Regioselectivity in acylation of oligosaccharides catalyzed by the metalloprotease thermolysin

Investigation of the acylation scope of carbohydrates by metalloprotease thermolysin immobilized on Celite as biocatalyst has been carried out. The reactions were performed in DMSO, a good solvent for carbohydrates, where the enzyme has previously shown its activity in transesterifications of sucrose, maltose and maltose-containing oligosaccharides. Surprisingly, no reaction was observed for glucose or the glucose-containing disaccharides, trehalose and lactose. In contrast, laurate monoesters of several sucrose-containing tri- and tetrasaccharides were synthetized through a one step transesterification using vinyl laurate as the acylating agent. Enzyme regioselectivity was accurately determined by HPLC/MS and the structure of the main regioisomers was established by a combination of NMR experiments. The preferred position of acylation in all cases was the 2-OH of the α-d-glucopyranose moiety linked 1→2 to the β-d-fructofuranose unit. These results correlate with the regioselectivity observed in the case of the disaccharide sucrose. A general carbohydrate binding motif for catalysis by thermolysin is proposed.     

Evaluation of Enzymatic Reactors for Large-Scale Panose Production

Panose is a trisaccharide constituted by a maltose molecule bonded to a glucose molecule by an α-1,6-glycosidic bond. This trisaccharide has potential to be used in the food industry as a noncariogenic sweetener, as the oral flora does not ferment it. Panose can also be considered prebiotic for stimulating the growth of benefic microorganisms, such as lactobacillus and bidifidobacteria, and for inhibiting the growth of undesired microorganisms such as E. coli and Samonella. In this paper, the production of panose by enzymatic synthesis in a batch and a fed-batch reactor was optimized using a mathematical model developed to simulate the process. Results show that optimum production is obtained in a fed-batch process with an optimum production of 11.23 g/l h of panose, which is 51.5% higher than production with batch reactor.     

Biocatalytic and chemical investigations in the synthesis of sucrose analogues

Herein, we report about the synthesis of sucrose analogues, obtained by two different approaches: a chemical and an enzymatic. The one step synthesis of the sucrose analogues with the exo-fructosyltransferase (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, which transfers the fructosyl residue of the substrate sucrose to the monosaccharide acceptors galactose, mannose, xylose and fucose, has been developed. Effects in the fructosylation by variation of the positions of the hydroxyl-groups in glycopyranoside acceptors have been studied in respect to their acceptor properties. In contrast, the chemical equivalent nonenzymatic organic synthesis of galacto-sucrose and manno-sucrose has been achieved including six synthetic steps.     

Effect of sodium tetraborate (borax) on the thermal properties of frozen aqueous sugar and polyol solutions

The effect of sodium tetraborate (Na(2)B(4)O(7), borax) on the thermal property of frozen aqueous sugar and polyol solutions was studied through thermal analysis. Addition of borax raised the thermal transition temperature (glass transition temperature of maximally freeze-concentrated solutes; T(g)') of frozen sucrose solutions depending on the borax/sucrose concentration ratios. Changes in the T(g)' of frozen mono- and disaccharide solutions suggested various forms of complexes, including those of a borate ion and two saccharide molecules. Borax exerted the maximum effect to raise the oligosaccharide and dextran T(g)'s at borax/saccharide molar ratios of approximately 1-2 (maltose and maltooligosaccharides), 2 (dextran 1060), 5 (dextran 4900), and 10 (dextran 10200). Further addition of borax lowered T(g)'s of the saccharide solutions. Borax also raised T(g) and T(g)' temperatures of frozen aqueous glycerol solutions. The decreased solute mobility in frozen solutions by the borate-polyol complexes suggested higher collapse temperature in the freeze-drying process and improved stability of biological systems in frozen solutions.     

Comparison of Pulsed Electrochemical Detection Modes Coupled with Microchip Capillary Electrophoresis

There is a need to develop analytical methods that are capable of rapidly measuring small biological markers in the field of metabolomics. Among others, carbohydrates play an important role biologically yet are traditionally hard to detect since they have no chromophore or fluorophore. In the present report, the first application of integrated pulsed amperometric detection (iPAD) coupled with microchip electrophoresis to the analysis of glucose, mannose, sucrose, maltose, glucosamine, lactose, maltotriose and galactose is demonstrated. iPAD is an electrochemical detection mode that can be used for direct detection of carbohydrates, amines and sulfur containing compounds. The effect of different solution parameters, including the buffer concentration, pH and the concentration of SDS on both separation and detection response was analyzed. In addition, a comparison study between PAD and iPAD was performed using glucose, glucosamine, sucrose and maltose as model carbohydrates.     

