Recombinant Capsicum annuum Acid beta-fructofuranosidase AIV-18

What is Beta-Fructofuranosidase and what are its primary biochemical functions?

Beta-Fructofuranosidase (EC 3.2.1.26) is an enzyme that catalyzes the hydrolysis of terminal non-reducing β-D-fructofuranoside residues in β-D-fructofuranosides. In Capsicum annuum (pepper), this enzyme plays crucial roles in carbohydrate metabolism, particularly in the degradation of sucrose and fructans. The primary reactions catalyzed by this enzyme include:

• β-D-fructofuranose(n) + H₂O → β-D-fructofuranose(n-1) + β-D-fructofuranose

• Sucrose + H₂O → α-D-glucose + β-D-fructofuranose

• 1-kestotriose + H₂O → D-fructofuranose + sucrose

• Fructan(n) + H₂O → β-D-fructofuranose + fructan(n-1)

These reactions are central to two major metabolic pathways in plants: sucrose degradation (via sucrose invertase) and fructan degradation. The enzyme's ability to both hydrolyze sucrose and catalyze transfructosylation reactions makes it particularly valuable in research and potential biotechnological applications.

How does Recombinant Capsicum annuum Beta-Fructofuranosidase differ from other plant and microbial sources?

Recombinant Capsicum annuum Beta-Fructofuranosidase differs from other sources primarily in its molecular structure, substrate specificity, and catalytic properties. While sharing the fundamental β-D-fructofuranosidase activity, different sources exhibit unique characteristics:

• Molecular structure: The gene encoding Capsicum annuum Beta-Fructofuranosidase (CA04g11820) is 1883 bp in length , whereas Beta-Fructofuranosidases from Aspergillus species show considerable variation in size and structure, such as those from A. luchuensis with 1887 bp encoding 628 amino acids .

• Catalytic efficiency: Microbial Beta-Fructofuranosidases, particularly from Aspergillus species, often demonstrate higher specific activities. For instance, AlFFase3 from A. luchuensis exhibits a specific activity of 771.2 U/mg towards sucrose , while other Aspergillus species such as A. terreus and A. sojae show even higher specific activities of 1985.7 U/mg and 1886.3 U/mg, respectively .

• pH and temperature optima: Different sources have distinct stability profiles. For example, AlFFase3 exhibits stability between pH 5.5 and 7.5, with maximal activity at pH 6.5 and 40°C , which may differ from the optimal conditions for Capsicum annuum Beta-Fructofuranosidase.

Understanding these differences is crucial when selecting the appropriate enzyme source for specific research applications.

What experimental methods are recommended for purifying recombinant Beta-Fructofuranosidase from expression systems?

Purification of recombinant Beta-Fructofuranosidase requires a systematic approach to maintain enzyme activity while achieving high purity. Based on successful protocols for similar enzymes, the following methodology is recommended:

• Expression system selection: For recombinant expression, E. coli systems with appropriate vectors such as pET-28a(+) have proven effective for Beta-Fructofuranosidase from other sources . Codon optimization of the gene sequence for the expression host can significantly improve yields.

• Affinity chromatography: Ni-IDA affinity chromatography is highly effective for purification of His-tagged Beta-Fructofuranosidase. This single-step purification can significantly increase specific activity, as demonstrated with AlFFase3 which showed an increase from 138.2 U/mg to 771.2 U/mg after purification .

• SDS-PAGE analysis: Molecular weight confirmation through SDS-PAGE is essential, using appropriate molecular weight standards (e.g., rabbit phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), etc.) .

• Activity assay: Enzyme activity can be determined by measuring glucose release using a glucose oxidase kit. One unit of Beta-Fructofuranosidase activity is typically defined as the enzyme amount that releases 1 μmol of glucose per minute under standard assay conditions .

• Protein concentration determination: The Lowry method with bovine serum albumin as a standard is commonly used for accurate protein quantification .

This systematic approach ensures both purity and activity preservation, which are critical for subsequent characterization and application studies.

What are the optimal methods for assessing the kinetic parameters of Recombinant Capsicum annuum Beta-Fructofuranosidase?

