Production Of Fructosyltransferase Research Articles

Fructosyltransferase production by Aspergillus oryzae BM-DIA using solid-state fermentation and the properties of its nucleotide and protein sequences.

Fructosyltransferase (FTase) catalyzes the transfer of a fructosyl group to a sucrose molecule or a fructooligosaccharide (FOS) when a FOS with a longer chain is formed. Production of FTase by two Aspergillus species and its mixture was exploited using solid-state fermentation (SSF) and employing agave sap as substrate. The maximum FTase activity (1.59 U/mL) by Aspergillus oryzae was obtained after 24h, using a temperature of 30°C, with an inoculum of 2 × 107 spores/mL. The nucleotide sequence coding for the fructosyltransferase showed 1494bp and encodes for a protein of 498 amino acids. The hypothetical molecular tertiary structure of Aspergillus oryzae BM-DIA FTase showed the presence of structural domains, such as a five-bladed beta-propeller domain characteristic of GH (glycoside hydrolase) and C terminal, which forms a beta-sandwich module. This study contributes to the knowledge of stability, compatibility, and genetic expression of Aspergillus oryzae BM-DIA under SSF bioprocess conditions for industrial production of fructosyltransferase.

Effect of agitation speed and aeration rate on fructosyltransferase production of Aspergillus oryzae IPT-301 in stirred tank bioreactor.

Fructooligosaccharides (FOS) are prebiotic substances that have been extensively incorporated in different products of food industry mostly for their bifidogenic properties and economic value. The main commercial FOS production comes from the biotransformation of sucrose and intracellular and extracellular microbial enzymes-fructosyltransferases (FTase). Aspergillus oryzae IPT-301 produces FTase. In order to increase its production, this study focuses on evaluating the effects of different agitation speed and aeration rates which affect yields in a stirred tank bioreactor. Agitation had more influence on cell growth than aeration. The maximum intracellular FTase activity and the volumetric productivity of total intracellular FTase were obtained at 800rpm and 0.75 vvm, and reached values of 2100 U g-1 and 667 U dm-3h-1, respectively. The agitation speed had a strong influence on the activity of extracellular FTase produced which reached the maximum amount of 53 U cm-3. The higher value of total activity obtained was 22,831 U dm-3 at 0.75 vvm and 800rpm. Aeration rates and agitation speed showed strong influence upon the growth and production of fructosyltransferase from Aspergillus oryzae IPT-301 in media containing sucrose as carbon source. The control of aeration rate and agitation speed can be a valuable fermentation strategy to improve enzyme production.

Investigation of Optimal Conditions for Production, Characterization, and Immobilization of Fructosyltransferase and β-Fructofuranosidase by Filamentous Fungi

This work's objective was the extracellular production, partial characterization, and immobilization of the enzymes fructosyltransferase (Ftase) and β-fructofuranosidadase (Ffase) by filamentous fungi. Aspergillus niger ATCC 9642 and Penicillium brasilianum were evaluated for the production of fructosyltransferase (Ftase) and β-fructofuranosidadase (FASE) enzymes. The A. niger presented the highest activity of FTase (24.86 µmol/min.mL) and FFase (28.68 µmol/min.mL) in medium composed of 20% sucrose, 0.5% yeast extract, 1% NaNO3, 0.05% MgSO4.7 H2O, 0.25% KH2PO4, 0.5% NH4Cl and 0.25% NaCl inoculated using 5x107spores/mL and incubated at 25°C, pH 5.5, 150 rpm for 48 h. Presenting optimum pH and temperature of 2.39 and 60°C. Thermal stability has shown that the enzyme FFase is more thermally stable when compared to FTase. Stability against different pHs showed similar behavior for FTase and FFase; the optimum pH being between 2.0 and 3.0. FTase and FFase showed storage stability in freezing and refrigeration temperature for approximately 400 h. The kinetic parameters, Km and Vmax, for the sucrose substrate were 24.60mM and 104.16 μmol/min.mL for FTase and 3.91mM and 20.24 μmol/min.mL for FFase. The immobilization process displayed a yield of 6744.66% for FFase and 3928.90% for FTase, with enzymatic activities of 364.79 U/g and 220.34 U/g, and 4 and 3 times reuse, respectively.