A mid-IR flow-through sensor for direct monitoring of enzyme catalysed reactions. Case study: Measurement of carbohydrates in beer

A novel mid-IR flow-through sensor for in situ monitoring of the enzymatic reaction of amyloglucosidase with carbohydrates was developed. Amyloglucosidase was immobilised on agarose beads with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and directly placed in a conventional IR flow-through cell. The carbohydrate content of various beer samples was then determined by following the enzymatic hydrolytic cleavage of carbohydrates to glucose with Fourier-transform infrared (FTIR) spectroscopy. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating the flow-through sensor in a sequential injection (SI) manifold. As the immobilised enzyme was directly probed by the IR beam, an in situ study of the enzymatic reaction was possible enabling determination of the Michaelis-Menten constant of the immobilised enzyme. A linear calibration curve was recorded using maltose standards in the range between 0.86 and 7.13 g L(-1). The proposed method was successfully applied to the determination of the carbohydrate content of four different beer samples by the standard addition method. Moreover experiments showed the possibility of monitoring in situ the immobilisation of the enzyme as well as a small organic acid (malic acid) on the agarose beads using EDC.     

Characterization of dextransucrases fromLeuconostoc mesenteroides NRRL B-1299

Leuconostoc mesenteroides NRRL B-1299 dextransucrase was fractionated into soluble (SGT) and insoluble (IGT) enzyme preparations differing by their dextran content. In spite of this, they displayed the same Km for sucrose (10 g/L) and the same activation energy (35 kJ/mol). But the presence of cells and insoluble dextran led to the IGT behaving like an immobilized enzyme: stabilization against thermal denaturation and diffusional limitations at low substrate concentrations were observed. On the other hand, the behavior of SGT was influenced by the presence, in the preparation, of soluble dextran that reduced enzyme inhibition by excess substrate. SGT and IGT present very different pH profiles. In the presence of 4 g/L of soluble dextran, IGT was activated and displayed the same susceptibility to pH as SGT. The activation of IGT was highly dependent on the nature of the acceptor added but also on the pH of the reaction medium. IGT and SGT synthesize both soluble and insoluble polymer containing α(l → 2), α(l → 3), and α(l → 6) linkages. A larger amount of insoluble dextran is elaborated by SGT. The polymer structures, examined by¹³C NMR spectrometry, revealed that they differ mainly by their α(l → 3) linkage content (from 0 to 11%). This linkage seems to be partly responsible for the dextran insolubility and can be completely eliminated by carrying out the synthesis of soluble polymer at pH 7.4 with SGT.     

Optimization of dilute-acid pretreatment of corn stover using a high-solids percolation reactor

We have previously demonstrated that pretreatment of corn stover with dilute sulfuric acid can achieve high digestibility and efficient recovery of hemicellulose sugars with high yield and concentration. Further improvement of this process was sought in this work. A modification was made in the operation of the percolation reactor that the reactor is preheated under atmospheric pressure to remove moisture that causes autohydrolysis. This eliminated sugar decomposition during the preheating stage and led to a considerable improvement in overall sugar yield. In addition, liquid throughput was minimized to the extent that only one reactor void volume of liquid was collected. This was done to attain a high xylose concentration in the hydrolyzate. The optimum reaction and operating conditions were identified wherein near quantitative enzymatic digestibilities are obtained with enzyme loading of 15 FPU/g glucan. With a reduced enzyme loading of 5 FPU/g glucan, the enzymatic digestibility was decreased, but still reached a level of 92%. Decomposition of carbohydrates was extremely low as indicated by the measured glucan and xylan mass closures (recovered sugar plus unreacted) which were 98% and 94%, respectively. The data obtained in this work indicate that the digestibility is related to the extent of xylan removal.     

Catalysis of the electrochemical oxidation of glucose by glucose oxidase and a single electron cosubstrate: kinetics in viscous solutions

Catalysis of the electrochemical oxidation of glucose by glucose oxidase with a single electron mediator (cosubstrate) may be used to transform mixtures of concentrated industrial sugars. How the high viscosity of such media may affect the enzymatic reaction and the transport of the mediator can be mimicked by addition of large concentrations of sucrose to glucose solutions. Cyclic voltammetry then provides a simple means of investigating the effect of an increased viscosity on the kinetics of the enzymatic reaction and the diffusion of the mediator. The diffusion coefficient of the mediator is decreased 10 times by addition of 1.6 M sucrose. At pH 8, in the presence of the same concentration of sucrose, the catalytic activity of the enzyme towards its substrate is only slightly affected. A 35% decrease of the glucose Michaelis constant is observed. The reaction of the reduced enzyme with the cosubstrate is six times slower and the mediator Michaelis constant undergoes a three-fold increase. It follows that glucose oxidase remains an efficient catalyst in such viscous media.     