Accurate determination of kinetic parameters is essential for characterizing recombinant Beta-Fructofuranosidase. The following methodological approach is recommended:

• Substrate concentration series: Prepare a range of sucrose concentrations (typically 5-500 mM) in appropriate buffer systems to determine Km and Vmax values.

• Standard reaction conditions: Based on similar enzymes, reactions should be performed under optimized conditions, typically at the enzyme's optimal pH and temperature (e.g., pH 6.5 and 40°C for similar Beta-Fructofuranosidases) .

• Reaction monitoring: Quantify the release of glucose using glucose oxidase kits or high-performance liquid chromatography (HPLC) for precise measurement of reaction progress .

• Data analysis: Use nonlinear regression analysis to fit data to the Michaelis-Menten equation, Lineweaver-Burk plots, or Eadie-Hofstee diagrams for calculation of kinetic parameters.

• Inhibition studies: Assess potential inhibitors using appropriate concentrations and determine inhibition constants and mechanisms (competitive, non-competitive, or uncompetitive).

• Temperature and pH effects: Systematically evaluate enzyme activity across temperature ranges (25-70°C) and pH values (3.0-10.5) using appropriate buffer systems such as sodium citrate (pH 3.0-6.0), MES (pH 5.0-6.5), sodium phosphate (pH 6.0-8.0), and others .

This comprehensive kinetic characterization provides fundamental insights into the enzyme's catalytic behavior and facilitates optimization of reaction conditions for various applications.

How can researchers evaluate the substrate specificity and transfructosylation activity of Beta-Fructofuranosidase?

Evaluating substrate specificity and transfructosylation activity requires specialized techniques to distinguish between hydrolytic and transferase activities:

• Substrate panel testing: Test the enzyme activity against various substrates beyond sucrose, including different fructooligosaccharides, raffinose, stachyose, and other potential substrates. Measure product formation using techniques such as HPLC, thin-layer chromatography (TLC), or mass spectrometry.

• Transfructosylation activity assessment: To evaluate transfructosylation capacity, incubate the enzyme with high concentrations of sucrose (typically 400-600 g/L) and monitor the formation of fructooligosaccharides (FOS) over time. The yield of FOS can be a significant indicator of transfructosylation efficiency - for reference, AlFFase3 from A. luchuensis demonstrated a FOS yield of up to 67% .

• Product profiling: Characterize the FOS products using analytical techniques such as high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

• Reaction optimization: Systematically vary reaction parameters (substrate concentration, temperature, pH, reaction time) to determine optimal conditions for transfructosylation versus hydrolysis activities.

• Competition experiments: Perform experiments with mixed substrates to evaluate preferential activity and potential competitive inhibition.

This methodological approach provides a comprehensive profile of the enzyme's catalytic versatility and potential for producing specific fructooligosaccharides, which is crucial for applications in functional food research.

What techniques are most effective for determining the structural properties of Recombinant Capsicum annuum Beta-Fructofuranosidase?

Structural characterization of Beta-Fructofuranosidase requires a multi-technique approach:

This comprehensive structural characterization is essential for understanding enzyme function at the molecular level and for rational engineering approaches to modify activity or stability.

How can researchers optimize expression systems for high-yield production of functional Recombinant Capsicum annuum Beta-Fructofuranosidase?

Optimizing expression systems for high-yield production requires addressing several key factors:

• Expression vector selection: For bacterial expression, vectors with strong inducible promoters (e.g., T7 promoter in pET systems) have proven effective for similar enzymes . For eukaryotic expression, vectors appropriate for fungi or yeast may provide better post-translational processing.

• Codon optimization: Codon optimization based on the expression host's codon usage bias is crucial for high-level expression. For instance, when expressing AlFFase3 in E. coli, codon optimization of the gene from A. luchuensis significantly improved expression levels .

• Expression host considerations:

  • E. coli: Suitable for rapid expression but may have limitations with eukaryotic post-translational modifications

  • Yeast systems (Pichia pastoris, Saccharomyces cerevisiae): Offer post-translational modifications and secretion capabilities

  • Filamentous fungi (Aspergillus, Trichoderma): Appropriate for industrial-scale production with native-like post-translational modifications

• Induction optimization: Systematic evaluation of induction parameters (inducer concentration, temperature, duration) is essential. For Beta-Fructofuranosidase, lower induction temperatures (15-25°C) often improve soluble protein yield.

