Fructosyltransferase Activity Research Articles

Fructooligosaccharides (FOS) Production by Microorganisms with Fructosyltransferase Activity

Fructans are fructose-based polymers, defined as fructooligosaccharides (FOS), when they possess a short chain. These molecules are highly appreciated in the food and pharmaceutical international market and have an increasing demand worldwide, mainly for their prebiotic activity and, therefore, for all their health benefits to those who consume them constantly. Thus, new natural or alternative FOS production systems of industrial scale are needed. In this regard, microorganisms (prokaryotes and eukaryotes) have the potential to produce them through a wide and diverse number of enzymes with fructosyltransferase activity, which add a fructosyl group to sucrose or FOS molecules to elongate their chain. Microbial fructosyltransferases are preferred in the industry because of their high FOS production yields. Some of these enzymes include levansucrases, inulosucrases, and β-fructofuranosidases obtained and used through biotechnological tools to enhance their fructosyltransferase activity. In addition, characterizing new microorganisms with fructosyltransferase activity and modifying them could help to increase the production of FOS with a specific degree of polymerization and reduce the FOS production time, thus easing FOS obtention. Therefore, the aim of this review is to compile, discuss, and propose new perspectives about the microbial potential for FOS production through enzymes with fructosyltransferase activity and describe the modulation of FOS production yields by exogenous stimuli and endogenous modifications.

Expression and characterization of a protease-resistant β-d-fructofuranosidase BbFFase9 gene suitable for preparing invert sugars from soybean meal

A novel gene (BbFFase9), with an ORF of 1557 bp that encodes β-d-fructofuranosidase from Bifidobacteriaceae bacterium, was cloned and expressed in Escherichia coli. The recombinant protein (BbFFase9) was successfully purified and showed a single band with a molecular mass of 66.2 kDa. This was confirmed as a β-d-fructofuranosidase and exhibited a high specific activity of 209.2 U/mg. Although BbFFase9 was a soluble protein, it exhibited excellent tolerance to proteases such as pepsin, trypsin, acidic protease, neutral protease and Flavourzyme®, indicating its potential applicability in different fields. BbFFase9 exhibited typical invertase activity, and highly catalyzed the hydrolysis of the α1↔2β glycosidic linkage in molecules containing fructosyl moieties but with no detectable fructosyltransferase activity. It was optimally active at pH 6.5 and 50 °C and stable between pH 6.0 and 9.0 at a temperature of up to 45 °C for 30 min BbFFase9 could also effectively hydrolyze galacto-oligosaccharides, which are a flatulence factor in soybean meal, thus releasing new types of product such as melibiose and mannotriose, or degrading them into invert sugars, the sweeter fructose and glucose. This study is the first to report the application of this type of β-d-fructofuranosidase.

Short-Chain Fructooligosaccharide Synthesis from Sugarcane Syrup with Commercial Enzyme Preparations and Some Physical and Antioxidation Properties of the Syrup and Syrup Powder.

Short-chain fructooligosaccharides (sc-FOS) are prebiotics beneficial to human health, which can be synthesized from raw material containing a high sucrose content. Sugarcane syrup (SS) without molasses removal contains sucrose as a major sugar and is a rich source of several bioactive compounds. The aim of this study is to investigate factors affecting sc-FOS synthesis from SS using commercial enzyme preparations containing fructosyltransferase activity as biocatalysts. sc-FOS content increased significantly as the sucrose concentration of SS in the reaction mixture increased up to 40% (w/v). Changes in carbohydrate compositions during the transfructosylating reaction of a pure sucrose solution and SS prepared from the two sugarcane cultivars Khon Kaen 3 and Suphanburi 50, catalyzed by Pectinex Ultra SP-L and Viscozyme L, showed similar profiles. Both enzymes showed a high ability to transfer fructosyl moieties to produce sc-FOS and a plateau of the total sc-FOS concentration was observed after 4 h of reaction time. For the pure sucrose solution and SS (Suphanburi 50), Viscozyme showed a superior ability to convert sucrose to Pectinex, with a higher sc-FOS yield (g FOS/100 g of initial sucrose), GF2 or 1-kestose yield (g GF2/g of initial sucrose) and GF3 or nystose yield (g GF3/g of initial sucrose). sc-FOS syrup (FOS SS) and the foam-mat-dried syrup powder prepared from SS and FOS SS, respectively, contained a high total phenolic content and possessed higher antioxidant activities than those prepared from pure sucrose, but contained lower calories.

