FTase Activity Research Articles

Improved production of fructooligosaccharides (FOS) using a mutant strain of Aspergillus oryzae S719 overexpressing β-fructofuranosidase (FTase) genes

Fructooligosaccharides (FOS) are widely used as bioactive ingredient in functional foods. In this study, we presented an efficient method to produce the FOS using a mutant strain of Aspergillus oryzae S719. This mutant strain hold an overexpressing β-fructofuranosidase (FTase) with high-level FOS synthetic activity and it was resistant to high osmotic pressure. At a sucrose concentration of 900 g/L, Aspergillus oryzae S719 produced 586.0 ± 4.7 g/L of FOS in a 50 L bioreactor during a 20 h transformation process, whose productivity was 2-fold higher compared to an initial sucrose concentration of 500 g/L. Scanning electron microscopy (SEM) revealed that the mycelia of Aspergillus oryzae S719 was loose and spherical and had a large number of vacuoles compared to the original strain Aspergillus oryzae ZT65. These morphological characteristics might have contributed to the improved FTase activity. In summary, this study demonstrated an efficient strategy for FOS production on an industrial scale.

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.

Fructosyltransferase and inulinase production by indigenous coprophilous fungi for the biocatalytic conversion of sucrose and inulin into oligosaccharides

There is a great interest in prebiotics consumptions eminence by their health promoting properties and economic market value. Filamentous fungi have been implicated as the key producers of fructosyltransferase (Ftase) and inulinase for synthesis of fructooligosaccharides (FOSs), which is used as a bioactive ingredient in functional foods. In this study, sixty-one indigenous coprophilous fungal strains were isolated, purified to monoculture and investigated for their potential use in biotransformation of sucrose and inulin into fructooligosaccharides (FOSs) and inulooligosaccharides (IOSs) by producing Ftase and inulinase enzymes, respectively. The molecular identification by 18S rDNA sequencing and morpho-taxonomic keys revealed that axenic fungal strains belonged to the genera Aspergillus, Neocosmospora, Trichoderma, Mucor and Fusarium. The crude enzyme extract from the isolates showed hydrolysis zones of 15–30 mm on the 2,3,5-triphenyltetrazolium chloride (TTC) and Lugol's iodine solution assay plates confirming the Ftase and inulinase activities, respectively. The submerged culture filtrates of eight fungal isolates showed high Ftase activity while six different fungal isolates exhibited high inulinase activity. The reaction products analysed with thin-layer chromatography and high-performance liquid chromatography coupled with refractive index detection indicated the presence of FOSs and IOSs. The strains isolated in this study have potential biotechnological implications to produce FOSs and IOSs.

Purification and biochemical characterization of an extracellular fructosyltransferase enzyme from Aspergillus niger sp. XOBP48: implication in fructooligosaccharide production.

An extracellular fructosyltransferase (Ftase) enzyme with a molar mass of ≈70kDa from a newly isolated indigenous coprophilous fungus Aspergillus niger sp. XOBP48 is purified to homogeneity and characterized in this study. The enzyme was purified to 4.66-fold with a total yield of 15.53% and specific activity of 1219.17 Umg-1 of protein after a three-step procedure involving (NH4)2SO4 fractionation, dialysis and anion exchange chromatography. Ftase showed optimum activity at pH 6.0 and temperature 50°C. Ftase exhibited over 80% residual activity at pH range of 4.0-10.0 and ≈90% residual activity at temperature range of 40-60°C for 6h. Metal ion inhibitors Hg2+ and Ag+ significantly inhibited Ftase activity at 1mmol concentration. Ftase showed K m, v max and k cat values of 79.51mmol, 45.04µmolmin-1 and 31.5min-1, respectively, with a catalytic efficiency (k cat/K m) of 396µmol-1min-1 for the substrate sucrose. HPLC-RI experiments identified the end products of fructosyltransferase activity as monomeric glucose, 1-kestose (GF2), and 1,1-kestotetraose (GF3). This study evaluates the feasibility of using this purified extracellular Ftasefor the enzymatic synthesis of biofunctional fructooligosaccharides.

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.

