Expression of an Aspergillus niger glucose oxidase in saccharomyces cerevisiae and its use to optimize fructo-oligosaccharides synthesis

Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of beta-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal beta-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained.     

The production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of fructosyltransferase and glucose oxidase

Summary A technique to produce high-content fructo-oligosaccharides by the mixed- enzyme system of fructosyltransferase and glucose oxidase was investigated. The mixed-enzyme reaction was carried out in a stirred tank reactor containing 40 %(w/v) sucrose with 10 unit of fructosyltransferase and 10 unit of glucose oxidase per gram sucrose for 25 h, at 40 °C and pH 5.5. Highly concentrated fructo-oligosaccharides up to 90 % was obtained by the mixed-enzyme system.     

Production of fructooligosaccharides in high yield using a mixed enzyme system of β-fructofuranosidase and glucose oxidase

A mixed enzyme system, with -fructofuranosidase (obtained from Aspergillus japonicus) and commercial glucose oxidase (Gluzyme, Novo Nordisk), produced fructooligosaccharides (FOS) in high yield from sucrose. The reaction was performed in an aerated stirred tank reactor controlled at pH 5.5 by a slurry of CaCO₃. Glucose, an inhibitor of -fructofuranosidase, produced in the reaction was converted by glucose oxidase to gluconic acid, which was then precipitated to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dry weight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate. Most of the FOS and sucrose was hydrolyzed to fructose in the mixed enzyme system with glucose oxidase and -fructofuranosidase from Asp. niger.     

Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum

Background

IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme.

Results

Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS.

Conclusions

Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.     

Novel fructooligosaccharide conversion from sugarcane syrup using a specialised enzymatic pH-stat bioreactor

Fructooligosaccharides (FOS) have gained significant attention for their prebiotic properties. Given that sugarcane syrup (SS) is sucrose-rich but with other nutritional benefits, its direct transformation into FOS may add value to this product. Therefore, the aims of this study were to develop FOS conversion from SS and to define the kinetic behaviour of FOS synthesized in a 1-L specialized pH-stat bioreactor (SPSB). The SS was composed of sucrose (58.93%) with considerable antioxidant capacities and Ƴ-aminobutyric acid. The developed SPSB process consisted of three stages: evaporation of sugarcane juice into syrup (68–75 °Brix) (stage 1), optimization of the Viscozyme L and SS mixture at different reaction temperatures (47–55 °C) (stage 2), and upscaling of the optimized reaction system under defined conditions in a 1 L-SPSB system (stage 3). In the 1 L-SPSB system, the enzymatic reaction yielded 32.22% of FOS from SS after a 6 h reaction, which is comparable with a pure system containing an equivalent concentration of 10% of sucrose as initial substrate with 39.55% yield. This result demonstrated the efficient conversion of SS into FOS, supporting the utilization of sugarcane juice for its health benefits.     

Enzymatic conversion of sucrose to kestose by fungal extracellular fructosyltransferase

Summary An extracellular fructosyltransferase (EC 2.4.1.9) fromAspergillus foetidus NRRL 337 was found capable of converting sucrose into a syrup containing more than 50% kestose. The production efficiencies ranged from 26 to 47%, depending oh sucrose concentration and the reaction time.     

Immobilization of fructosyltransferase by chitosan and alginate for efficient production of fructooligosaccharides

The effective system of reusing mycelial fructosyltransferase (FTase) immobilized with two polymers, chitosan and alginate were evaluated for continuous production of fructooligosaccharides (FOS). The alginate beads were successfully developed by maintaining spherical conformation of using 0.3% (w/v) sodium alginate with 0.1% (w/v) of CaCl₂ solution for highest transfructosylating activity. The characteristics of free and immobilized FTase were investigated and results showed that optimum pH and temperature of FTase activity were altered by immobilized materials. A successive production of FOS by FTase entrapped alginate beads was observed at an average of 62.96% (w/w) up to 7 days without much losing its activity. The data revealed by HPLC analysis culminate 67.75% (w/w) of FOS formation by FTase entrapped alginate beads and 42.79% (w/w) by chitosan beads in 36 h of enzyme substrate reaction.     

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) gluconate 6-phosphatase (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.Abbreviations GOD glucose oxidase - GD glucose dehydrogenase - PP pentose phosphate - EM Embden-Meyerhof - TCA tricarboxylic acid     

Fructosyltransferase activity of commercial enzyme preparations used in fruit juice processing

Summary Of twenty-two commercial fungal enzyme preparations used in fruit juice processing examined, Pectinex Ultra SP-L, was found to possess the highest activity of fructosyltransferase (44.8 units/ml). The enzyme preparation converted sucrose into a high fructooliogosaccharide syrup containing 42.3 % kestose, 17.2% nystose, 10.6% sucrose, 27.8% glucose, and 2.1% fructose. The efficiency was 69% based the amount of sucrose consumed.     

Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^-3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^?3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Continuous production of high-content fructooligosaccharides by a complex cell system

A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high-content fructooligosaccharides (FOS) in a continuous process initiated by a batch process. The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with beta-fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high-content FOS was discharged continuously from a MF module. The complex cell system was run at 30 degrees C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5-7% glucose and 8-10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7-day continuous production process with a dilution rate of 0.04 h(-1), and the volumetric productivity for total FOS was more than 160 g L(-1) h(-1).     

Isoglucose production from raw starchy materials based on a two-stage enzymatic system

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.     

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.     

Metabolism of Fructooligosaccharides by Lactobacillus paracasei 1195

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [³H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) α-1,2 linked to two, three, or four fructose (F) units (GF₂, GF₃, and GF₄, respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF₂ and GF₃ was rapid, whereas little GF₄ uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.     

Monitoring and control of enzymic sucrose hydrolysis using on-line biosensors

Summary Previously reported flow microcalorimeter devices for enzymic reaction heat measurement, enzyme thermistors, have here been extended with systems for on-line sample treatment. Glucose analysis was performed by intermittent flow injections of 50 l samples through such an enzyme thermistor device containing immobilized glucose oxidase and catalase. Sucroce analysis was performed by allowing diluted samples to continuously pass through an additional enzyme thermistor containing immobilized invertase. The reaction heats were recorded as temperature changes in the order of 10–50 m°C for concentrations of 0.05–0.30 M glucose or sucrose present in the original non-diluted samples.The performance of this system was investigated by its ability to follow concentration changes obtained from a gradient mixer. The system was applied to monitoring and controlling the hydrolysis of sucrose to glucose and fructose in a plug-flow reactor with immobilized invertase. The reactor was continuously fed by a flow of scurose of up to 0.3 M (100 g/l). Glucose and remaining sucrose were monitored in the effluent of the column. By using flow rate controlled feed pumps for sucrose and diluent the influent concentration of sucrose was varied while the overall flow rate remained constant.On-line control of the effluent concentration of lucose and sucrose was achieved by a proportional and integral regulator implemented on a microcomuter. Preset concentration of glucose in the effluent could be maintained over an extended period of time espite changes in the overall capacity of the invertase reactor. Long delay times in the sensor system and the enzyme column made it necessary to carefully tune the control parameters. Changes of set-point value and temperature disturbances were used to verify accuracy of controlling performance.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

The Pasting and Gel Textural Properties of Corn Starch in Glucose,Fructose and Maltose Syrup

The pasting and gel textural properties of corn starch in syrup at different concentrations were investigated by Rapid Visco Analyzer (RVA) and Texture profile analysis (TPA) tests. The results showed that the pasting temperatures of corn starch greatly increased, especially at higher sugar concentration. Increasing concentration of syrup caused an increase in peak, trough and final viscosity of corn starch. Peak viscosity and the disintegration rate of starch increased in the following order: fructose syrup> maltose syrup> glucose syrup. Increasing syrup concentration to 13%, 25% and 50% resulted in a lower retrogradation rate than the control. When the maltose syrup concentration increased to 50%, the retrogradation rate decreased to 14.30% from 33.38%. The highest hardness was observed when the syrup concentration was 25%. There was a particular low hardness when the concentration of syrup was 50%. The springiness of starch gels in syrup was similar at different concentrations.     

Batch production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase

The production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase was investigated. The mixed-enzyme reaction was carried out in a stirred tank reactor containing 0.7 l of sucrose solution with coupled β-fructofuranosidase and glucose oxidase for 25 h. The optimum conditions for the mixed-enzyme reaction were as follows: pH, 5.5; temperature, 40°C; sucrose concentration, 400 g/l; agitation speed, 550 rpm; oxygen flow rate, 0.7 l/min; enzyme dosage, 10 units of β-fructofuranosidase with the combination of 15 units of glucose oxidase per gram sucrose. Under optimum conditions, high-content fructo-oligosaccharides up to 98% were obtained with complete consumption of sucrose and glucose by the mixed-enzyme system. Compared with the fructo-oligosaccharides produced by the β-fructofuranosidase, the high-content fructo-oligosaccharides produced by the mixed-enzyme system showed a significant difference with respect to sugar composition; i.e., a higher content of nystose was accumulated and only a trace amount of fructofuranosyl nystose was detected.     

Microbial production of fructosyltransferases for synthesis of pre-biotics

Fructooligosaccharides (FOS) are prebiotic substances found in several vegetable or natural foods. The main commercial production of FOS comes from enzymatic transformation of sucrose by the microbial enzyme fructosyltransferase. The development of more efficient enzymes, with high activity and stability, is required and this has attracted the interest of biotechnologists and microbiologists with production by several microorganisms being studied. This article reviews and discusses FOS chemical structure, enzyme characteristics, the nomenclature, producer microorganisms and enzyme production both in solid state fermentation and submerged cultivation.