Evidence for Light and Tissue Specific Regulation of Genes Involved in Fructan Metabolism in Agave tequilana.

Plant Glycoside Hydrolase Family 32 (PGHF32) contains the fructosyltransferases and fructan exohydrolase enzymes responsible for fructan metabolism, in addition to closely related vacuolar and cell wall acid invertases. Agave species produce complex and dynamic fructan molecules (agavins) requiring 4 different fructosyltransferase activities (1-SST, 1-FFT, 6G-FFT and 6-SFT) for their synthesis. Combined analysis of RNAseq and genome data for A. tequilana led to the characterization of the genes encoding 3 fructosyltransferases for this species and support the hypothesis that no separate 6-SFT type enzyme exists in A. tequilana, suggesting that at least one of the fructosyltransferases identified may have multiple enzymatic activities. Structures for PGHF32 genes varied for A. tequilana and between other plant species but were conserved for different enzyme types within a species. The observed patterns are consistent with the formation of distinct gene structures by intron loss. Promoter analysis of the PGHF32 genes identified abundant putative regulatory motifs for light regulation and tissue-specific expression, and these regulatory mechanisms were confirmed experimentally for leaf tissue. Motifs for phytohormone response, carbohydrate metabolism and dehydration responses were also uncovered. Based on the regulatory motifs, full-length cDNAs for MYB, GATA, DOF and GBF transcription factors were identified and their phylogenetic distribution determined by comparison with other plant species. In silico expression analysis for the selected transcription factors revealed both tissue-specific and developmental patterns of expression, allowing candidates to be identified for detailed analysis of the regulation of fructan metabolism in A. tequilana at the molecular level.

Fructooligosaccharides production by immobilized Pichia pastoris cells expressing Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase.

Fructooligosaccharides (FOSs)—fructose-based oligosaccharides—are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Screening of microorganisms from pineapple waste for fructooligosaccharides production

Fructooligosaccharides (FOS) are one of the well-known low caloric value sweeteners with prebiotic properties that promote positives effects on consumer’s health. They are synthetically produced by transfructosylation of sucrose via microbial enzymes which are β-fructofuranosidases (FFase) (EC 3.2.1.26) and fructosyltransferase (FTase) (EC 2.4.1.9). Despite the large number of microbial FTases that are produced, the yield of FOS is low and has poor stability, thus, only a few of them have the potential for industrial application. Research for a new source of microbial enzyme for FOS production becomes necessary due to the high demand for FOS in the pharmaceutical and food industry. Fruit waste such as pineapple waste can be an alternative source of microbial enzyme for FOS production beside can be recycled as FOS substrate. This will reduce the dumping and open burning of these waste which eventually will lead to environmental pollution. This paper presents an experimental study of microbial screening from pineapple waste that can catalyze FOS production. Three different parts of pineapple waste were used in this study which are peels, pulps, and leaves. From screening, all the five isolated bacteria which belong to gram-positive groups did possess both hydrolytic and fructosyltransferase activity with bacteria isolated from leaves showed the highest fructosyltransferase activity which is 0.91 U/ml. Bacterial identification using sequencing of 16S rRNA showed that the isolated bacteria is from the genus Bacillus sp.

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.