Trypsin-catalyzed synthesis of dipeptide containing alpha-aminoisobutyric acid using p- and m-(amidinomethyl)phenyl esters as acyl donor

Two series of inverse substrates, p- and m-(amidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)amino acid, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide. An alpha-aminoisobutyric acid containing dipeptide was especially obtained in satisfactory yield. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. The optimum condition for the coupling reaction was studied by changing the organic solvent, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.     

液相色谱法测定蚕卵中的碳水化合物

本文报道了蚕卵样品的前处理方法,它用于提取净化其中的水溶性糖和糖醇;采用Waters μ-Bondapak Carbohydrate柱,麦芽糖为内标,定量分析蚕卵内果糖、葡萄糖、山梨醇、蔗糖和海藻糖的含量。回收率在87%以上。     

Simultaneous quantitation of mono-, di-and trisaccharides as their TMS ether oxime derivatives by GC-MS: I. In model solutions

Summary This paper describes the separation and quantitation of mono- di- and trisaccharides in model solutions. Optimized conditions for identification and determination of common sugar constituents in several natural matrices by GC-MS, from one solution by one injection, are given. Applying a 52 min temperature program to a 30 m capillary column and taking advantage of the stable ratios of the syn- and anti oximes eluting either alone or together with one or two other anomers: provides the basis of calculation for co-eluting compounds. Saccharides (5–200 ng), such as fructose, glucose, sucrose, trehalose, cellobiose, maltose, turanose, gentiobiose, palatinose, melibiose, isomaltose, erlose, raffinose, melezitose, maltotriose, panose and isomaltotriose, in the presence of each other in various ratios, have been measured as silyloxime compounds with a reproducibility of ≤5 R.S.D. %. The possibility of measuring all these saccharides as their TMS methoximes is also presented. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Selectivity of methyl-fructoside synthesis with Β-fructofuranosidase

Enzyme synthesis of methyl fructoside was studied usingΒ-fructofuranosidase fromSacharomyces cerevisiae and sucrose and methanol as substrates. Taking into account the inhibition and deactivation effects of methanol on the enzyme, a system with 4.9M (20%, v/v) methanol was selected. At this alcohol level, 35% of sucrose is converted to fructoside at low or high substrate concentrations. The effect of enzyme concentration, pH, and temperature on both the synthesis and the hydrolysis of the fructoside was investigated. It was found that if the reaction proceeds at pH 6.0, 4‡C and/or 0.014 mg/mL (3 U/mL) of Β-fructofuranosidase at varying sucrose concentrations, methyl fructoside may be obtained with a minimum loss of the fructoside at the end of the reaction.     

Production and use of glucosyltransferases fromLeuconostoc mesenteroides NRRL B-1299 for the synthesis of oligosaccharides containing α-(1→2) linkages

Glucosyltransferase activities, produced by batch culture ofLeuconostoc mesenteroides NRRL B-1299, were recovered both in the culture supernatant (SGT) and associated with the insoluble part of the culture (IGT). A total glucosyltransferase activity of 3.5 U/mL was measured in batch culture. The enzymes from the supernatant were purified 313 times using aqueous two-phase partition between dextran and PEG phases, yielding a preparation with 18.8 U/mg protein. It was shown that both SGT and IGT preparations catalyze acceptor reactions and transfer the glucose unit from sucrose onto maltose to produce glucooligosaccharides. Some of the glucooligosaccharides synthesized (L_n series) contain α-(l→6) osidic linkages and a maltose residue at the reducing end. They were completely hydrolyzed by glucoamy-lase and dextranase. The other glucooligosaccharides synthesized (Bn series) resisted the action of these enzymes. The tetrasaccharide of this series has been characterized by¹³C NMR. Its structure was determined as 2–O–α–D–glucosylpanose. The oligosaccharides synthesized by the maltose acceptor reaction with the SGT and IGT preparations only differed in the relative amounts in which they were produced. The difference may arise from diffusional limitations appearing when the insoluble catalyst is used. Under the assay conditions, the glucanase resistant oligosaccharide yield was 35% with both glucosyltrans-ferase preparations.     

Synthesis of Rapeseed Biodiesel Using Short-Chained Alkyl Acetates as Acyl Acceptor

In this study, we conducted experiments using a response surface methodology to determine the optimal reaction conditions for the enzymatic synthesis of biodiesel from rapeseed oil and short-chained alkyl acetates, such as methyl acetate or ethyl acetate, as the acyl acceptor at 40 °C. Based on our response surface methodology experiments, the optimal reaction conditions for the synthesis of biodiesel were as follows: methyl acetate as acyl acceptor, catalyst concentration of 16.50%, oil-to-methyl acetate molar ratio of 1:12.44, and reaction time of 19.70 h; ethyl acetate as acyl acceptor, catalyst concentration of 16.95%, oil-to-ethyl acetate molar ratio of 1:12.56, and reaction time of 19.73 h. The fatty acid ester content under the above conditions when methyl acetate and ethyl acetate were used as the acyl acceptor was 58.0% and 62.6%, respectively. The statistical method described in this study can be applied to effectively optimize the enzymatic conditions required for biodiesel production with short-chained alkyl acetates.     