• Solubility enhancement strategies:

  • Fusion tags (e.g., MBP, SUMO, thioredoxin)

  • Co-expression with chaperones

  • Periplasmic targeting in bacterial systems

• Scale-up considerations: Fermentation parameters including media composition, dissolved oxygen levels, and pH control need careful optimization when transitioning from laboratory to larger-scale production.

These optimization strategies can significantly improve the yield of functional Beta-Fructofuranosidase for research applications.

What are the key considerations when designing site-directed mutagenesis experiments to modify the catalytic properties of Beta-Fructofuranosidase?

Site-directed mutagenesis is a powerful approach for modifying enzyme properties, but requires careful experimental design:

• Target residue selection based on:

  • Sequence alignment with well-characterized Beta-Fructofuranosidases from other sources

  • Structural analysis of the active site and substrate-binding regions

  • Phylogenetic analysis to identify conserved versus variable residues

  • Computational prediction of functional residues

• Mutagenesis strategy:

  • Conservative substitutions (maintaining similar physicochemical properties) for initial structure-function analysis

  • Non-conservative substitutions to significantly alter activity or specificity

  • Alanine scanning to identify essential catalytic residues

  • Creation of chimeric enzymes by swapping domains between Beta-Fructofuranosidases from different sources

• Screening methodology:

  • High-throughput colorimetric assays for rapid identification of variants with altered activity

  • Detailed kinetic analysis of promising variants

  • Thermal stability assessment using differential scanning fluorimetry

  • Substrate specificity profiling using multiple substrates

• Validation experiments:

  • Determination of kinetic parameters (Km, kcat, kcat/Km) for wild-type and mutant enzymes

  • pH and temperature profiles to assess changes in optimal conditions

  • Product profile analysis to evaluate changes in transfructosylation versus hydrolysis ratio

• Structural analysis:

  • Structural determination of key mutants to understand the molecular basis of altered properties

  • Molecular dynamics simulations to predict the effect of mutations on enzyme dynamics

This systematic approach to mutagenesis enables rational engineering of Beta-Fructofuranosidase with tailored catalytic properties for specific research applications.

How do temperature and pH stability profiles influence experimental design when working with Recombinant Capsicum annuum Beta-Fructofuranosidase?

Temperature and pH stability profiles are critical parameters that significantly influence experimental design when working with enzymes:

• Stability profiling methodology:

  • Temperature stability: Incubate enzyme samples at various temperatures (25-70°C) for defined time periods, then measure residual activity under standard conditions

  • pH stability: Pre-incubate enzyme in buffers spanning pH 3.0-10.5 for specific durations, then assay for remaining activity at optimal pH

  • Combined effects: Establish stability matrices examining both parameters simultaneously

• Impact on reaction conditions:

  • Optimal conditions for activity may differ from optimal stability conditions

  • For Beta-Fructofuranosidases, pH stability typically ranges from pH 5.5-7.5, with maximum activity around pH 6.5 at 40°C (based on similar enzymes)

  • Reaction duration must be optimized based on stability at the selected temperature and pH

• Considerations for experimental design:

  • Buffer selection: Different buffer systems for different pH ranges (sodium citrate for pH 3.0-6.0, MES for pH 5.0-6.5, sodium phosphate for pH 6.0-8.0, etc.)

  • Temperature control precision requirements

  • Need for stabilizing additives in prolonged reactions

  • Enzyme concentration adjustments based on stability profile

• Special considerations for transfructosylation reactions:

  • Transfructosylation often requires extended reaction times, making stability crucial

  • Higher substrate concentrations may provide stabilizing effects

  • Product inhibition becomes more significant in longer reactions

• Storage and handling recommendations:

  • Optimal storage conditions (temperature, buffer, additives)

  • Freeze-thaw stability considerations

  • Guidelines for working stock preparation

Understanding these stability parameters ensures reproducible results and maximizes enzyme performance in both analytical and preparative applications.

