Production, Kinetic/Thermodynamic Study, and Evaluation of the Influence of Static Magnetic Field on Kinetic Parameters of ß-Fructofuranosidase from Aspergillus tamarii Kita UCP 1279 Produced by Solid-State Fermentation.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and can be used in the production of invert sugar and fructo-oligosaccharides (FOS). This last is an important prebiotic extensively used in the food industry. In the present study, the FFase production by Aspergillus tamarii Kita UCP 1279 was assessed by solid-state fermentation using a mixture of wheat and soy brans as substrate. The FFase presents optimum pH and temperature at 5.0-7.0 and 60 °C, respectively. According to the kinetic/thermodynamic study, the FFase was relatively stable at 50 °C, a temperature frequently used in industrial FOS synthesis, using sucrose as substrate, evidenced by the parameters half-life (115.52 min) and D-value (383.76 min) and confirmed by thermodynamic parameters evaluated. The influence of static magnetic field with a 1450 G magnetic flux density presented a positive impact on FFase kinetic parameters evidenced by an increase of affinity of enzyme by substrate after exposition, observed by a decrease of 149.70 to 81.73 mM on Km. The results obtained indicate that FFases present suitable characteristics for further use in food industry applications. Moreover, the positive influence of a magnetic field is an indicator for further developments of bioprocesses with the presence of a magnetic field.

Production, immobilization and application of invertase from new wild strain Cunninghamella echinulata PA3S12MM.

AIMS: The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. METHODS AND RESULTS: The maximum production was verified in 8 days of cultivation at 28°C supplemented with 10 g L-1 apple peel, reaching 1054.85 U ml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15 ml of sucrose 500 g L-1 in a fixed bed reactor (total volume of 31 ml) produced 24.44 µmol min-1 of glucose and fructose at a residence time of 30 min and a conversion factor of 0.5. CONCLUSIONS: The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.

Extraction and purification of Aspergillus tamarii ß-fructofuranosidase with transfructosylating activity using aqueous biphasic systems (PEG/phosphate) and magnetic field.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and fructo-oligosaccharides' production which are of great interest for the food industry. FFase from Aspergillus tamarii URM4634 was extracted using PEG/Phosphate Aqueous Biphasic Systems (ABS), and the impact of magnetic field on the extraction behavior was evaluated. A 24-full experimental design was employed to study the influence of molar mass of PEG, concentrations of PEG and phosphate and pH on the selected response variables, i.e., partition coefficient (K), purification factor (PF), activity yield (Y) and selectivity (S). The influence of magnetic field during partition and NaCl concentration on the same responses was also studied. The best results of FFase extraction without magnetic field (K = 0.50, PF = 4.05, Y = 72.66% and S = 0.06) were observed at pH 8.0 using 12.5% (w/w) PEG 400 and 25% (w/w) NaH2PO4/K2HPO4. Application of the magnetic field allowed improving the performance, with the best results being obtained at the longest distance between magnets (lowest magnetic field) and absence of NaCl (K = 0.93, PF = 4.22, Y = 83.79% and S = 0.09). The outcomes obtained demonstrate that ABS combination with low intensity magnetic field can be used as an efficient FFase pre-purification method.

Cunninghamella echinulata PA3S12MM invertase: Biochemical characterization of a promiscuous enzyme.

The Cunninghamella echinulata PA3S12MM fungus is a great producer of invertases in a growth medium supplemented by apple peels. The enzyme was purified 4.5 times after two chromatographic processes, and it presented a relative molecular mass of 89.2 kDa. The invertase reached maximum activity at pH of 6 and at 60°C, in addition to presenting stability in alkaline pH and thermal activation at 50°C. The enzymatic activity increased in the presence of Mn2+ and dithiothreitol (DTT), while Cu2+ and Z2+ ions inhibited it. Also, DTT showed to protect enzymatic activity. The apparent values for Km , Vmáx , and Kcat for the sucrose hydrolysis were, respectively, 173.8 mmol/L, 908.7 mmol/L min-1 , and 1,388.79 s-1 . The carbohydrate content was of 83.13%. The invertase presented hydrolytic activity over different types of glycosidic bonds, such as α1 â†” 2ß (sucrose), α1 â†’ 4 (polygalacturonic acid), α1 â†’ 4 and α1 â†’ 2 (pectin), and α1 â†” 1 (trehalose), indicating that the enzyme is multifunctional. Thus, the biochemical properties showed by the C. echinulata PA3S12MM suggest a broad industrial application, such as in the biomass hydrolysis or in the food industry. PRACTICAL APPLICATIONS: Invertases are hydrolytic enzymes employed in several industrial sectors. Given their great importance for the economy and several industrial sectors, there is a growing interest in microorganisms producing this enzyme. The analysis of the biochemical properties of invertase in C. echinulata PA3S12MM suggest applications in the food industry. Due to its increased hydrolytic activity, the hydrolysis process of the sucrose may employ invertase for the production of invert sugar. The stability at alkaline pH suggests an application in the development of enzymatic electrodes for the quantification of sucrose in food and beverage. The multifunctional activity may work in the biomass hydrolysis or saccharification of by-products for the extraction of fermentable sugars. The high level of invertase N-linked glycosylation of invertase grants this enzyme thermal stability at high temperatures, in addition to resistance against the action of proteases, which are desirable characteristics for the application of this enzyme in industrial processes.

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

OBJECTIVE: 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. RESULTS: 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 800 rpm and 0.75 vvm, and reached values of 2100 U g-1 and 667 U dm-3 h-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 800 rpm. CONCLUSION: 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.

Production of ß-fructofuranosidase with transfructosylating activity by Aspergillus tamarii URM4634 Solid-State Fermentation on agroindustrial by-products.

Eight different Aspergillus strains were tested for their ability to produce ß-fructofuranosidase (FFase) by Solid-State Fermentation. The Aspergillus tamarii URM4634 strain was selected as the most performant and tested on six different agroindustrial by-products. Soy, wheat and oat brans, which allowed for the highest hydrolytic (UH) and transfructosylating (UTF) activities, were tested individually or in mixtures according to a simplex-centroid mixture design in order to investigate their effects on FFase production at different times. The best results in terms of both enzyme activities were obtained with only soy bran. The influence of substrate, moisture and sucrose levels on FFase production was evaluated, and the highest UH and UTF activities were 229.43 ± 4.88 and 66.93 ± 3.02 U/mL, respectively. The obtained results indicate that A. tamarii FFase may be a biocatalyst with great potential for industrial applications such as sugar inversion and fructo-oligosaccharides production.

Biochemical characterization and kinetic/thermodynamic study of Aspergillus tamarii URM4634 ß-fructofuranosidase with transfructosylating activity.

This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 ß-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.

Low-resolution structure, oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis.

Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.

A novel ß-fructofuranosidase in Coleoptera: Characterization of a ß-fructofuranosidase from the sugarcane weevil, Sphenophorus levis.

ß-fructofuranosidases or invertases (EC 3.2.1.26) catalyze the hydrolysis of sucrose into fructose and glucose. ß-fructofuranosidases have been widely described in microorganisms, but were not known in the animal kingdom until very recently. There are studies reporting lepidopteran ß-fructofuranosidases, but no ß-fructofuranosidase gene sequence or encoding transcript has previously been identified in beetles. Considering the scarcity of functional studies on insect ß-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize a ß-fructofuranosidase transcript identified in a cDNA library from the sugarcane weevil, Sphenophorus levis (Curculionidae). To validate that the ß-fructofuranosidase sequence (herein denominated Sl-ß-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-ß-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. The Sl-ß-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-ß-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-ß-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-ß-fruct to be an acidic ß-fructofuranosidase. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as ß-fructofuranosidase, indicating the presence of a Sl-ß-fruct isoform or a ß-fructofuranosidase from insect intestinal microbiota. Moreover, it was found that α-glucosidases do not act on sucrose hydrolysis. Phylogenetic analyses indicated this enzyme to be similar to enzymes found in other coleopteran and lepidopteran ß-fructofuranosidases, but also closely similar to bacterial enzymes, suggesting potential horizontal gene transfer. Despite this, the enzyme seems to be restricted to different groups of bacteria, which suggests distinct origin events. The present study expands the concept of the occurrence of ß-fructofuranosidase in insects. Despite the few descriptions of this gene in the animal kingdom, it is possible to state that ß-fructofuranosidase is crucial to the establishment of some insects throughout their evolutionary history, especially members of the Lepidoptera and Coleoptera clades.

Utilization of molasses and sugar cane bagasse for production of fungal invertase in solid state fermentation using Aspergillus niger GH1.

Agro-industrial wastes have been used as substrate-support in solid state fermentation for enzyme production. Molasses and sugarcane bagasse are by-products of sugar industry and can be employed as substrates for invertase production. Invertase is an important enzyme for sweeteners development. In this study, a xerophilic fungus Aspergillus niger GH1 isolated of the Mexican semi-desert, previously reported as an invertase over-producer strain was used. Molasses from Mexico and Cuba were chemically analyzed (total and reducer sugars, nitrogen and phosphorous contents); the last one was selected based on chemical composition. Fermentations were performed using virgin and hydrolyzate bagasse (treatment with concentrated sulfuric acid). Results indicated that, the enzymatic yield (5231 U/L) is higher than those reported by other A. niger strains under solid state fermentation, using hydrolyzate bagasse. The acid hydrolysis promotes availability of fermentable sugars. In addition, maximum invertase activity was detected at 24 h using low substrate concentration, which may reduce production costs. This study presents an alternative method for invertase production using a xerophilic fungus isolated from Mexican semi-desert and inexpensive substrates (molasses and sugarcane bagasse).

Mutagenesis of Aspergillus Oryzae IPT-301 to improve the production of ß-Fructofuranosidase.

Aspergillus oryzae IPT-301, previously reported as a ß-fructofuranosidase producing microorganism, was successfully mutated using UV irradiation at 253.7 nm followed by the screening of survivors resistant to certain stress conditions. Strains were first subjected to the ß-fructofuranosidase activity assay using a portion from the colony grown in Petri dish as the enzyme source. Seven mutants with ß-fructofuranosidase activity values relative to the parent culture between 140 - 190% were selected from survivors grown at temperature of 40ºC or 0.018% (w/v) sodium dodecyl sulfate concentration. They were cultivated on a rotary shaker to characterize mycelium and extracellular fructosyltransferase activities. Three mutants named IPT-745, IPT-746 and IPT-748 showed the highest amount of mycelium activity whose values increased 1.5 - 1.8 fold, compared with the parent strain. It was found that more than 55% of total enzyme activity (mycelium- plus extracellular- activity) from these strains was detected in the mycelium fraction. Only one mutant, IPT-747, exceeded the amount of extracellular enzyme exhibited by the parent strain (1.5 times). This mutant also showed the highest value of total fructosyltransferase activity.