) was tested at various temperatures and reaction times. The best results were obtained with a combination of a primary alcohol and diisopropylethylamine, while aspartimide ring opening by secondary alcohols occurred only at high temperatures. The optimal conditions were also applied to solid-phase peptide synthesis.     

Synthesis of alkyl-alpha-L-rhamnosides by water soluble alcohols enzymatic glycosylation

The synthesis of alkyl-alpha-rhamnosides by alpha-rhamnosidase was studied using rhamnose and rhamnosides, particularly the flavonoid naringin, as glycosylation agents, and water soluble alcohols as acceptors. The reaction products were analyzed by HPLC chromatography and identified by 13C y 1H NMR. The glycosylation of alcohols by reverse hydrolysis was maximum for 40% methanol, 30% ethanol, 10% propanol and 20% isopropanol. Under optimum conditions the yield of rhamnose to alkyl-alpha-rhamnoside transformation decreased from 68% for methyl-alpha-rhamnoside to 10% for isopropyl-alpha-rhamnoside. The time course of rhamnosylations produced using naringin as the donor was comparable with that of the reverse hydrolysis obtained at the same molar concentration of the donor. The flavonoids and their derivatives remaining in the solution after the glycosylation were removed by ion exchange QEAE chromatography at pH 10. These results indicate that both, reverse hydrolysis and glycosylation by naringin are acceptable procedures for the enzymatic synthesis of short chain length alkyl-alpha-L-rhamnosides.     

Transglucosylation of tertiary alcohols using cassava beta-glucosidase

We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from p-nitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis.     

Role of hydrophobic residues in the aglycone binding subsite of a GH39 β-xylosidase in alkyl xylosides synthesis

The GH39 β-xylosidase from Bacillus halodurans (BhXyl39) was previously reported to catalyze the synthesis of alkyl xylosides from donors such as pNP β-d-xylopyranoside or xylobiose and from aliphatic alcohols acting as acceptors with chain length inferior to five carbons. In the present study, the role played by aromatic residues present in the aglycone binding subsite of BhXyl39 in the hydrolysis and transglycosylation reactions of the enzyme was investigated. In this way, site-directed mutagenesis was carried out in order to highlight the role of three targeted hydrophobic residues F116, F167 and Y284. These residues were replaced by alanine to decrease the steric hindrance or were mutated into serine to evaluate the impact of the presence of a polar residue into the aglycone binding subsite of BhXyl39. Taking into account kinetic parameters and yields of transglycosylation, the function of each mutated residue in the catalytic mechanism was studied. Results concerning transglycosylation reactions in the presence of pentan-1-ol and octan-1-ol indicated that yields of transglycosylation were impacted both by the position and the nature of the mutated residues. These results were consistent with molecular docking performed with both acceptors which notably confirms that among the three targeted residues, F116 represents the most interesting one for mutagenesis to increase the transglycosylation reactions in presence of long chain alcohols.     

Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum

Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.     

Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.     

Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

A kinetic study of almond-β-glucosidase catalysed synthesis of hexyl-glycosides in low aqueous media: Influence of glycosyl donor and water activity

A variety of alkyl and aryl glycosides were investigated as substrates for almond β-glucosidase catalysed synthesis of hexyl-β- -glycosides in low aqueous hexanol media. The rate-limiting step in the organic media was determined to be the glycosylation of the enzyme. The kinetic constants V_max, K_m (glycosyl donor) and V_max/K_m were all influenced by the water activity and they all increased in value with increasing water activity. The increase in V_max/K_m was mainly determined by the increase in V_max and a plot of log(V_max/K_m) versus water activity resulted in a straight line with similar slopes for all glycosides but with different absolute values and thus the most reactive substrate p-nitrophenyl glucoside was the best one in the entire water activity range studied (0.53–0.96). The preference for the two competing acceptors, hexanol and water, was not affected by the aglycon part of the glucoside. Surprisingly, the ratio between trans glycosylation and hydrolysis increased with increasing water activity. A decrease in water activity caused an increase in equilibrium yield of hexyl glycoside, as expected, but was not beneficial for the kinetically controlled yield.     