Immobilization of a commercial Aspergillus aculeatus enzyme preparation with fructosyltransferase activity in chitosan beads: A kinetic/thermodynamic study and fructo-oligosaccharides continuous production in enzymatic reactor

Pectinex Ultra SP-L, a food-grade enzymatic preparation with high transfructosylating activity, was covalently immobilized on chitosan beads. The highest immobilization yield (95.9%) was obtained using 4.0% glutaraldehyde for 60min and 80rpm at 25±1°C. The immobilized biocatalyst showed good operational stability, being able to retain, after the third cycle of reuse, no less than 100.0 and 73.9% of starting hydrolytic and transfructosylating activities, respectively. Results of residual activity experiments allowed estimating, for irreversible thermal inactivation of both free and immobilized enzyme, the activation energy (E*d=234.3 and 242.2kJ·mol−1), enthalpy (234.4≤ΔH*d≤234.2kJ·mol−1 and 239.4≤ΔH*d≤239.3kJ·mol−1), entropy (381.2≤ΔS*d≤379.8J·mol−1·K−1 and 390.4≤ΔS*d≤389.7J·mol−1·K−1) and Gibbs free energy (109.3≤ΔG*d≤103.9kJ·mol−1 and 111.3≤ΔG*d≤103.6kJ·mol−1). The use of the immobilized enzyme in a packed bed reactor allowed obtaining, after 100min, a fructo-oligosaccharide mixture containing more than 25gL−1 1-kestose.

Purification and biochemical characterization of an extracellular fructosyltransferase-rich extract produced by Aspergillus tamarii Kita UCP1279

Abstract The market for prebiotics is gaining strength and is growing greatly in recent years since the consumers demand are changing and are highly influenced by the increasing consumption of healthier products. The fructosyltransferase (FTase) mainly produced by filamentous fungi is responsible for the synthesis of fructooligosaccharides which are widely used as a bioactive ingredient in functional foods. The objectives of this work were to select the best Aspergillus strains fructosyltransferase producer, purify FTase using ion-exchange chromatography coupled to FPLC under Superdex-G75 and to produce fructooligosaccharides. Six strains of Aspergillus sp. were studied, and A. tamarii Kita was highlighted because of the greater fructosyltransferase activity. At the production stage, the maximum activity of crude extract (1629.03 U/gds) was obtained under the following conditions: wheat bran 3.0g, sucrose concentration at 20%, 106 spore/mL, 70% moisture, 12h in the presence of light, at 30 °C for 48h. Superdex-G75 was able to purify the enzyme with high activity, and after SDS-PAGE under zymography conditions, one band of protein was observed at 89.7 kDa. The specific activity of the final purified material was 112,629.03 U/gds with purification ratio of 49 times and yield of 36%. FTase presented optimum temperature at 60 °C and pH 5.0, respectively. Together, these results indicated that the FTase of Aspergillus tamarii Kita is an excellent candidate for the industrial production of FOS.

Fructo-oligosaccharides production by an Aspergillus aculeatus commercial enzyme preparation with fructosyltransferase activity covalently immobilized on Fe3O4–chitosan-magnetic nanoparticles

Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe3O4–chitosan- magnetic nanoparticles. Immobilization carried out according to a 23-full factorial design where glutaraldehyde concentration, activation time and time of contact between enzyme and support were selected as the independent variables and immobilization yield as the response. The highest immobilization yield (94.84%) was obtained using 3.0% (v/v) glutaraldehyde and activation and contact times of 180 and 30 min, respectively. The immobilized biocatalyst, which showed for both hydrolytic and transfructosylating activities optimum pH and temperature of 7.0 and 60 °C, respectively, retained 70 and 86% of them after 6 cycles of reuse. A kinetic/thermodynamic study focused on thermal inactivation of the immobilized construct indicated high thermostability at temperatures commonly used for fructo-oligosaccharides (FOS) production. Maximum FOS concentration obtained in lab-scale experiments was 101.56 g L−1, with predominant presence of 1-kestose in the reaction mixture. The results obtained in this study suggest that the immobilized-enzyme preparation may be effectively exploited for FOS production and easily recovered from the reaction mixture by action of a magnetic field.