How can Recombinant Capsicum annuum Beta-Fructofuranosidase be utilized for the synthesis of structurally defined fructooligosaccharides?

Beta-Fructofuranosidase's transfructosylation activity makes it valuable for synthesizing defined fructooligosaccharides (FOS) with specific structures:

• Reaction optimization for FOS synthesis:

  • High substrate concentrations (typically 400-600 g/L sucrose) favor transfructosylation over hydrolysis

  • Temperature and pH optimization specific to transfructosylation (may differ from optimal hydrolytic conditions)

  • Time-course monitoring to determine optimal reaction duration for desired FOS profile

• Product diversity control:

  • Beta-Fructofuranosidases can produce various FOS structures including 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4)

  • Reaction conditions can be tuned to favor specific chain lengths and linkage patterns

  • The yield of FOS can reach up to 67% under optimized conditions, as demonstrated with similar enzymes

• Analytical methods for FOS characterization:

  • High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

  • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

  • Nuclear magnetic resonance (NMR) spectroscopy for detailed structural elucidation

• Purification strategies for synthesized FOS:

  • Size-exclusion chromatography

  • Charcoal column fractionation

  • Preparative HPLC

• Assessment of FOS bioactivity:

  • Prebiotic potential evaluation using in vitro fermentation models

  • Growth promotion of beneficial bacteria such as Bifidobacterium and Lactobacillus species

  • Structure-function relationship studies comparing different FOS structures

This application of Beta-Fructofuranosidase enables researchers to produce well-defined FOS structures for various glycobiology studies, including investigations of structure-activity relationships and prebiotic mechanisms.

What research approaches can be used to compare the transfructosylation efficiency of Recombinant Capsicum annuum Beta-Fructofuranosidase with other sources?

Comparative analysis of transfructosylation efficiency requires standardized methodologies:

• Standardized reaction conditions:

  • Fixed substrate concentration (e.g., 500 g/L sucrose)

  • Equivalent enzyme units (based on hydrolytic activity)

  • Consistent temperature, pH, and reaction duration

  • Identical analytical methods for product quantification

• Performance parameters to measure:

  • Maximum FOS yield (% of total sugars)

  • Transfructosylation/hydrolysis ratio

  • FOS production rate

  • Product spectrum (distribution of different FOS chain lengths)

  • Enzyme stability under reaction conditions

• Molecular basis comparison:

  • Structural alignment of active sites

  • Identification of key residues that differ between high and low transfructosylation efficiency enzymes

  • Creation of chimeric enzymes to identify domains responsible for transfructosylation specificity

• Kinetic analysis of transfructosylation:

  • Determination of kinetic parameters specific to transfructosylation (as distinct from hydrolysis)

  • Competitive substrate experiments to evaluate acceptor preferences

This systematic comparison provides valuable insights into the structural determinants of transfructosylation efficiency and guides enzyme engineering efforts.

How can protease resistance profiles be evaluated for Beta-Fructofuranosidase in research applications?

Protease resistance is an important characteristic for enzymes used in various research and potential biotechnological applications:

• Standardized protease resistance assay:

  • Incubate purified Beta-Fructofuranosidase with various proteases (e.g., trypsin, pepsin, Proteinase K, acidic protease, neutral protease, alkaline proteinase, and Flavourzyme) at defined concentrations (e.g., 10 U/mL)

  • Standard incubation conditions (e.g., 37°C for 30 min)

  • Measure residual Beta-Fructofuranosidase activity using standard assay conditions

  • Calculate percent activity retention compared to untreated enzyme control

• Detailed protease resistance profile:

  • Time-course experiments to determine degradation kinetics

  • Dose-response curves with varying protease concentrations

  • SDS-PAGE analysis to visualize proteolytic fragments

• Structural basis of protease resistance:

  • Identification of potential protease cleavage sites through in silico analysis

  • Site-directed mutagenesis to modify susceptible sites

  • Analysis of glycosylation patterns that may contribute to protease resistance

• Comparative resistance analysis:

