Enzymatic Synthesis of Polyol- and Masked Polyol- Glycosides using β-Glycosidase of Sulfolobus Solfataricus

The use of commercially available mesophilic glycosidases in the enzymatic synthesis of glycosides of different types is a well established method suffering from some drawbacks such as a poor yield. Substrates with three or four hydroxyl groups have been subjected to enzymatic glucosylation using crude homogenate of the thermophilic archaeon Sulfolobus solfataricus containing a β-glycosidase activity able to transfer glucose, galactose and fucose from different donors. The stereochemistry of this reaction was interpreted in terms of interaction with a possible “glucose” active site of the enzyme. In addition masked or protected derivatives of tetritols and some simple unsaturated alcohols were glycosylated yielding glycosides in yields very competitive with those obtained using mesophilic enzymes, examples of further chemical manipulation of these compounds were reported. When using a scarce amount of acceptor, a reasonable amount of products could be obtained by adding different aliquots of donor at time intervals.     

Enzymatic Synthesis of Carbohydrate Derivatives Using β-Glycosidase of Sulfolobus Solfataricus

Enzymatic synthesis of different β-D-glycosides was obtained using as biocatalyst immobilized cells, crude homogenate, and homogeneous native and recombinat β-glycosidase activity of the thermophilic archaeon Sulfolobus solfataricus. In particular our investigation was concerned with the selectivity in the glycosylation of hydroxybenzyl alcohols, salicin, 1,2-propanediol, and more complex polyols as well as the use of immobilized cells for the synthesis of hexyl-β-D-glucoside. The aromatic glucosides obtained by enzyme-catalyzed transglucosylation were used for kinetic studies of purified Sulfolobus solfataricus enzyme in the hydrolysis reaction.     

Enzymic Preparation of Benzyl β-D-Glucopyranoside

Benzyl β-D-glucopyranoside was prepared by an enzyme-catalysed direct reaction between D-glucose, or better cellobiose, and benzyl alcohol in the presence of a minimum amount of water. The enzyme β-glucosidase was used in the immobilized form (adsorbed onto macroporous polyethylene terephthalate or covalently bound on polyglycidyl methacrylate), enabling multiple application.     

Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme.     

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.     

Enzymatic Synthesis of Thioglucosides Using Almond β-glucosidase

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The β-glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond β-glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond β-glucosidase. The substrate specificity of the almond β-glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond β-glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT.     

Biotransformations of α,β-Epoxyalcohols Catalyzed by Epoxide Hydrolases

Microsomal and cytosolic fractions of mammalian livers were screened for their capacity to resolve racemic mixtures of trans -2,3-epoxy-l-alkanols. The epoxide hydrolase activities showed some specificity for the 2S, 3S enantiomers which were attacked at the proximate carbon atom. The best resolutions were observed with guinea pig liver microsomal enzymes.     

Temporal Changes of Acid Phosphatase, Arylsulphatase, β-Galactosidase and β-N-ACETYL-d-Glucosaminidase Activities in Subcellular Fractions of Rat Liver

In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β-N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values.     

Modification of subunit interaction in membrane-bound acid β-glucosidase from Gaucher disease

The radiation inactivation method has been used to determine the molecular mass of membrane-bound acid β-glucosidase (EC 3.2.1.21) in situ, in normal human spleen and in that of two patients with type I Gaucher disease: the molecular mass in Gaucher spleen is about double (125 000 ± 8900) of that found in the normal spleen (67 000 ± 7700) which is compatible with the existence of subunit coupling in the muted acid β-glucosidase. From the results, we conclude that subunit interaction is altered in mutant acid β-glucosidase and that this may be due to a direct effect of the mutation.     

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.     

Inhibition of protein synthesis by the β-subunit of spectrin

The 220 kDa β-subunit of erythroid cell spectrin is a potent inhibitor of protein synthesis in lysates from rabbit reticulocytes. On the basis of weight of protein added to a lysate reaction mixture, it has about half the inhibitory activity of highly purified heme-regulated eIF-2 kinase. Inhibition appears to be at the level of peptide initiation but does not involve a kinase that phosphorylates eIF-2 on its -subunit.     

Synthesis of Oligosaccharides by β-Fructofuranosidase in Biphasic Medium Containing Organic Solvent as Bulk Phase

The effect of four organic solvents on β-fructofuranosidase mediated synthesis of oligosaccharides from sucrose were investigated. Amongst the solvents examined, butyl acetate proved to be the best for oligosaccharide synthesis. Starting with the equivalent of 44.6 g/L of sucrose, 247 U of enzyme and 91.6% (by vol.) of butyl acetate results in the production of 8.8 g/L of oligosaccharides within 30 min, with trisaccharides constituting more than 60% of the oligosaccharides. The efficiency for conversion of sucrose to oligosaccharides is greater than 19%, and this exceeds the 11.6% (in 24 h) previously achieved with 1271 U of the same enzyme in aqueous medium. Use of butyl acetate as the bulk phase therefore modifies the reaction environment in favour of enhanced and accelerated rate of oligosaccharide synthesis by this β-fructofuranosidase.     

