Reverse Hydrolysis Reaction Research Articles

Studies on the Syntheses of Oligosaccharides Using Glycosidases.

I have studied the enzymatic syntheses of oligosaccharides composing the sugar chains of glycoconjugates using glycosidases by two types of reactions: reverse hydrolysis and transglycosylation. In the reverse hydrolysis reaction, oligosaccharides having a variety of linkages were obtained by a batch method. On the other hand, it has been clarified that glycosidase having a different substrate specificity and from a different origin gives a different oligosaccharide with respective regioselectivity by the column method. In the transglycosylation reaction, the oligosaccharide was synthesized regioselectively according to the substrate specificity of the glycosidase used.

52] Enzymatic synthesis of [14C]ceramide, [14C]glycosphingolipids, and ω-aminoceramide

Publisher Summary This chapter isolates a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids and tentatively names it “sphingolipid ceramide N-deacylase” (SCDase). SCDase is found to catalyze the hydrolysis and its reverse reaction as well, under different conditions. Under acidic conditions including a high concentration of Triton X-100, SCDase catalyzes the hydrolysis reaction efficiently. In contrast, at neutral pH with a low concentration of the detergent, the reverse reaction tends to be favored. It is possible therefore to utilize the desired reaction of SCDase if the conditions are changed. This chapter describes the synthesis of [ 14 C]ceramide, [ 14 C]glycosphingolipids, and ω-aminoceramide by the reverse hydrolysis (condensation) reaction of SCDase. Because the specificity of the enzyme is very specific to the amino group of sphingosine bases, no by-products are generated during the reaction. The fact that a wide range of sphingolipid species and sample amounts (from the nanomole to millimole level) can be used is significantly advantageous.

Reverse hydrolysis reaction of chitin deacetylase and enzymatic synthesis of β- d-GlcNAc-(1→4)-GlcN from chitobiose

We found that a chitin deacetylase from Colletotrichum lindemuthianum could acetylate free amino sugar residues into N-acetylated forms in the presence of 3.0 M sodium acetate. The result was analyzed using a β- N-acetylhexosaminidase-coupled assay system with p-nitrophenyl 2-amino-2-deoxy-β- d-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β- d-glucopyranoside as the substrate, and the liberation of p-nitrophenol was observed as a consequence of enzymatic N-acetylation of the glucosamine residue at the nonreducing end of the substrate. The chitin deacetylase also acetylated chitobiose and chitotetraose as substrates, which was evidenced by the decrease in the amount of free amino sugar residues in the chitooligosaccharides. The reaction product of chitobiose after the acetylation reaction was exclusively 2-acetamido-2-deoxy-β- d-glucopyranosyl-(1→4)-2-amino-2-deoxy- d-glucose [GlcNAcGlcN], the structure of which was determined by FABMS and NMR analyses. This study offers a novel method for enzymatic N-acetylation of amino sugars, and especially with chitobiose as substrate, a selectively N-acetylated product, GlcNAcGlcN, can be synthesized.

Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins.

Sphingolipid ceramide N-deacylase is an enzyme capable of hydrolyzing the N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis of fluorescence-labeled sphingolipids utilizing the condensation reaction of the enzyme. N-TFAc-aminododecanoic acids were efficiently condensed by the enzyme to the lyso-forms of GM1 and sphingomyelin in glycine buffer (pH 10). The reaction products, N-TFAc-amino-GM1 and sphingomyelin, were obtained with overall yields of 60%. The purified products were identified to be omega-amino-GM1 and omega-amino-sphingomyelin, respectively, by TLC and FAB-MS or ESI-LC/MS analysis after removal of the N-TFAc by mild alkaline treatment. NBD-labeled GM1 and sphingomyelin were prepared from omega-amino-GM1 and omega-amino-sphingomyelin by coupling with 4-fluoro-NBD. These fluorescence-labeled substrates, C12-NBD-GM1 and C12-NBD-sphingomyelin, were hydrolyzed by endoglycoceramidase and sphingomyelinase, respectively, to produce NBD-dodecanoylsphingosines, but were resistant to hydrolysis by sphingolipid ceramide N-deacylase. C12-NBD-sphingomyelin was found to be a better substrate than the commercially available C6-NBD-sphingomyelin for the assay of sphingomyelinase from various sources. We also describe a new method to detect GM1-binding proteins using fluorescence-labeled GM1.

