Purification of Aspergillus tamarii mycelial fructosyltransferase (m-FTase), optimized FOS production, and evaluation of its anticancer potential.

In the present study, generation of prebiotic fructooligosaccharides (FOS) using Aspergillus tamarii FTase was optimized by applying response surface methodology. Optimal FOS (251 g L-1 ) was generated at 28.4°C, pH 7.0 and 50% (w/v) sucrose leading to 1.97-fold yield enhancement. The m-FTase was purified using ultrafiltration followed by HiTrap Q HP anion exchange chromatography resulting in 2.15-fold purified FTase with 12.76 U mg-1 specific activity. Purified FTase (75 kDa) had Km and Vmax values of 1049.717 mM and 2.094 µmol min-1  mg-1 , respectively. FOS incorporation led to upregulation of caspase 3, caspase 9, and Bax genes suggesting mitochondrial apoptosis activation in cancer cells. The study describes characteristics of purified FTase from A. tamarii, production optimization of FOS and unravels the role of FOS in anticancer activity against HT-29 cells. PRACTICAL APPLICATION: This study provides detailed insights of kinetic and thermodynamic characteristics of purified FTase, a prebiotic FOS-generating enzyme. Moreover, the role of the apoptotic genes involved in anticancer activity, and the prebiotic potential of FOS is also investigated. These findings are important in the context of FOS applications, and the optimized production strategies make it useful for industrial application.

Production, properties, and applications of fructosyltransferase: a current appraisal.

BACKGROUND: Fructosyltransferases (FTases) are drawing increasing attention due to their application in prebiotic fructooligosaccharide (FOS) generation. FTases have been reported to occur in a variety of microorganisms but are predominantly found in filamentous fungi. These are employed at the industrial scale for generating FOS which make the key ingredient in functional food supplements and nutraceuticals due to their bifidogenic and various other health-promoting properties. SCOPE AND APPROACH: This review is aimed to discuss recent developments made in the area of FTase production, characterization, and application in order to present a comprehensive account of their present status to the reader. Structural features, catalytic mechanisms, and FTase improvement strategies have also been discussed in order to provide insight into these aspects. KEY FINDINGS AND CONCLUSIONS: Although FTases occur in several plants and microorganisms, fungal FTases are being exploited commercially for industrial-scale FOS generation. Several fungal FTases have been characterized and heterologously expressed. However, considerable scope exists for improved production and application of FTases for cost-effective production of prebiotic FOS.HIGHLIGHTSFructosyltrasferase (FTase) is a key enzyme in fructo-oligosaccharide (FOS) generationDevelopments in the production, properties, and functional aspects of FTasesMolecular modification and immobilization strategies for improved FOS generationFructosyltransferases are innovation hotspots in the food and nutraceutical industries.

Exo-inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production.

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.

Characterization of biogenically synthesized silver nanoparticles for therapeutic applications and enzyme nanocomplex generation.

The present study describes green synthesis of silver nanoparticles (AgNPs) and inulin hydrolyzing enzyme nanocomplexes (ENC) using Azadirachta indica (Ai) and Punica granatum (Pg) leaf extracts. Surface topology and physico-chemical characteristics of AgNPs were studied using surface plasmon resonance (SPR), FTIR, SEM, AFM and EDX analyses. Particle size analysis using dynamic light scattering and AFM studies revealed that Ai-AgNPs (76.4 nm) were spherical in shape having central bigger nano-regime with smaller surroundings while Pg-AgNPs (72.1 nm) and ENCs (Inulinase-Pg-AgNPs ~ 145 nm) were spherical particles having smooth surfaces. Pg-AgNPs exhibited significant photocatalysis of a thiazine dye, methylene blue. Both Ai- and Pg-AgNPs showed selective antibacterial action by inhibiting pathogenic Bacillus cereus, while the probiotic Lactobacillus strains remained unaffected. Ai-AgNPs showed potential anti-biofilm effect (30% viability) on B. cereus biofilms. Pg-AgNPs showed anti-cancer effect against human colon cancer cell lines (Caco-2) resulting in 40% cell death in 48 h. Enzymes (inulinase, L-asparaginase and glucose oxidase) were successfully immobilized onto nanoparticles together with the biogenic synthesis of AgNPs and recyclability of the Inulinase-Pg-AgNPs complex was demonstrated. The study elaborates characteristics of green synthesized nanoparticles and their potential applications as anti-cancer, antibacterial and antioxidant nano drugs that could be used in food and nutraceutical industries. Enzyme immobilization on AgNPs without any toxic cross-linker opens up newer possibilites in enzyme-nanocomplex research.

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.