N-Nitroso-β-lactams as β-lactamase inhibitor

N-Nitroso-β-phenyl-β-lactam has been found to be a specific inhibitor of β-lactamase. N-Nitroso--phenyl-β-lactam, by contrast, was virtually ineffective although a transient inhibition of short duration was observed. The acyl enzyme derived from the β-phenyl isomer is presumably involved in a cross-linking reaction, whereas that from the -phenyl isomer was quenched by spontaneous hydrolysis without formation of a covalent bond. No inhibitory effect of the β-phenyl isomer on chymotrypsin has been observed.     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 *

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei)TK1501 β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86.63kDa和675.56U/mg。最适作用温度和pH分别为30℃和6.5。 Mg ²⁺和Ca ²⁺对β-葡糖苷酶酶活抑制作用最小,Cu ²⁺几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_max分别为1.44mmol/L和58.32mmol/(L·s),催化系数k_cat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501 β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

Proteolytic modification of membrane-associated phospholipase C-β by μ-calpain enhances its activation by G-protein βγ subunits in human platelets

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-β (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-β was found to be activated to a greater extent by brain G-protein βγ subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with μ-calpain of the intact PI-PLC-β (150 kDa) caused a marked augmentation of its activation by βγ subunits. This enhanced PLC activation by βγ subunits was due to truncation by μ-calpain, producing a 100 kDa PI-PLC, but not by another protease,thrombin.     

Kinetics of Parallel Electron Transfer from β-Carotene to Phenoxyl Radical and Adduct Formation Between Phenoxyl Radical and β-Carotene

Phenoxyl radicals generated by laser flash photolysis were found to react with β-carotene with concomitant β-carotene bleaching in two parallel reactions with similar rates: (i) formation of a β-carotene adduct with a (pseudo) first order rate constant of 1-1.5 ± 10⁴ s^-1 with absorption maximum around 800 nm, and (ii) formation of a β-carotene radical cation with a (pseudo) first order rate constant of 2-3 ± 10⁴ s^-1 with absorption maximum around 920 nm. Both β-carotene radicals decay on a similar time scale and have virtually disappeared after 100 ms, the β-carotene adduct by a second order process. Oxygen had no effect on β-carotene bleaching or radical formation and decay. The reduction of phenoxyl radicals by β-carotene may prove important for an understanding of how β-carotene acts as an antioxidant.     

Hyperproduction of thermostable β-glucosidase by Sporotrichum (Chrysosporium) thermophile

The effect of the growth of temperature, pH, carbon source, nitrogen supplementation and inoculum size were examined in shake-flask-scale studies to determine the optimum conditions for β-glucosidases production by Sporotrichum (Chrysosporium) thermophile. Wheat bran and sugar-beet pulp were selected as the best carbon sources and (NH₄)₂SO₄, NH₄Cl and KNO₃ as the best nitrogen supplementation. Ten liter fermentations were carried out to study the kinetics of product formation. It was found that S. thermophile is able to produce high thermostable extracellular cellobiase and aryl-β-glucosidase. Very high aryl-β-glucosidase (PNPG) activities in the range from 30 to 40 U ml⁻¹ and cellobiase activities of 2,45 U ml⁻¹ in the 3-day batch fermentations were obtained. The K_m for aryl-β-glucosidase and its thermal properties were also estimated.     

Enzymatic production of γ-L-glutamyltaurine through the transpeptidation reaction of γ-glutamyltranspeptidase from Escherichia coli K-12

γ-L-Glutamyltaurine is a naturally occurring peptide and known to have several physiological functions in mammals. This paper describes a new method for the enzymatic production of γ-L-glutamyltaurine from L-glutamine and taurine through the transpeptidation reaction of γ-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12. The optimum conditions for the production of γ-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine and 0.2 U/ml γ-glutamyltranspeptidase, pH 10, and 1-h incubation at 37°C. Forty-five mM γ-L-glutamyltaurine was obtained, the yield being 22.5%. γ-L-Glutamyltaurine was purified on Dowex 1 × 8 and C₁₈ columns, and identified by means of NMR and a polarimeter.     

Effects of Acetonitrile-Water Mixtures on α-Chymotrypsin Catalyzed Dipeptide Synthesis

-Chymotrpysin (EC 3.4 21.1) was immobilized by deposition on celite and subsequent cross-linking with glutaraldehyde. The effects of different mixtures of aqueous buffer and acetonitrile on the immobilized preparation were evaluated using a dipeptide synthesis as model reaction. The initial reaction rate at 6-95% of water increased with increasing water content. The maximum yield of peptide had two maxima; the first one at 6% of water (92%) and the second one at 80% of water (39%). The presence of two maxima was due to severe enzyme inactivation at intermediate water contents (50-60%). The immobilisation procedure slowed the inactivation of -chymotrypsin. Cross-linked enzyme was inactivated to a lesser extent than both free enzyme and enzyme that had been deposited on celite. The increased resistance to inactivation was, however, not sufficient to make peptide synthesis attractive at intermediate water contents (50-60%). In order to obtain good peptide yields, low water contents (below 10%) should be used.