Enzymatic Synthesis of Hexyl Glycosides from Lactose At Low Water Activity and High Temperature Using Hyperthermostable β-glycosidases

Optimization of hexyl-g-glycoside synthesis from lactose in hexanol at low water activity and high temperature was investigated using g-glycosidases from hyperthermophilic organisms: Sulfolobus solfataricus (LacS) and Pyrococcus furiosus (CelB). The method for water activity adjustment by equilibration with saturated salt solutions was adapted for use at high temperature. The influence of enzyme immobilization (on XAD-4, XAD-16, or Celite), addition of surfactants (AOT or SDS), substrate concentration, water activity, and temperature (60-90°C) on enzymatic activity and hexyl-g-glycoside yield were examined. Compared to other g-glycosidases in lactose conversion into alkyl glycoside, these enzymes showed high activity in a hexanol one-phase system and synthesized high yields of both hexyl-g-galactoside and hexyl-g-glucoside. Using 32 λg/l lactose (93 λmM), LacS synthesized yields of 41% galactoside (38.1 λmM) and 29% glucoside (27.0 λmM), and CelB synthesized yields of 63% galactoside (58.6 λmM) and 28% glucoside (26.1 λmM). With the addition of SDS to the reaction it was possible to increase the initial reaction rate of LacS and hexyl-g-galactoside yield (from 41 to 51%). The activity of the lyophilized enzyme was more influenced by the water content in the reaction than the enzyme on solid support. In addition, it was concluded that for the lyophilized enzyme preparation the enzymatic activity was much more influenced by the temperature when the water activity was increased. A variety of different glycosides were prepared using different alcohols as acceptors.     

Screening of various glycosidases for the synthesis of octyl glucoside

Thirteen glycosidases of microbial origin and almond beta-glycosidase were assayed in octanol/DMF (80:20, v/v), using a combination of hydrolysis, transglycosylation, and condensation reactions, in order to assess their potential for the production of alkyl glucosides. The two mesophile enzymes were highly impaired by the organic media. Three of the 11 thermophile enzymes gave interesting results in the hydrolysis and transglycosylation reactions, but they were highly inhibited by glucose. This made their use in a condensation reaction less interesting than the use of almond beta-glucosidase, which has a lower activity but shows less inhibition by the glucose.     

Kinetically controlled synthesis of ampicillin with immobilized penicillin acylase in the presence of organic cosolvents

Penicillin acylase (PA) is used in the industrial production of 6-amino penicillanic acid (6-APA). However, by proper control of reaction medium, the enzyme can be used in the reverse synthesis of β-lactam antibiotics from the corresponding β-lactam nuclei and suitable acyl donors. Under thermodynamically controlled strategy, the use of organic cosolvents can favor synthesis over hydrolysis by lowering water activity and favoring the non-ionic reactive species. Under kinetically controlled strategy using activated acyl donors, organic solvents can favor synthesis by depressing hydrolytic reactions. Results are presented on the synthesis of ampicillin from phenylglycine methyl ester and 6-APA with immobilized Escherichia coli PA in the presence of organic cosolvents. Several solvents were tested in terms of enzyme stability and solubility of substrates. Ethylene glycol, glycerol, 1–2 propanediol and 1–3 butanediol were selected accordingly and ampicillin synthesis was performed in all of them. Best results in terms of yield and productivity were obtained with ethylene glycol, with which further studies were conducted. Variables studied were enzyme to limiting substrate ratio, acyl acceptor to acyl donor ratio, organic solvent concentration, pH and temperature. Experimental design based on a two-level fractional factorial design was conducted. pH was determined as the most sensitive variable and was further optimized. The best conditions for ampicillin synthesis in terms of productivity, within the range of values studied for those variables, were pH 7.4, 28°C, 36 U_S PA/mmol 6-APA, 3 mol PGME/mol 6-APA and 45 % (v/v) ethylene glycol concentration. Productivity was 7.66 mM ampicillin/h, which corresponds to a specific productivity of 7.02 μmol ampicillin/h U_S at 55 % yield. Productivity was lower than in buffer but product yield was higher because of the much lower relative hydrolysis rates.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for β-galactosidase activity, were used for the synthesis of 1-hexyl-β- -galactoside and 1-octyl-β- -galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6–36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week.     

α-Amylase-catalysed synthesis of alkyl glycosides

Alkyl glycosides were synthesised from starch and alcohols using Aspergillus oryzae α-amylase as catalyst. In the degradation of starch by α-amylase, the alcohols competed with water as glycosyl acceptors. In the reaction with methanol, methyl maltoside and methyl maltotrioside were the main alcoholysis products. Conversion of 45 g/l starch in 30% methanol resulted in a product mixture containing 26 mM maltooligosaccharides and 3.6 mM methyl glycosides. With ethanol, propanol and butanol, alkyl maltosides and alkyl maltotetraosides were detected, and with benzyl alcohol, benzyl glycosides having two, three or five glucose units were formed. No alcoholysis reaction occurred with hexanol or octanol. In conclusion, α-amylase is promising for the one-step synthesis of alkyl glycosides having more than one monosaccharide unit, which are difficult to synthesise in other ways.