Applying structural transition theory to describe enzyme kinetics in heterogeneous systems

Enzyme action was investigated by assuming the occurrence of different states of enzyme-substrate affinities. These states were considered to involve enzyme species with distinct abilities to form reaction product. The results obtained showed strong agreement with the experimental data for the action of peroxidase. This approach provides a powerful tool for predicting the kinetic behavior of other enzymatic processes in conditions not described before. An additional feature of this approach is the ability to characterize processes at any enzyme-substrate concentration ratio, including high enzyme-substrate ratios and enzyme inhibition by substrate or product. This proposal can also be used in systems with heterogeneity concerning the investigated enzyme.     

Flow-injection determination of sugars with immobilized enzyme reactions and chemiluminescence detection

Glucose is determined by reaction with gluocose oxidase to produce hydrogen peroxide which is quantified via a chemiluminescence reaction with luminol. Sucrose, maltose, lactose and fructose are determined by enzymatic conversion to glucose (using invertase, amyloglucosidase, lactase. and glucose isomerase, respectively) and subsequent determination of the glucose, All enzymes are immobilized on controlled-pore glass and contained in flow-through reactors. For glucose, sucrose, and maltose the linear log-log working range 0.2 μM-1 mM, with a detection limit of 0.1 μM; for lactose and fructose the linear working range is 3 μM-1 mM with a detection limit of 1 μM. Assay time is 2 min.     

Determination of carbohydrates and sweeteners in foods and biologically active additives by high-performance liquid chromatography

A method for the determination of large amounts of carbohydrates (glucose, lactose, maltose, mannose, sucrose, and fructose) and sweeteners (xylitol and sorbitol) by reversed-phase liquid chromatography with refractive index detection without derivatization has been developed. The limits of determination for glucose, fructose, and sucrose in liquid samples were 0.1 g/L, and for xylite, lactose, maltose, mannose, and sorbite, 1 g/L. In solid samples the limits of determination for glucose, fructose, and sucrose were 0.1%, and for xylite, lactose, maltose, mannose, and sorbite, 0.6%. The method is applicable to the analysis of samples of wine, juice, honey, cookies, dairy products, and biologically active additives. The developed method for the determination of carbohydrates and sweeteners in foods and biologically active additives was certified in the Mendeleev Institute for Metrology (St. Petersburg).     

Enzymatic synthesis of phosphoric monoesters with alkaline phosphatase in reverse hydrolysis conditions

Title compounds were synthesized on a preparative scale using alkaline phosphatase, orthophosphoric monoester phosphohydrolase B.C. 3.1.3.1, in reverse hydrolysis conditions. Optimization for one of the 25 phosphoryl acceptors investigated (glycerol) shows that up to 55% synthesis yield can be obtained using a large excess of substrate, conditions in which the enzymatic activity remains high. From the results obtained with different phosphoryl group donors, phosphate, pyrophosphate and polyphosphates and with enzymes of different sources, it comes up that the best results are obtained with pyrophosphate and with the weakly purified calf intestine alkaline phosphatase. The extent of enzymatic hydrolysis of the donor can be reduced owing to the existence of two different pH optima for the two reactions, phosphorylation and hydrolysis. The synthesis can be also performed using inert co-solvents which allow to reduce the amount of acceptor used, as long as Zn⁺⁺ is added to the reaction medium. The results are discussed in terms of the catalytic mechanism of alkaline phosphatase.     

Synthesis of Cephalexin in Aqueous Medium with Carrier-bound and Carrier-free Penicillin Acylase Biocatalysts

The use of very high substrate concentrations favors the kinetically controlled synthesis of cephalexin with penicillin acylase (PA) not only by Michaelian considerations, but also because water activity is depressed, so reducing the rates of the competing reactions of product and acyl donor hydrolysis. Commercial PGA-450, glyoxyl agarose immobilized (PAIGA) and carrier-free cross-linked enzyme aggregates of penicillin acylase (PACLEA) were tested in aqueous media at concentrations close to the solubility of nucleophile and at previously determined enzyme to nucleophile and acid donor to nucleophile ratios. The best temperature and pH were determined for each biocatalyst based on an objective function considering conversion yield, productivity, and enzyme stability as evaluation parameters. Stability was higher with PAIGA and specific productivity higher with PACLEA, but best results based on such objective function were obtained with PGA-450. Yields were stoichiometric and productivities higher than those previously reported in organic medium, which implies significant savings in terms of costs and environmental protection. At the optimum conditions for the selected biocatalyst, operational stability was determined in sequential batch reactor operation. The experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium.