Production Of Β-fructofuranosidase Research Articles

Production of β-fructofuranosidase from<i>Aspergillus flavus</i> IBK-02 using orange peel as a low-cost substrate: Optimization through response surface methodology

Production of β-fructofuranosidase from<i>Aspergillus flavus</i> IBK-02 using orange peel as a low-cost substrate: Optimization through response surface methodology

Sequential cultivation method for β-fructofuranosidase production from Aspergillus tamarii URM4634, evaluation of their biochemical and kinetic/thermodynamic characteristics, and application on sucrose hydrolysis.

The present study focused on evaluating the sequential fermentation (SF) method for FFase production from Aspergillus tamarii URM4634 using soybean bran as substrate. The SF was performed using soybean bran as substrate at 72h and 30°C and the maximum hydrolytic activity (44.00 U mL-1), corresponding to an increase of 2.98-fold to about SmF using sucrose as substrate. Already the maximum transfructosylating activity was 26.10 U mL-1. The FFase presents maximum hydrolytic activity at pH 5.0-6.0 and transfructosylating at pH 6.0 and 60°C for both enzyme activities. The enzyme showed a typical hydrolytic kinetic profile evidenced by more affinity by sucrose hydrolysis reaction than the fructosyl transfer one. From kinetic and thermodynamic data of thermal denaturation, it was observed that the enzyme presents suitable at 55°C, evidenced by the large half-life (990.21min) and D values (3289.41min). The maximum release of reducing sugars (8.45g L-1) was obtained in hydrolysis of 20% sucrose during 180min. The results obtained for FFase production by SF proved that this method can be used satisfactorily for sucrose-degrading enzymes and can contribute to the development of the SF technique to produce different industrial-interest enzymes.

Thermoacidophilic β-fructofuranosidase induced with soybean meal produced by a new Aspergillus flavus isolated from the Atlantic Forest

High production of β-fructofuranosidase was obtained with the new fungus Aspergillus flavus isolated from the Atlantic Forest under solid state fermentation supplemented with soybean meal (53.36 U/ml). This beta-fructofuranosidase obtained after six days of cultivation with soybean meal was partially purified by DEAE-Sephadex ion exchange chromatographic columns, with a final yield of 19%, with an apparent molecular mass of 37 KDa by SDS-PAGE. The optimum pH and temperature for enzyme activity were 5.0 and 60 °C, respectively. The enzyme showed stability with 70% residual activity after 12 hours in the acid pH range (5.0 and 5.5), while at temperatures from 45 ºC to 55 ºC the enzyme showed stability greater than 50%. Therefore, the characteristics of this thermoacidophilic β-fructofuranosidase from A. flavus presents potential for use in several biotechnological processes.

Evaluation of an Edibleİinsect (Locusta migratoria) as a Substrate for Microbial β-fructofuranosidase Production

Rising population in the world causes reduction on present food resources. Investigators have been looking for sustainable, nutritional and alternative food-stocks. Insects have been consumed as snacks or as food supplement in many countries, but they are still not attractive food resource worldwide. In the current study, β-fructofuranosidase enzyme production by Galactomyces geotrichum TS61 (GenBank accession: MN749818) strain was investigated using an edible insect (Locusta migratoria) as substrate. β-fructofuranosidase is a valuable enzyme in food industry. Taguchi L16 design of experiment (DOE) was employed to achieve an effective statistical optimization process, including three factors (concentration of locust powder, concentration of sucrose and initial pH) with four levels. The optimized conditions were determined as 40 g/L locust powder, 30 g/L sucrose and 6.0 pH. The analysis of variance results showed that locust powder had more effect on the enzyme production than sucrose and pH. At the end of the optimization process, approximately 4-fold higher β-fructofuranosidase production (40.91 U/mL) was obtained when compared with unoptimized experimental run (9.91 U/mL). Consequently, powdered insects may serve as an effective supplement for valuable enzyme production in food industry.