Some biochemical, catalytic, thermodynamic and kinetic properties of purified fructosyltransferase from wild and improved mutant-type Aureobasidium pullulans NAC8

A low molecular weight intracellular and extracellular fructosyltransferase was purified from the wild (Wt) and improved mutant-type (Mt) Aureobasidium pullulans and its characteristics and thermodynamic properties determined. The Wt had been previously genetically modified by chemical mutagenesis. The purified fructosyltransferases from the Wt and Mt had subunit molecular weights between 13.4 and 17.0 kDa, respectively. The pH of the purified fructosyltransferase from the Wt and Mt were within 4.0–5.5. From the KM values, the fructosyltransferase from Mt showed higher affinity for sucrose than Wt fructosyltransferase. The optimum temperature obtained for the Mt intracellular and extracellular fructosyltransferase were 80 and 70 °C, respectively, while the Wt extracellular and intracellular fructosyltransferase was 60 °C. Most metals enhanced fructosyltransferase activity in a concentration-dependent manner except for mercury. Triton X-100 and Tween-80 enhanced the fructosyltransferase activity. Organic solvents such as acetone, ethanol, methanol, toluene and dichloromethane enhanced the enzyme activity while dimethylformamide inhibited the enzyme. From the thermodynamic and kinetic parameters ( Mt fructosyltransferase was more stable to thermal inactivation than the Wt fructosyltransferase. Hence, it can be concluded that, after strain improvement of the Wt, the purified Mt fructosyltransferase was more stable to organic solvents, surfactants, and thermal denaturation, etc. compared to the Wt. This makes Mt fructosyltransferase more useful mostly in the food industry than the Wt.

PRODUCCIÓN DE FRUCTOOLIGOSACÁRIDOS CON DOS NUEVAS ENZIMASβ-FRUCTOFURANOSIDASA RECOMBINANTES DE LEVADURAS AISLADAS DELA FERMENTACIÓN DEL MEZCAL

The gene Cap inv32 from Candida apicola and Tde inv32 from Torulaspora delbrueckii were identified as β-fructofuranosidase belonging to glycoside hydrolases family 32, with an opening reading frame of 1593 base pairs and 1743 base pairs, and a molecular weight of 59 kDa and 65 kDa, for Cap Inv32 and Tde Inv32 correspondingly. These enzymes contained the motif characteristics of the GH32 and showed an identity of 40 % (Cap Inv32) and 52.4 % (Tde Inv32) with an invertase of S. cerevisiae. The fructosyltransferase activity was tasted obtaining for Cap Inv32 the quantity of FOS 12.4 gL-1 and 9.03 gL-1 with 420 gL-1 and 700 gL-1, the production of FOS with Tde Inv32 was 14.61 gL-1 (420 gL-1) and 13.3 gL-1 (700 gL-1). Cap Inv32 and Tde Inv32 are β-fructofuranosidase with fructosyltransferase activity, with potential to industrial applications, focused in FOS production.

Microbial Fructosyltransferase: Production by Submerged Fermentation and Evaluation of pH and Temperature Effects on Transfructosylation and Hydrolytic Enzymatic Activities

Fructosyltransferases (FTase, E.C. 2.4.1.9) are enzymes that catalyze transfructosylation reactions obtaining, as final product, fructose oligomers. In terms of industrial production, the use of microbial enzymes is interesting, especially those produced by Aspergillus sp. The aim of the work was to study the production of FTase by submerged fermentation and evaluation of pH and temperature effects on fructosyltransferase activity. The enzyme was produced by Aspergillus oryzae IPT 301 in a 10-liter bioreactor with growth medium containing sucrose as main carbon source. The assay was performed at 800 rpm, 30 °C, 0.75 vvm and pH 4.5. Transfructosylation (At) and hydrolytic (Ah) activities were determined in the temperature range from 35 to 65 °C and pH range from 3.5 to 6.0. It was observed that mycelial is increases with temperature, holding the maximum value at 50 – 65 oC, while the optimum pH value were 5.0. The optimum temperature for extracellular At ranged from 55 to 65 °C and the optimum pH ranged from 5.0 to 6.0. Furthermore, the optimum temperature for mycelial Ah was 65 °C and optimum pH 4.5 – 5.0. For extracellular Ah, the optimum temperature and optimum pH were 55 to 65 °C and 3.5 to 6.0, respectively.

New β-galactosidase producers with potential for prebiotic synthesis

β-Galactosidases (EC 3.2.1.23) are interesting enzymes with potential application in the pharmaceutical and food industry. In this work, a screening study was carried out to identify new fungal sources of β-galactosidase. A total of 50 fungi were evaluated using a chromogenic test performed in agar plates. The most promising microorganisms were validated as effective β-galactosidase producers under submerged fermentation conditions. The crude β-galactosidases were characterized regarding their optimal pH (3.0–5.5) and temperature (45–65 °C). All enzymes showed ability to synthesize lactose-based prebiotics, namely lactulose (maximal yield 3.3%) and a galacto-oligosaccharide (GOS) (maximal yield 20%). Additionally, some enzymatic extracts with fructosyltransferase activity allowed to produce other type of prebiotics, namely fructo-oligosaccharides (FOS). This work, reports for the first time the simultaneous synthesis of different mixtures of GOS (2–15% yield and 0.07–0.5 g/L·h−1 productivity) and FOS (4–30% yield and 0.1–1 g/L·h−1 productivity) by crude extracts exhibiting dual enzymatic activity.

Artificial Neural Network-Assisted Spectrophotometric Method for Monitoring Fructo-oligosaccharides Production

Short-chain fructo-oligosaccharides (FOS) are considered as low-calorie carbohydrates with prebiotic function. They can be produced from sucrose by fructosyltransferase activity, resulting in a mixture of saccharides with different chain lengths. Current practice for carbohydrate analysis involves the use of time-costly and off-line chromatographic procedures. This study is dedicated to the development of an artificial neural network (ANN) model for predicting carbohydrate composition from the direct measurement of UV spectra. A total of 182 samples were generated by operating an enzyme membrane reactor (EMR) under both optimal and suboptimal settings. The concentration data determined by HPLC and corresponding absorbance readings were used to train a two-layer feedforward neural network. The optimized model was then validated by using new observations that were not involved in the training. The model explained 98, 97, and 88% of the variation in the composition of the new observations regarding the main components sucrose, kestose, and glucose with a mean squared error of prediction of 6.59, 3.40, and 2.81, respectively. The results indicate that the proposed UV-ANN method has a great potential to be used for the real-time monitoring of the bioconversion.

Prebiotic Oligosaccharides: Special Focus on Fructooligosaccharides, Its Biosynthesis and Bioactivity.

The bacterial groups in the gut ecosystem play key role in the maintenance of host's metabolic and structural functionality. The gut microbiota enhances digestion processing, helps in digestion of complex substances, synthesizes beneficial bioactive compounds, enhances bioavailability of minerals, impedes growth of pathogenic microbes, and prevents various diseases. It is, therefore, desirable to have an adequate intake of prebiotic biomolecules, which promote favorable modulation of intestinal microflora. Prebiotics are non-digestible and chemically stable structures that significantly enhance growth and functionality of gut microflora. The non-digestible carbohydrate, mainly oligosaccharides, covers a major part of total available prebiotics as dietary additives. The review describes the types of prebiotic low molecular weight carbohydrates, i.e., oligosaccharides, their structure, biosynthesis, functionality, and applications, with a special focus given to fructooligosaccharides (FOSs). The review provides an update on enzymes executing hydrolytic and fructosyltransferase activities producing prebiotic FOS biomolecules, and future perspectives.

β-galactosidase from Aspergillus lacticoffeatus: A promising biocatalyst for the synthesis of novel prebiotics