Screening of low cost agricultural wastes to maximize the fructosyltransferase production and its applicability in generation of fructooligosaccharides by solid state fermentation

Production of fructosyltransferase by Aspergillus flavus NFCCI 2364 was exploited in solid state fermentation using low cost agricultural wastes for their ability to generate fructooligosaccharides. Among sixteen screened agro wastes, sugar cane bagasse was remarked as the most promising substrate suited for excellent growth and adequate production of fructosyltransferase (FTase) in 96 h of fermentation. The fermentation attempted in one variable executed yeast extract 0.2 g/gds (gram per gram dry substrate) as the most applicable nitrogen source and inoculum size 1 × 108 spores/gds resulted FTase activity of 197.10 U/gds (unit per gram dry substrate). The recuperated FTase activity of 423.18 U/gds was executed by rotable central composite design RCCD) approach of response surface methodology by validated process variables viz. pH 5.0, substrate particle size 2.5 mm, substrate moisture ratio 1:1.4 and substrate quantity of 7.0 g respectively. The study signifies excellent stability and compatibility of A. flavus NFCCI 2364 using sugarcane baggase as an economical substrate for industrial production of fructosyltransferase.

Enhanced production of fructosyltransferase in Aspergillus oryzae by genome shuffling.

To breed Aspergillus oryzae strains with high fructosyltransferase (FTase) activity using intraspecific protoplast fusion via genome-shuffling. A candidate library was developed using UV/LiCl of the conidia of A. oryzae SBB201. By screening for enzyme activity and cell biomass, two mutants (UV-11 and UV-76) were chosen for protoplast fusion and subsequent genome shuffling. After three rounds of genome recombination, a fusion mutant RIII-7 was obtained. Its FTase activity was 180Ug-1, approximately double that of the original strain, and RIII-7 was genetically stable. In fermentation culture, FTase activity of the genome-shuffled strain reached a maximum of 353 Ug-1 using substrate-feeding method, and this value was approximately 3.4-times higher than that of the original strain A. oryzae SBB201. Intraspecific protoplast fusion of A. oryzae significantly enhanced FTase activity and generated a potentially useful strain for industrial production.

Enhancement of fructosyltransferase and fructooligosaccharides production by A. oryzae DIA-MF in Solid-State Fermentation using aguamiel as culture medium

The aim of this work was to improve the production of fructosyltransferase (FTase) by Solid-State Fermentation (SSF) using aguamiel (agave sap) as culture medium and Aspergillus oryzae DIA-MF as producer strain. SSF was carried out evaluating the following parameters: inoculum rate, incubation temperature, initial pH and packing density to determine the most significant factors through Box-Hunter and Hunter design. The significant factors were then further optimized using a Box-Behnken design and response surface methodology. The maximum FTase activity (1347U/L) was obtained at 32°C, using packing density of 0.7g/cm3. Inoculum rate and initial pH had no significant influence on the response. FOS synthesis applying the enzyme produced by A. oryzae DIA-MF was also studied using aguamiel as substrate.

Production of fructosyltransferase by Penicillium simplicissimum in batch culture

Seventeen (17) strains of macro-fungi were screened for fructosyltransferase (FTase) production, of which Penicillium simplicissimum was selected for further work. FTase production was carried out in shake flask cultured at 30°C and 250 rpm for 48 h. For optimization of one factor-at-a-time method, effects of initial sucrose concentration, fermentation time, pH, inoculum size and agitation speed were studied on growth and extracellular FTase activity. These fermentation parameters were then optimized using response surface methodology (RSM) coupled with a central composite design (CCD). 20 g/l sucrose concentration, 36 h fermentation time, pH 6, 15 ml inoculum size and 150 rpm agitation speed were the optimum parameters obtained from RSM. The experimental values were in good agreement with predicted values within -0.057 to 0.023 errors. Nevertheless, the biomass and FTase (extracellular and intracellular) activities increased from 4.38 - 12.16 g/l, 92.76 - 99.46 IU/ml and 60.73 - 69.98 IU/ml in the unoptimized parameters to 14.16 g/l, 118.86 IU/ml (extracellular) and 71.98 IU/ml (intracellular) in the parameters optimized by RSM, respectively. Keywords: Fructosyltransferase, Penicillium simplicissimum , response surface methodology, fructooligosaccharides. African Journal of Biotechnology , Vol 13(46) 4294-4307