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

BackgroundFilamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications.ResultsA novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%.ConclusionsThis PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

Upcycling of carrot discards into prebiotics (fructooligosaccharides) as high value food ingredients

A great amount of carrots is discarded daily in the word because they do not meet the quality standards imposed by consumer market. The aim of this work was to evaluate the feasibility of using carrot discards as substrate of solid-state fermentation to produce β-fructofuranosidase by Aspergillus niger and its subsequent use to catalyze the synthesis of fructooligosaccharides (prebiotics) using carrot juice. Results indicated that carrot bagasse has adequate nutrients for the growth of A. niger, but supplementation with a source of nitrogen is required to obtain a high production of β-fructofuranosidase (90.82 U/mL). Maximum specific activity (251 U/mg) was obtained after 3 days of fermentation at 30 ºC, using an inoculum of 106 conidia/ mL. β-fructofuranosidase was successfully used to synthesize fructooligosaccharides using carrot juice as substrate, raising the possibility for future development of a natural functional beverage. Tweetable abstractA great amount of carrots is discarded daily. This paper evaluates the use of carrot bagasse to produce β-fructofuranosidase and its subsequent application in carrot juice to produce a natural beverage with prebiotics.

Optimization of β-Fructofuranosidase Production from Agrowaste by Aspergillus carbonarius and Its Application in the Production of Inverted Sugar.

SUMMARYResearch backgroundMicrobial β-fructofuranosidases are widely employed in food industry to produce inverted sugar or fructooligosaccharides. In this study, a newly isolated Aspergillus carbonarius PC-4 strain was used to optimize the β-fructofuranosidase production in a cost-effective process and the sucrose hydrolysis was evaluated to produce inverted sugars.Experimental approachOptimization of nutritional components of culture medium was carried out using simplex lattice mixture design for 72 and 120 h at 28 °C. One-factor-at-a-time methodology was used to optimize the physicochemical parameters. Crude enzyme was used for sucrose hydrolysis at different concentrations.Results and conclusionsThe optimized conditions of enzyme production were achieved from cultivations containing pineapple crown waste (1.3%, m/V) and yeast extract (0.3%, m/V) after 72 h with an enzyme activity of 9.4 U/mL, obtaining R2=91.85%, R2adjusted=85.06%, highest F-value (13.52) and low p-value (0.003). One-factor-at-a-time used for optimizing the physicochemical conditions showed optimum temperature (20 °C), pH (5.5), agitation (180 rpm) and time course (72 h) with a 3-fold increase of enzyme production. The invertase-induced sucrose hydrolysis showed the maximum yield (3.45 mmol of reducing sugars) using 10% of initial sucrose concentration. Higher sucrose concentrations caused the inhibition of invertase activity, possibly due to the saturation of substrate or formation of sucrose aggregates, making it difficult for the enzyme to access sucrose molecules within the created clusters. Therefore, a cost-effective method was developed for the invertase production using agroindustrial waste and the produced enzyme can be used efficiently for inverted sugar production at high sucrose concentration.Novelty and scientific contributionThis study presents an efficient utilization of pineapple crown waste to produce invertase by a newly isolated Aspergillus carbonarius PC-4 strain. This enzyme exhibited a good potential for inverted sugar production at high initial sucrose concentration, which is interesting for industrial applications.