β-galactosidase (EC 3.2.1.23) are interesting enzymes able to catalyze lactose hydrolysis and transfer reactions to produce lactose-based prebiotics with potential application in the pharmaceutical and food industry. In this work, Aspergillus lacticoffeatus is described, for the first time, as an effective β-galactosidase producer. The extracellular enzyme production was evaluated in synthetic and alternative media containing cheese whey and corn steep liquor. Although β-galactosidase production occurred in all media (expect for the one composed solely by cheese whey), the highest enzymatic activity values (460U/mL) were obtained for the synthetic medium. Ochratoxin A production in synthetic medium was also evaluated and 9days of fermentation was identified as a suitable fermentation time to obtain a crude extract enzyme with mycotoxin concentration below the legal comparable value established for wine and grape juices (2ng/mL). The optimal pH and temperature for the crude extract enzyme was found in the range of 3.5–4.5 and 50–60°C, respectively. The β-galactosidase activity was reduced in the presence of Ba2+, Fe2+, Li+, K+ and galactose, while additives (except for ascorbic acid) and detergents exhibited a positive effect on enzymatic activity. This enzyme was able to catalyze the synthesis of prebiotics, namely lactulose (2.5g/L) and a galacto-oligosaccharide (trisaccharide, 6.3g/L), either when whole cells or crude enzyme was used as biocatalyst. The lactulose production using fungal whole cells is herein reported for the first time. Additionally, A. lacticoffeatus was also found to produce an enzyme with fructosyltransferase activity and other prebiotics, namely fructo-oligosaccharide 1-kestose (2.4g/L).

English

In order to obtain a native isolate with high β-fructofuranosidase activity (FFase), a preliminary selection of 44 isolates, predominantly formed by Aspergillus niger (72%), was performed among  167 isolates of Aspergillus native to the Peruvian coast (Casa grande, Cartavio, Paramonga and Tacama). In addition, genetic diversity was studied using molecular markers, Inter Simple Sequence Repeats (ISSR). The FFase activity of these 44 isolates was compared and the isolate which showed the highest fructosyltransferase (FTase) activity was identified molecularly at the species level by DNA sequencing. Cluster analysis indicated 16 sub-genetic groups of which 11 of them were morphologically identified as Aspergillus niger. Samples from Paramonga showed the highest genetic diversity as explained by the Shannon diversity index (I= 0.21). No positive association was found between the genetic diversity of populations and FFase activity. Isolates PR-151, PR-144 and PR-142 showed the highest FFase activity in 5 consecutive evaluation generations, PR-142 being the most active with a total FFase activity of 11,248 (U.L-1) and 6.17 g/L of biomass indicating a great biotechnological potential for the synthesis of prebiotics. The molecular identification at the species level confirmed that PR-142 belongs to A. niger lineage.   Key words: β-Fructofuranosidase, fructosyltransferase, Aspergillus, Inter Simple Sequence Repeat (ISSR), fructooligosaccharides.

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.

Sequence and structure-based prediction of fructosyltransferase activity for functional subclassification of fungal GH32 enzymes.

Sucrolytic enzymes catalyse sucrose hydrolysis or the synthesis of fructooligosaccharides (FOSs), a prebiotic in human and animal nutrition. FOS synthesis capacity differs between sucrolytic enzymes. Amino-acid-sequence-based classification of FOS synthesizing enzymes would greatly facilitate the insilico identification of novel catalysts, as large amounts of sequence data lie untapped. The development of a bioinformatics tool to rapidly distinguish between high-level FOSs synthesizing predominantly sucrose hydrolysing enzymes from fungal genomic data is presented. Sequence comparison of functionally characterized enzymes displaying low- and high-level FOS synthesis revealed conserved motifs unique to each group. New light is shed on the sequence context of active site residues in three previously identified conserved motifs. We characterized two enzymes predicted to possess low- and high-level FOS synthesis activities based on their conserved motif sequences. FOS data for the enzymes confirmed our successful prediction of their FOS synthesis capacity. Structural comparison of enzymes displaying low- and high-level FOS synthesis identified steric hindrance between nystose and a long loop region present only in low-level FOS synthesizers. This loop is proposed to limit the synthesis of FOS species with higher degrees of polymerization, a phenomenon observed among enzymes displaying low-level FOS synthesis. Conserved sequence motifs surrounding catalytic residues and a distant structural determinant were identifiers of FOS synthesis capacity and allow for functional annotation of sucrolytic enzymes directly from amino acid sequence. The tool presented may also be useful to study the structure-function relationships of β-fructofuranosidases by identifying mutations present in a group of closely related enzymes displaying similar function.