Production of fructosyltransferase in mechanically stirred and air-lift bioreactors

AbstractAureobasidium pullulans cells were cultivated in a 3 dm3 stirred bioreactor to optimize the production of fructosyltransferase (FTase). Batch experiments were focused on the influence of the initial sucrose concentration (150–350 g dm−3), sodium nitrate concentration (10–25 g dm−3), and stirring rate (180–540 min−1) on the process. It was found that the FTase specific activity per cell mass was positively influenced by the conditions that impeded the cell growth such as higher sucrose concentrations or lower oxygen transfer rates. Higher content of inorganic nitrogen substrate slightly increased the overall FTase production. The presence of fructose moiety-containing saccharides in the cultivation medium was observed to be indispensable for FTase production. An addition of a concentrated sucrose solution in the 40th cultivation hour therefore essentially resumed the FTase production. Finally, scale-up experiments were performed in 12 dm3 and 100 dm3 mechanically stirred bioreactors and in 20 dm3 and 60 dm3 air-lift bioreactors.

Production of fructosyltransferase by a local isolate ofAspergillus nigerin both submerged and solid substrate media

Screening of microorganisms capable of producing the enzyme fructosyltransferase (FTase) that could be used for the production of fructooligosaccharides (FOS) led to the isolation of a strain of Aspergillus niger. The fungus produced extracellular FTase in both submerged fermentation (SmF) using chemically-defined medium, and solid state fermentation (SSF) using agricultural by-products such as kola nut pod and ripe plantain peel. Maximum enzyme activity of 24.49 Uml−1 was obtained in SmF after 48 h of fermentation, while maximum enzyme activities of 20.77 and 27.77 U g−1 were obtained in SSF using ripe plantain peel and kola nut pod, respectively. The enzyme was used to prepare fructooligosaccharides (FOS), with the maximum yield of 33.24% FOS, consisting of kestose and nystose produced by FTase of kola nut pod fermentation.The safety of prepared FOS was investigated using albino rats. The rats were grouped and fed orally with 2500 mg kg−1 bw/day of FOS, 5000 mg kg−1 bw/day FOS, 2500 mg kg−1 bw/day of ho...

Production of fructosyltransferase by Aureobasidium sp. ATCC 20524 in batch and two-step batch cultures

A comparison of fructosyltransferase (EC 2.4.1.9) production by Aureobasidium sp. ATCC 20524 in batch and two step batch cultures was investigated in a 1-l stirred tank reactor using a sucrose supply of 200 g/l. Results showed that the innovative cultivation in two step of Aureobasidium sp. produced more fructosyltransferase (FFase) than the single batch culture at the same sucrose concentration with a maximal enzyme production of 523 U/ml, which was 80.5% higher than the one obtained in the batch culture. The production of fructooligosaccharides (FOSs) was also analyzed; their concentration reached a maximum value of 160 g/l the first day in the two-step culture and 127 g/l in the single-batch mode. The use of the two-step batch culture with Aureobasidium sp. ATCC 20524 in allowing the microorganism to grow up prior to the induction of sucrose (second step), proved to be a powerful method for producing fructosyltransferase and FOSs.

Optimization of Medium Constituents for the Production of Fructosyltransferase (Ftase) by Bacillus subtilis Using Response Surface Methodology

Optimization of Medium Constituents for the Production of Fructosyltransferase (Ftase) by Bacillus subtilis Using Response Surface Methodology

Effect of substrate feeding on production of fructosyltransferase by Penicillium purpurogenum

Penicillium purpurogenum was found to produce both extracellular and intracellular fructosyltransferase. The organism could also produce sucrose hydrolytic enzyme. Sucrose was found to be the best carbon source for fructosyltransferase production. Maximum intracellular and extracellular fructosyltransferase production was observed after 3rd and 4th day of cultivation, respectively. The enzyme activity was optimum at temperature of 55oC and pH 5.5. The addition of amino acids, like leucine induced slightly the extracellular fructosyltransferase production, where as histidine and leucine had little inductive effect on intracellular fructosyltransferase production. Enhanced production of fructosyltransferase by Penicillium purpurogenum was observed when sucrose content was restored by additional sucrose feeding to the cultivation medium during production period.