Enzymatic-catalyzed synthesis of alkylglycosides in monophasic and biphasic systems. II. The reverse hydrolysis reaction

The β-glucosidase catalyzed synthesis of butylglucoside through the condensation of glucose and butanol was carried out in monophasic and biphasic systems. Water was indispensable for the enzyme activity. In the case of the monophasic system, product concentration increased with the increase of water content, but at high levels of water the equilibrium shifted towards hydrolysis and therefore the butylglucoside concentration decreased. An optimal water content of about 8–13% (v/v) was found. However, in the case of biphasic system, the increase of the aqueous/butanol phase ratio (v/v) was accompanied by a decrease of the butylglucoside concentration. An analysis of the causes of the equilibrium shift is given.

Surface chemistry effects on the processing and superplastic properties of nanocrystalline oxide ceramics

The unusual bulk behavior of nanoparticle and nanograined systems often originates in surface chemistry effects. Three examples are used to illustrate this point. In the first, newly precipitated nanocrystalline titania is washed with ethanol, and the mixture of these two supposedly inert substances causes the titania to lose its anatase crystal structure and become amorphous. This phenomenon is attributed to a reverse hydrolysis reaction at the particle surface. In the second example, nanocrystalline ZrO 2-3mol%Y 2O 3 is observed to partially dissolve on exposure to pH-adjusted water, due to the formation of soluble hydroxides at the particle surface. A major consequence of the dissolution is the formation of large, hard, multiparticle agglomerates on subsequent drying. In the final example, ZrO 2-3mol%Y 2O 3 particles are intentionally surface-doped with submonolayer levels of Cu-containing ions from ammoniacal solutions. The ceramics fabricated from such powders exhibit superplastic strain rates 100 or so times faster than in comparable undoped systems, due to the dopant’s role in lowering of the activation energy for diffusion along grain boundaries.

C-Terminal Peptide Amidation Catalyzed by Orange Flavedo Peptide Amidase

The reverse reaction of amide hydrolysis can be achieved with the peptide amidase derived from oranges [Eq (1); Z=benzyloxycarbonyl]. The C-terminal carboxy group of the peptide is directly converted into an amide group by condensation with an ammonium salt. The amidation of peptides is of major interest since the biological activity of proteohormones and peptides is strongly influenced by the presence of a C-terminal amide group.

Enzymatic synthesis of 14C-glycosphingolipids by reverse hydrolysis reaction of sphingolipid ceramide N-deacylase: detection of endoglycoceramidase activity in a seaflower.

This paper describes the synthesis of 14C-labeled glycosphingolipids using the reverse hydrolysis reaction (condensation) of sphingolipid ceramide N-deacylase. It was found that 50-70% of 14C-fatty acids were incorporated into various lyso-glycosphingolipids when a mixture of lyso-glycosphingolipids and fatty acids was incubated at 37 degrees C with 1 mU of the enzyme for 20 h in 1 ml of 25 mM phosphate buffer, pH 6.0-7.0, containing 0-0.1% Triton X-100. The optimum concentration of lyso-glycosphingolipids was 100-400 microM depending on the species of lyso-form when [14C]stearic acid was used at the concentration of 100 microM. Free 14C-fatty acids and lyso-glycosphingolipids were separated from the synthesized 14C-glycosphingolipids by using a Sep-Pak Plus Silica and a Sep-Pak CM or a QMA cartridge, respectively. After treatment of 14C-glycosphingolipids with endoglycoceramidase or sphingolipid ceramide N-deacylase, digestion products were clearly separated from the parent glycosphingolipids on TLC and determined using an image analyzer with a sensitivity 100 times higher than that using non-radiolabeled substrates. Using this method, we found endoglycoceramidase activity in a seaflower, Condylactis sp., for the first time.

Effective biocatalytic transgalactosylation in a supercritical fluid using a lipid-coated enzyme

A lipid-coated β-D-galactosidase is soluble and acts as an efficient trangalactosylation catalyst (the reverse hydrolysis reaction) in supercritical carbon dioxide (scCO2).

Intrinsic nucleoside diphosphate kinase-like activity as a novel function of 14-3-3 proteins

14-3-3 proteins play a role in many cellular functions as molecular chaperone and adapter proteins: they bind to and modulate several proteins involved in cell proliferation and differentiation, and also function ATP-dependently in targeting of precursors to mitochondria. We show here that 14-3-3 purified from a human lymphoblastoma and also its recombinant τ isoform exhibited intrinsic nucleoside diphosphate (NDP) kinase-like activity. 14-3-3 proteins preferentially catalyzed the transfer of the γ-phosphate group from ATP, dATP or dGTP to all nucleoside diphosphates and this transfer involved acid-labile phosphoenzyme intermediates. They also simultaneously catalyzed the reverse reaction of ATP hydrolysis. These properties of 14-3-3 are similar to those of NDP kinase, but not to those of adenylate kinase.