Relationship between pellet formation by Aspergillus oryzae strain KB and the production of β-fructofuranosidase with high transfructosylation activity

Aspergillus oryzae KB produces two β-fructofuranosidases (F1 and F2). F1 has high transfructosylation activity (Ut) to produce fructooligosaccharides. F2 has high hydrolysis activity (Uh), releasing glucose and fructose. It is desirable to selectively produce F1, which can be used for production of fructooligosaccharides. Here, the relationship between filamentous pellet size and selective production of F1 in liquid culture was investigated. Our finding revealed that: (i) The mean particle size of pellets (5.88 ± 1.36 mm) was larger, and the ratio of Ut to Uh was improved (Ut/Uh = 5.0) in 10% sucrose medium compared with 1% sucrose medium (pellet size = 2.60 ± 0.37 mm; Ut/Uh = 0.96). (ii) The final culture pH of the 1% sucrose medium was 8.7; on controlling the pH of 1% sucrose medium at 5.0, increased pellet size (9.69 ± 2.01 mm) and Ut/Uh (7.8) were observed. (iii) When 3% glycerin was used as carbon source, the pellet size decreased to 1.09 ± 0.33 mm and Ut/Uh was 0.57. (iv) In medium containing 1% sucrose, the pellet size was dependent on the number of spores used in the culture inoculum, but, in these experiments, Ut/Uh was almost constant (1.05 ± 0.08). Collectively, the data show that the value of Ut/Uh is proportional to the pellet size when liquid culture of A. oryzae strain KB is performed in some conditions (such as in the presence of high sucrose concentration, low pH, or added Tween surfactant), but in other conditions Ut/Uh is independent of pellet size.

Production of β-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 β-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.

Enhanced β-fructofuranosidase biosynthesis by Rhodotorula glutinis using Taguchi robust design method

The aim of this study was to produce β-fructofuranosidase enzyme by Rhodotorula glutinis SO28, using sugar beet (Beta vulgaris) as carbon source due to its high sucrose content and easy availability. β-Fructofuranosidase production was carried out in submerged fermentation. Taguchi orthogonal array (OA) design of experiment (DOE) method was employed for optimization process of β-fructofuranosidase production by R. glutinis SO28. An OA layout of L16 was constructed with five influential factors on β-fructofuranosidase biosynthesis namely, carbon source (sugar beet), initial pH, incubation temperature, agitation speed and incubation time. The average results of β-fructofuranosidase yield obtained from the determined 16 batches were processed with Minitab® 16.2.3 software at “larger is better” as quality character. The results showed that the maximum β-fructofuranosidase activity was obtained as 21.11 ± 0.47 U/mL, which was close to the predicted result (21.78 ± 0.43 U/mL). Consequently, sugar beet can be suggested as an economical substrate for β-fructofuranosidase production. Besides, use of Taguchi DOE enhanced enzyme activity about 3-fold when compared with unoptimized condition.

Comparison of constitutive and inducible β-fructofuranosidase production by recombinant Pichia pastoris in fed-batch culture using defined and semi-defined media

Short-chain fructooligosaccharides produced from sucrose by transfructosylation using β-fructofuranosidase (FFase), an industrially important enzyme, finds application in pre-biotics, sweeteners and confectionary products. Using recombinant Pichia pastoris, the influence of replacing the commonly-used Invitrogen® medium with a semi-defined medium for FFase production under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) and alcohol oxidase (AOX) promoters was investigated. Replacing the trace metals (PTM1) solution with yeast extract resulted in a 54.3% decrease in FFase volumetric activity under control of the AOX promoter, suggesting a distinct requirement for trace metals for recombinant protein synthesis during methanol induction, given that the biomass yield on methanol decreased by only 10%. The same medium adjustment had no effect on enzyme production under GAP promoter control, although AOX promoter control resulted in double the FFase volumetric activity compared to glycerol-fed cultures. Decreasing basal salts by half did not affect the cultures, but alleviated precipitation during sterilisation. Optimisation of the glycerol feed rate and dissolved oxygen tension in DO-stat fed-batch fermentations using the semi-defined medium resulted in 17% increase in volutmetric activity of FFase expressed under the GAP promoter. This study highlighted the influence of carbon source and trace metals on heterologous protein production by P. pastoris using constitutive and inducible promoters.

Why sucrose is the most suitable substrate for pullulan fermentation by Aureobasidium pullulans CGMCC1234?