Production of fructosyl transferase by Aspergillus oryzae CFR 202 in solid-state fermentation using agricultural by-products.

Fructosyl transferase (FTase) production by Aspergillus oryzae CFR 202 was carried out by solid-state fermentation (SSF), using various agricultural by-products like cereal bran, corn products, sugarcane bagasse,cassava bagasse (tippi) and by-products of coffee and tea processing. The FTase produced was used for the production of fructo-oligosaccharides (FOS), using 60% sucrose as substrate. Among the cereal bran used, rice bran and wheat bran were good substrates for FTase production by A. oryzae CFR 202. Among the various corn products used, corn germ supported maximum FTase production, whereas among the by-products of coffee and tea processing used, spent coffee and spent tea were good substrates, with supplementation of yeast extract and complete synthetic media. FTase had maximum activity at 60 degrees C and pH 6.0. FTase was stable up to 40 degrees C and in the pH range 5.0-7.0. Maximum FOS production was obtained with FTase after 8 h of reaction with 60% sucrose. FTase produced by SSF using wheat bran was purified 107-fold by ammonium sulphate precipitation (30-80%), DEAE cellulose chromatography and Sephadex G-200 chromatography. The molecular mass of the purified FTase was 116.3 kDa by SDS-PAGE. This study indicates the potential for the use of agricultural by-products for the efficient production of FTase enzyme by A. oryzae CFR 202 in SSF, thereby resulting in value addition of those by-products.

Enhanced production of fructosyltransferase and glucosyltransferase by substrate-feeding cultures of Aureobasidium pullulans

Enhanced production of both fructosyltransferase and glucosyltransferase from Aureobasidium pullulans was achieved as a result of substrate-feeding experiments designed to enhance enzyme production. Supplying of sucrose significantly increased both the final concentration and specific activity of fructosyltransferase, which were about 25–47% higher than in batch culture. Glucosyltransferase production was likewise markedly promoted as a result of maltose feeding by 16–41% compared with batch operation.

Effects of metabolic inhibitors on extracellular fructosyltransferase production in Actinomyces viscosus.

Extracellular fructosyltransferase (levansucrase; EC 2.4.1.10) production in Actinomyces viscosus T14AV was demonstrated to occur concomitantly with cellular growth. The inhibition of both cellular ribonucleic acid and protein synthesis resulted in no further accumulation of enzyme activity. The antibiotic sodium clofibrate differentially inhibited the production of fructosyltransferase by strain T14AV. Furthermore, the antibiotic preferentially inhibited [14C]acetate incorporation into cellular lipid, but did not affect protein synthesis. In addition, no inhibition of fructosyltransferase production was observed upon the addition of the fatty acid acid synthesis inhibitor cerulenin. On the other hand, extracellular fructosyltransferase production was apparently stimulated in the presence of the cell wall synthesis inhibitors penicillin, amphomycin, and tunicamycin. These results are discussed in terms of the mechanism of extracellular protein production in A. viscosus.

Effect of cobalt on the growth of soybeans in the absence of supplied nitrogen

Production of fructosyltransferase by Aspergillus flavus NFCCI 2364 was exploited in solid state fermentation using low cost agricultural wastes for their ability to generate fructooligosaccharides. Among sixteen screened agro wastes, sugar cane bagasse was remarked as the most promising substrate suited for excellent growth and adequate production of fructosyltransferase (FTase) in 96 h of fermentation. The fermentation attempted in one variable executed yeast extract 0.2 g/gds (gram per gram dry substrate) as the most applicable nitrogen source and inoculum size 1 × 108 spores/gds resulted FTase activity of 197.10 U/gds (unit per gram dry substrate). The recuperated FTase activity of 423.18 U/gds was executed by rotable central composite design RCCD) approach of response surface methodology by validated process variables viz. pH 5.0, substrate particle size 2.5 mm, substrate moisture ratio 1:1.4 and substrate quantity of 7.0 g respectively. The study signifies excellent stability and compatibility of A. flavus NFCCI 2364 using sugarcane baggase as an economical substrate for industrial production of fructosyltransferase.