14C]Ceramide Synthesis by Sphingolipid CeramideN-Deacylase: New Assay for Ceramidase Activity Detection

Sphingolipid ceramideN-deacylase (SCDase) is an enzyme which hydrolyzes theN-acyl linkage between fatty acid and sphingosine in ceramide of various glycosphingolipids and sphingomyelin. Recently the enzyme was found to catalyze the hydrolysis and its reverse reaction under different conditions. We report here an innovative method for synthesis of radioisotope-labeled ceramide with high specific activity using the reverse hydrolysis reaction of SCDase. More than 80% of free fatty acid was transferred to sphingosine when 1 nmol [14C]stearic acid and 2 nmol sphingosine were incubated with 5 μU SCDase at 37°C for 20 h in 10 μl of 25 mmphosphate buffer, pH 7.0, containing 0.3% Triton X-100. Free [14C]fatty acid and sphingosine were easily separated from synthesized [14C]ceramide by using a Sep-Pak Plus Silica and a Sep-Pak CM cartridge, respectively. We also developed a sensitive assay method for ceramidase using the [14C]ceramide prepared. The method consists of separation of the [14C]fatty acid released from [14C]ceramide by the action of enzyme on thin-layer chromatography followed by analysis and quantification with an imaging analyzer (BAS1000). This method was sensitive and qualitative and enabled the detection of ceramidase activity in invertebrates for the first time as well as in several human cancer cell lines.

Reverse hydrolysis reaction in aqueous medium without any cosolvent

The synthesis of L-tyrosine fructosyl ester, from fructose and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by α-chymotrypsin in water without cosolvent. The effect of fructose concentration and temperature for the transesterification reaction were determined on both specific activities and product yield. The influence of the presence of fructose has been studied regarding α-chymotrypsin and L-tyrosine fructosyl ester stabilities. It appeared that an increase of temperature enhanced enzyme activity but slumped the product yield because of the very weak stability of tyrosine fructosyl ester.

Role of methanol on the catalytic behavior of β-glucosidase from Fusarium oxysporum

Fusarium oxysporum β-glucosidase catalyses the transglycosylation and reverse hydrolysis reaction in an aqueous solution, in the presence of a second nucleophile stronger than water, such as methanol. In transglycosylation, the K m values of cellobiose for the trans reaction and hydrolysis were 138.2mM and 356.5mM respectively and the competitive role of methanol was shown. The K m value of methanol was calculated to be 1.41M. In the reverse hydrolysis the influence of the donor concentration, up to 90% w/v glucose, was investigated and the competitive role of methanol as a nucleophilic acceptor of the glycosyl moiety was also demonstrated.

Glycosylation employing bio-systems: from enzymes to whole cells

This contribution will highlight chemoenzymatic approaches to the rather complex task of stereospecific and regiospecific glycosylation. Advantages and problems associated with the application of enzymes from carbohydrate metabolism as organic reagents in aqueous and non-aqueous solvent systems are discussed. One chapter will report on the use of glycosidases in reverse hydrolysis and tranglycosylation reactions. Another section focuses on the syntheses with simple as well as more complex and delicate glycosyl-transferases including cofactor regeneration. Further, effective combination of both degrading and synthesising enzyme systems, and finally glycosylations employing cells with direct access to their complete enzyme equipment are treated.

Bioconversion of 7-Aminocephalosporanic Acid by Intact Rhodotorula glutinis Cells

When 7-aminocephalosporanic acid (7-ACA) was used as a single carbon source in the enrichment culture medium for screening 7-ACA-degrading microorganisms, pink yeast colonies appeared frequently, and these were identified as Rhodotorula glutinis. These intact R. glutinis cells converted (i) 7-ACA to deacetyl-7-ACA (7-ADACA) and (ii) monochloroacetyl-7-ACA to monochloroacetyl-7-ADACA at sufficiently high levels to be of commercial interest. Acetylation of 7-ADACA to 7-ACA, the reverse reaction of hydrolysis in an organic medium with methyl acetate as an acetyl donor, was also demonstrated.