  • Some Beta-Fructofuranosidases, such as AlFFase3 from A. luchuensis, demonstrate remarkable resistance to common proteases

  • This resistance profile can be compared with that of Recombinant Capsicum annuum Beta-Fructofuranosidase to identify unique characteristics

• Applications leveraging protease resistance:

  • Development of immobilized enzyme systems with extended operational stability

  • Potential for development of oral enzyme delivery systems if gastrointestinal protease resistance is demonstrated

  • Enhanced stability in complex reaction environments containing proteases

This methodological approach provides valuable information about the enzyme's robustness in various experimental conditions and potential applications in complex biological systems.

What are the methodological considerations for immobilizing Recombinant Capsicum annuum Beta-Fructofuranosidase for continuous FOS production?

Enzyme immobilization can significantly enhance the practical utility of Beta-Fructofuranosidase for continuous FOS production:

• Immobilization method selection:

  • Covalent binding to activated supports (e.g., epoxy-activated, glutaraldehyde-activated)

  • Adsorption on ion-exchange resins or hydrophobic carriers

  • Entrapment in polymeric matrices (alginate, polyacrylamide)

  • Cross-linked enzyme aggregates (CLEAs)

  • Affinity immobilization via engineered tags

• Support selection criteria:

  • Mechanical stability for continuous operation

  • Porosity appropriate for substrate/product diffusion

  • Chemical stability under reaction conditions

  • Cost-effectiveness for research applications

  • Biocompatibility for potential food-related applications

• Immobilization parameter optimization:

  • Enzyme loading (mg protein/g support)

  • Buffer composition and pH during immobilization

  • Immobilization time and temperature

  • Cross-linking degree (if applicable)

• Performance evaluation:

  • Activity recovery (% of initial activity retained after immobilization)

  • Operational stability (activity retention over multiple cycles)

  • Thermal and pH stability changes upon immobilization

  • Kinetic parameter alterations (apparent Km, Vmax)

  • FOS production profile compared to free enzyme

• Reactor design considerations:

  • Packed bed reactors

  • Fluidized bed reactors

  • Membrane reactors

  • Residence time optimization

  • Flow rate effects on transfructosylation/hydrolysis ratio

This methodological approach supports the development of efficient immobilized Beta-Fructofuranosidase systems for continuous FOS production in research settings.

How can researchers design experiments to evaluate the potential synergistic effects between Beta-Fructofuranosidase and other carbohydrate-active enzymes?

Investigating enzyme synergy requires systematic experimental design:

This systematic investigation of enzyme synergy can lead to novel applications in carbohydrate research and prebiotic development.

What future research directions could address the current knowledge gaps regarding Recombinant Capsicum annuum Beta-Fructofuranosidase?

Several promising research directions could address knowledge gaps:

• Structural biology investigations:

  • High-resolution crystal structure determination

  • Structure-function relationship studies through site-directed mutagenesis

  • Computational modeling of substrate binding and catalytic mechanisms

  • Molecular dynamics simulations to understand conformational flexibility

• Comparative genomics and evolution:

  • Comprehensive phylogenetic analysis of Beta-Fructofuranosidases across plant species

  • Investigation of gene duplication and functional diversification

  • Analysis of adaptive evolution in different plant lineages

  • Comparative expression profiling under various physiological conditions

• Engineering for enhanced properties:

  • Rational design for improved thermostability

  • Directed evolution for altered product specificity

  • Engineering of the transfructosylation/hydrolysis ratio

  • Development of chimeric enzymes with novel properties

• Novel analytical methods development:

  • Real-time monitoring of transfructosylation reactions

  • High-throughput screening methods for mutant libraries

  • Advanced structural characterization of enzyme-substrate complexes

  • Computational tools for predicting transfructosylation outcomes

• Biological role exploration:

  • Investigation of the physiological role in Capsicum annuum

  • Developmental regulation of expression

  • Response to biotic and abiotic stresses

  • Potential roles beyond carbohydrate metabolism

These research directions would significantly advance our understanding of Recombinant Capsicum annuum Beta-Fructofuranosidase and expand its potential applications in glycobiology research.