This paper studies the metabolic pathway of sucrose in pullulan fermentation by Aureobasidium pullulans. Because of its high pullulan production, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234 (36.3g/L). Compared to other carbon sources, A. pullulans cells reached the stationary phase more quickly in the presence of sucrose. The specific sugar types and concentrations occurring during pullulan fermentation were detected using High Performance Liquid Chromatography (HPLC). HPLC results revealed that sucrose did not simply break down into glucose and fructose in the medium employed. Kestose (22.69g/L) also accumulated during early stages of fermentation (24h), which reduced the osmotic pressure of the extracellular fluid and diminished the inhibition of cell growth and pullulan production. β-Fructofuranosidase activity strongly depended on the carbon source. Sucrose was the best inducer of β-fructofuranosidase production. However, β-fructofuranosidase production did not directly and/or proportionally correlate with the growth of A. pullulans cells.

Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

BackgroundKluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins.ResultsThe recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively).ConclusionsK. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures.

Media optimization for β-Fructofuranosidase production by Aspergillus oryzae

β-Fructofuranosidase production by Aspergillus oryzae IPT301 was maximized in shake flasks. Response Surface Methodology (RSM) involving Small Central Composite Design was adopted to evaluate the fructosyltransferase (FTase) activity by changing three medium component concentrations: sucrose, urea and yeast extract. The optimal set of conditions for maximum fructosyltransferase production was as follows: sucrose 320.5 g/L, urea 7.13 g/L and yeast extract 2.11 g/L. In this optimal condition, the following improvements were achieved: an increase of 48.8% in cell growth, 112% and 62% in micelial and free FTase activities, respectively, 62.8% in the ratio of fructosyltransferase/hydrolytic activities for enzyme linked to mycelium and 67.5% for free enzyme.

PROPERTIES AND APPLICATION OF Β-FRUCTOFURANOSIDASE FROM ASPERGILLUS JAPONICUS 3.3556

ABSTRACT In this study, β-fructofuranosidase from Aspergillus japonicus was purified to homogeneity according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis by (NH4)2SO4 precipitation and gel filtration on sephadex G-100. The molecular mass of FFase from Aspergillus japonicus 3.3556 was estimated to be 89 kDa. Parameters like temperature, pH, and metal ions had different effects on FFase activity. The result showed that Fe3+ and Mg2+ were the inhibitors of FFase. The enzyme was completely stable in the pH range from 5 to 7 with the optimum pH of 5.5. The enzyme was stable in the temperature range from 40 to 60C with the optimum temperature of 50C. The Km and Vmax values for FFase were determined to be 59.88 mmol/L and 0.9421 µmol/mL/min, respectively. FFase activity increased with the increase of sucrose concentration. FFase could produce 1-kestose and nystose from sucrose by fructosyl-transferring activity. PRACTICAL APPLICATIONS The fructooligosaccharides (FOS) have recently attracted special attention as food additives for their favorable functional properties, which include low calorie and noncariogenic sweeteners, prevention of dental caries, relief of constipation, decrease of total cholesterol and lipid in serum, promotion of animal growth and consecutive improvement of the intestinal microflora, acting as a growth factor for bifidobacteria. FOS can be produced by transferring one to three molecules of fructose to the fructose residue in sucrose through the action of β-fructofuranosidase (FFase, EC 3.2.1.26) with high transfructosylating activity obtained from plants and microorganisms. Aspergillus Japonicus was found to be very useful for industrial production of β-Fructofuranosidase, which could produce 1-kestose and nystose from sucrose.

Mutagenesis of Aspergillus oryzae IPT-301 to improve the production of β-fructofuranosidase

Aspergillus oryzae IPT-301, previously reported as a β-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 β-fructofuranosidase activity assay using a portion from the colony grown in Petri dish as the enzyme source. Seven mutants with 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.