Enzymatic glucosylation of hydrophobic alcohols in organic medium by the reverse hydrolysis reaction using almond‐β‐D‐glucosidase

Alkyl beta-D-glucosides were synthesized from D-glucose and alcohols by reverse hydrolysis using the commercially available almond beta-D-glucosidase in 9:1 (v/v) acetonitrile-water medium. The main characteristics of this enzyme-catalyzed glucosylation were established by using 2-hydroxybenzyl alcohol. The reaction is entirely regio- and stereoselective. The solvent plays a fundamental role because, by decreasing the water concentration in the medium, the shift of the reaction equilibrium toward synthesis is realized without using an excessive amount of alcohol. Nevertheless, a minimum amount of water is necessary to maintain the enzyme activity. In contrast to the use of the enzyme in aqueous medium, the pH of the added water in acetonitrile did not influence the synthesis. Using this procedure, we have conducted systematic glucosylation of numerous alcohols and we have investigated enzyme specificity and alcohol reactivity. The enzyme has a pronounced affinity for the alcohols containing a phenyl group, and enantioselectivity for the aglycon is obtained with 1-phenylethyl alcohol. Moreover, by using almond beta-D-glucosidase it was also possible to synthesize alkyl beta-D-galactosides. (c) 1995 John Wiley & Sons, Inc.

Monte Carlo simulation of the sol-gel process for the hydrolysis and polycondensation of tin tetra-n-butoxide

The sol-gel process for the hydrolysis and polycondensation of tin tetra-n-butoxide in butanol was followed by viscosity measurements. The precipitation of tin-containing ultrafine particles was observed without any variation in the solution viscosity in the absence of diethylene glycols, while the viscosity increase was made without any precipitation by the addition of diethylene glycol. The induction period and the rate of the viscosity increase after the induction period was independent of the diethylene glycol content but depended upon the water content. The Monte Carlo simulation data were consistent with data for the viscosity experiment. The simulation was based on a kinetic model of the slow hydrolysis of tin tetra-n-butoxide, the reverse reaction of hydrolysis, and the polycondensation of the hydroxy derivatives.

Lipase made active in hydrophobic media by coupling with polyethylene glycol

AbstractLipases from various microorganisms were chemically modified with polyethylene glycol derivatives: 2,4‐bis[O‐methoxypoly(ethylene glycol)]‐6‐chloro‐s‐triazine (activated PEG2, a chain‐shaped polymer) and copolymer of polyoxyethylene allyl methyl diether and maleic anhydride (activated PM, a comb‐shaped polymer). Because each polymer is amphipathic, the modified lipases become soluble not only in aqueous solution but also in hydrophobic media. They exhibit potent catalytic actions for ester synthesis and ester exchange reactions, the reverse reaction of hydrolysis, in transparent organic solvents and also in oily substrates without organic solvents. With PEG2‐lipases, macrocyclic lactone and gefarnate (geranyl farnesylacetate) were synthesized in high yields from 16‐hydroxy‐hexadecanoic acid ethyl ester and from farnesylacetic acid and geraniol in organic solvents, respectively. The modified lipase catalyzed the esterification preferentially with the (R)‐isomer of secondary alcohols. Because the ester synthesis reactions with modified lipase proceeded in the transparent benzene system, the kinetic parameters (Michaelis constant and maximum velocity) were obtained by reciprocal plotting according to the Michaelis equation. With comb‐shaped polymer as modifier, PM‐lipase catalyzed effectively the reverse reaction of hydrolysis in organic solvents. The properties of each modified lipase are discussed in relation to those of the nonmodified lipase.

A New Peptidase Enzyme Useful for Peptide and Amino Acid Derivative Synthesis by Reverse Hydrolysis Reactions

Annals of the New York Academy of SciencesVolume 613, Issue 1 p. 201-206 A New Peptidase Enzyme Useful for Peptide and Amino Acid Derivative Synthesis by Reverse Hydrolysis Reactions D. AURIOL, D. AURIOL BioEurope 31400 Toulouse, FranceSearch for more papers by this authorF. PAUL, F. PAUL BioEurope 31400 Toulouse, FranceSearch for more papers by this authorP. MONSAN, P. MONSAN BioEurope 31400 Toulouse, FranceSearch for more papers by this author D. AURIOL, D. AURIOL BioEurope 31400 Toulouse, FranceSearch for more papers by this authorF. PAUL, F. PAUL BioEurope 31400 Toulouse, FranceSearch for more papers by this authorP. MONSAN, P. MONSAN BioEurope 31400 Toulouse, FranceSearch for more papers by this author First published: December 1990 https://doi.org/10.1111/j.1749-6632.1990.tb18161.xCitations: 8AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume613, Issue1Enzyme Engineering 10December 1990Pages 201-206 RelatedInformation

Immobilization of Porcine-Pancreatic Lipase on Silica Support to Study Lipolysis and Reverse Hydrolysis Reaction

Abstract Porcine-pancreatic lipase has been immobilized on epoxypropylsilanized PartiSphere-5. The hydrolysis of olive oil on this column provided oleic acid as the major reaction product. The reversal of the hydrolysis reaction was tested by esterification of acetic acid and benzoic acid under nonaqueous conditions.