Fructooligosaccharides synthesized by fructosyltransferase from an indigenous coprophilous Aspergillus niger strain XOBP48 exhibits antioxidant activity

Fructooligosaccharides (FOS) produced via sucrose biotransformation by partially purified Fructosyltransferase (Ftase) from an indigenous coprophilous Aspergillus niger strain XOBP48 (AnXOBP48) are reported to have antioxidant and nutraceutical properties in the present study. The Ftase activity on sucrose yielded FOS identified as monomeric glucose, 1- kestose (GF2), and 1,1– kestotetraose (GF3) which were further purified and quantified using HPLC-RI system. The antioxidant activities of purified FOS at concentrations of 1, 1.5, 3, 6, 12, 24 and 48 μg/ml were determined by applying three experimental models and comparing their properties with FOS standards and vitamin C. The free radical scavenging activity measured by 1,1 - diphenyl-2-picryl hydroxyl (DPPH) assay, ferric reducing antioxidant power (FRAP) assay and nitric oxide (NO) radical inhibition assay, yielded IC50 values of 2.6 μg/ml, 3.9 μg/ml and 3.4 μg/ml, 3.8 μg/ml and 0.69 μg/ml, 0.74 for purified GF2 and GF3, respectively. The free radical scavenging and inhibition activities showed a concentration dependent antioxidant activity of purified FOS with no significant differences as compared to the standards p < 0.01 and vitamin C. In conclusion, the results demonstrated that purified FOS could be exploited in biotechnological applications and/or as to be used as functional ingredients in commercial products such as the production of nutraceutical compounds due to their potential antioxidant properties.

Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides

An efficient system for biotransformation of sucrose to fructooligosaccharides (FOS) was obtained using Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (m-FTase). Zymographic analysis confirmed mycelial localization of the FTase (36 U/g) and lyophilized fungal pellets were used for bioconversion. m-FTase had molecular weight ∼75 kDa with optimum activity at pH 7.0 and 20 °C. FOS production after parametric optimization (sucrose − 50% w/v, m-FTase dose − 4.5% w/v, inoculum age − 48 h and incubation time − 24 h) reached 325 g/L (55% yield) with 14% residual sucrose, 25% glucose and 6% fructose. FTase activity was enhanced after pre-treatment with organic solvents and SDS. FOS was purified in a single step using gel filtration matrix, Bio-Gel P2. FOS was characterized using Diffusion ordered spectroscopy-Nuclear Magnetic Resonance (1H DOSY-NMR) and Fourier-transform infrared spectroscopy (FTIR). Continuous generation of FOS was achieved using recyclable mycelia upto 10 consecutive cycles.

English

A strain of Aspergillus niger PR-142 native to northern coast of Peru was subjected to successive processes of mutagenesis by ultraviolet light (UV) irradiation at 253.7 nm to increase the production of fructooligosaccharides (FOS). An initial selection was made by considering the mutants with increased invertase activity followed by the measurement of β-fructosyltransferase (FTase) activity both in mycelium and extracellular environment. Five selected mutants, which showed increased values of mycelium invertase activity (ranging from 101 to 128% as compared to the parent strain) at 40°C and sodium dodecylsulfate 0.15 (w/v), were grown in a fermentative medium in 50 mL conical tubes on a rotary shaker, and their FTase activity was determined. The 6-M69 mutant showed the most active mycelium activity of 1.5 fold as compared to the parent strain. When the same reaction was performed between 1 to 4 h, at the 3rd h, the mycelium FTase activity significantly increased up to 7 and 3 times in the mutant and parental strain, respectively. Finally, 4 mutants and the parental PR-142 were genetically characterized using inter simple sequence repeat polymerase chain reaction (ISSR-PCR) molecular markers. This analysis showed a significant 33% polymorphic bands between the parent and mutant markers, and 20 bands were unique to the mutants. Key words: Aspergillus, mutagenesis, β-fructosyltransferase, fructooligosaccharides, inter simple sequence repeat polymerase chain reaction (ISSR-PCR).

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.

Discovery and preliminary structure–activity relationship studies on tecomaquinone I and tectol as novel farnesyltransferase and plasmodial inhibitors

Biological screening of a library of synthesized benzo[c]chromene-7,10-dione natural products against human farnesyltransferase (FTase) has identified tecomaquinone I (IC50 of 0.065±0.004μM) as being one of the more potent natural product inhibitors identified to date. Anti-plasmodial screening of the same library against a drug-resistant strain of Plasmodium falciparum identified the structurally-related dichromenol tectol as a moderately active growth inhibitor with an IC50 3.44±0.20μM. Two novel series of analogues, based on the benzo[c]chromene-7,10-dione scaffold, were subsequently synthesized, with one analogue exhibiting farnesyltransferase inhibitory activity in the low micromolar range. A preliminary structure–activity relationship (SAR) study has identified different structural requirements for anti-malarial activity in comparison to FTase activities for these classes of natural products. Our results identify tecomaquinone I as a novel scaffold from which more potent inhibitors of human and parasitic FTase could be developed.

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.

Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris

In this work, the cDNA encoding fructosyltransferase (FTase) from Aspergillus niger YZ59 (CICIM F0901) was obtained and expressed in the methylotrophic yeast Pichia pastoris strain GS115. The yield of recombinant FTase in a 5-L fermentor reached 1020.0 U/mL after 96 h of induction, which was 1160.4 times higher that of native FTase from A. niger YZ59. The specific activity of recombinant FTase was 6.8×10(4) U/mg. The optimum temperature and pH of the recombinant FTase were 55 °C and 5.5, respectively. The recombinant FTase was stable below 40 °C and at pH from 3.0 to 10.0. Using sucrose as the substrate, the Km and Vmax values of recombinant FTase were 159.8 g/L and 0.66 g/(L min), respectively. The turnover number (kcat) and catalytic efficiency (kcat/Km) of recombinant FTase was 1.1×10(4) min(-1) and 68.8 L/(g min), respectively. The recombinant FTase was slightly activated by 5mM Ni(2+), Mg(2+), K(+), Fe(3+), or Mn(2+), but inhibited by all other metal ions (Na(+), Li(+), Ba(2+), Ca(2+), Zn(2+), and Cu(2+)). The highest yield of fructooligosaccharides for purified FTase reached approximately 343.3 g/L (w/v). This is the first study reporting the heterologous expression of FTases from A. niger in P. pastoris. This study plays an important role in the fructooligosaccharide synthesis industry by recombinant FTases.

English

Fructooligosaccharides are gaining importance by leaps and bounce every day due to their low caloric value and accompanying health benefits for the consumer. Hence, newer potential sources for the fructosyltransferase (Ftase) enzyme are thoroughly scrutinized. The present work was carried out for preliminary and secondary screening of the microbial isolates for determination of the Ftase producing potential. Three classes of microorganisms&rsquo; viz. molds, yeasts and bacterial isolates were qualitatively screened. The preliminary screening revealed molds as the most potent group exhibiting greater zone of hydrolysis in the range of 0.30 &plusmn; 0.10 to 2.58 &plusmn; 0.10 cm. The secondary screening of 20 selected mold isolates was performed in liquid batch culture. Four isolates identified as Aspergillus niger, Aspergillus flavus, Aspergillus stallus and Aspergillus versicolor exhibited higher fructosyltransferase : invertase ratio which is most critical for the synthesis of fructooligosaccharides due to higher transferase activity and low hydrolytic activity. A. niger exhibited highest Ftase activity, 36.88 &plusmn; 0.23 IU/mg. A. flavus produced 21.45 &plusmn; 0.33 IU/mg of Ftase. A. stallus produced 18.09 &plusmn; 0.14 IU/ mg and A. versicolor was found to produce 23.78 &plusmn; 0.12 IU/mg of Ftase without any cultural medium optimization. Key words: Prelimnary screening, secondary screening, fructosyltransferase, fructooligosaccharides.

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

Quantitative determination of cellular farnesyltransferase activity: towards defining the minimum substrate reactivity for biologically relevant protein farnesylation.

Prenylation is a post-translational modification wherein an isoprenoid group is attached to a protein substrate by a protein prenyltransferase. Hundreds of peptide sequences are in vitro substrates for protein farnesyltransferase (FTase), but it remains unknown which of these sequences can successfully compete for in vivo prenylation. Translating in vitro studies to predict in vivo protein farnesylation requires determining the minimum reactivity needed for modification by FTase within the cell. Towards this goal, we developed a reporter protein series spanning several orders of magnitude in FTase reactivity as a calibrated sensor for endogenous FTase activity. Our approach provides a minimally invasive method to monitor changes in cellular FTase activity in response to environmental or genetic factors. Determining the reactivity "threshold" for in vivo prenylation will help define the prenylated proteome and identify prenylation-dependent pathways for therapeutic targeting.

Recycling of cell culture and efficient release of intracellular fructosyltransferase by ultrasonication for the production of fructooligosaccharides

Production of fructooligosaccharide (FOS) through efficient cultivation of biotransformation process by fructosyltransferase (FTase) was evaluated by two new isolates, Aspergillus niger and Aspergillus flavus NFCCI 2364. The saccharide consumption revealed lag phase of A. niger in 10h which were smaller extent than A. flavus of 14h. For the recycling of cell culture, the pellet cells were continuously reused after 24h of submerged fermentation by these microorganisms in which FTase activity remains stable in four consecutive cycles in A. niger and six cycles in A. flavus. When freshly prepared pellets were sonicated for efficient release of intracellular FTase, the best transformation reaction was performed at 20W acoustic power giving conversion yield of FOS 61.43% (w/w) by A. niger and 70.44% (w/w) by A. flavus respectively. This study was shown that the two fungal isolates can serve as veritable source of intracellular FTase for industrial production of FOS.

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 36h of enzyme substrate reaction.

Progress in Making Ras as a Druggable Target.

Progress in Making Ras as a Druggable Target.