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

Aspergillus oryzae IPT-301, previously reported as a β-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 β-fructofuranosidase activity assay using a portion from the colony grown in Petri dish as the enzyme source. Seven mutants with β-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.

β-Fructofuranosidase production by repeated batch fermentation with immobilized Aspergillus japonicus

The fungus Aspergillus japonicus ATCC 20236 was immobilized in vegetal fiber and used in repeated batch fermentations of sucrose (200 g/l) for the production of beta-fructofuranosidases (FFase). The assays were performed during eight consecutive cycles that were completed in a total period of 216 h. After each 24-h cycle of fermentation (except for the first cycle, which lasted 48 h), the fermented broth was replaced by fresh medium, and the FFase activity was determined in the replaced medium. The average value of FFase activity was a constant 40.6 U/ml at the end of the initial seven cycles, but had decreased by 22% at the end of the eighth cycle. Concurrent with these high and constant FFase values, the hydrolyzing activity of this enzyme increased during the cycles, while the transfructosylating activity decreased. As a consequence, the maximum production of fructooligosaccharides of 134.60 g/l observed in the initial 30 h of fermentation (first cycle) had gradually decreased by the end of the subsequent cycles, reaching approximately 23% of this value during cycles 4-8. Based on these results, we conclude that the present immobilization system has a great potential for application in a semi-continuous process for the production of FFase, but further studies are necessary to maintain the FFase transfructosylation activity at high levels during the overall process.

Colonization of Aspergillus japonicus on synthetic materials and application to the production of fructooligosaccharides

The ability of Aspergillus japonicus ATCC 20236 to colonize different synthetic materials (polyurethane foam, stainless steel sponge, vegetal fiber, pumice stones, zeolites, and foam glass) and to produce fructooligosaccharides (FOS) from sucrose (165 g/L) is described. Cells were immobilized in situ by absorption, through direct contact with the carrier particles at the beginning of fermentation. Vegetal fiber was the best immobilization carrier as A. japonicus grew well on it (1.25 g/g carrier), producing 116.3 g/L FOS (56.3 g/L 1-kestose, 46.9 g/L 1-nystose, and 13.1 g/L 1-β-fructofuranosyl nystose) with 69% yield (78% based only in the consumed sucrose amount), giving also elevated activity of the β-fructofuranosidase enzyme (42.9 U/mL). In addition, no loss of material integrity, over a 2 day-period, was found. The fungus also immobilized well on stainless steel sponge (1.13 g/g carrier), but in lesser extents on polyurethane foam, zeolites, and pumice stones (0.48, 0.19, and 0.13 g/g carrier, respectively), while on foam glass no cell adhesion was observed. When compared with the FOS and β-fructofuranosidase production by free A. japonicus, the results achieved using cells immobilized on vegetal fiber were closely similar. It was thus concluded that A. japonicus immobilized on vegetal fiber is a potential alternative for high production of FOS at industrial scale.

Improved productivity of β-fructofuranosidase by a derepressed mutant of Aspergillus niger from conventional and non-conventional substrates

Wild-type cultures of Aspergillus niger produced a basal level of β-fructofuranosidase on glucose of 1 IU l−1 h−1. In contrast, a catabolite-derepressed mutant strain of the same organism produced a markedly higher level (25 IU l−1 h−1) of this enzyme when grown on the same carbon source. Wheat bran induced both the wild type (252 IU l−1 h−1) and the mutant strain (516 IU l−1 h−1) to produce 252- to 516-fold higher levels of this enzyme than was observed with the wild-type grown on glucose and was the best carbon source. When corn steep liquor served as a nitrogen source, the wild-type organism showed a higher activity of enzyme on monosaccharides and disaccharides comparable to that produced by corncobs in the basal medium and that mutant was a potentially improved (> 2-fold) organism for the production of β-fructofuranosidase on all carbon sources. Enhanced substrate consumption and product formation kinetic parameters suggest that the mutant organism may be exploited for bulk production of this useful enzyme.