Recombinant Pinus halepensis Glucan endo-1,3-beta-glucosidase

What is Glucan endo-1,3-beta-glucosidase in Pinus halepensis and what are its main functions?

Glucan endo-1,3-beta-glucosidase (EC 3.2.1.39) from Pinus halepensis (Aleppo pine) is an enzyme that catalyzes the hydrolysis of 1,3-beta-glucosidic linkages in 1,3-beta-D-glucans. This enzyme functions as an endo-hydrolase, cleaving β-linkages at internal sites along the polysaccharide chain and releasing smaller oligosaccharides . It is also known by alternative names such as (1→3)-beta-glucan endohydrolase, (1→3)-beta-glucanase, and Beta-1,3-endoglucanase .

In plant systems, this enzyme plays crucial roles in various physiological processes, particularly in defense mechanisms against fungal pathogens, as the enzyme can degrade 1,3-beta-glucans, which are major components of fungal cell walls . The enzyme is particularly important for delaying the growth of pathogenic fungi and decreasing disease-related damage in plants .

How is the recombinant form of Pinus halepensis Glucan endo-1,3-beta-glucosidase produced and what are its key specifications?

The recombinant form of Pinus halepensis Glucan endo-1,3-beta-glucosidase (Product Code: CSB-BP307750PKI, UniProt No.: P85483) is produced using a baculovirus expression system . This system is chosen for its ability to produce eukaryotic proteins with proper folding and post-translational modifications.

Key specifications include:

• Sequence: TYNNNLIR

• Expression Region: 1-8

• Purity: >85% as determined by SDS-PAGE

• Protein Length: Full-length protein

• Source organism: Pinus halepensis (Aleppo pine)

The recombinant protein requires proper handling for optimal activity, including:

• Storage at -20°C, or -80°C for extended storage

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) for long-term storage

• Avoiding repeated freezing and thawing cycles

How does the structure of Glucan endo-1,3-beta-glucosidase relate to its catalytic function?

Glucan endo-1,3-beta-glucosidase functions as an endo-hydrolase that cleaves β-1,3-glycosidic bonds at random sites along glucan chains. The enzyme's structure facilitates this function through specific domains:

The catalytic domain contains acidic amino acid residues (typically glutamic acid) that participate in the hydrolysis reaction through acid-base catalysis. These residues are positioned to interact with the β-1,3-glucan substrate in a manner that allows for the breaking of glycosidic bonds .

While the specific structural details of the Pinus halepensis enzyme are not fully described in the search results, studies on homologous enzymes from other species suggest that these endo-glucanases typically have a substrate-binding cleft that can accommodate several glucose units of the β-1,3-glucan chain. This structural arrangement facilitates the endolytic mode of action, where the enzyme cleaves internal glycosidic bonds rather than removing terminal glucose units .

What are the optimal storage and handling conditions for maintaining the activity of recombinant Pinus halepensis Glucan endo-1,3-beta-glucosidase?

For optimal storage and handling of recombinant Pinus halepensis Glucan endo-1,3-beta-glucosidase, researchers should follow these evidence-based protocols:

Long-term storage:

• Store at -20°C, or -80°C for extended storage periods

• The lyophilized form has a shelf life of approximately 12 months at these temperatures

• The liquid form has a reduced shelf life of approximately 6 months

Reconstitution procedure:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Prepare working aliquots to avoid repeated freeze-thaw cycles

Working conditions:

• Working aliquots can be stored at 4°C for up to one week

• Repeated freezing and thawing significantly reduces enzyme activity and should be avoided

• The enzyme's stability is affected by buffer ingredients, storage temperature, and the intrinsic stability of the protein itself

What experimental approaches can be used to assess the enzymatic activity of Glucan endo-1,3-beta-glucosidase?

Several methodological approaches can be employed to assess the enzymatic activity of Glucan endo-1,3-beta-glucosidase:

Spectrophotometric assays:

• Reducing sugar assay: Measure the release of reducing sugars (glucose or oligosaccharides) from laminarin or other β-1,3-glucan substrates using colorimetric methods such as DNS (3,5-dinitrosalicylic acid) or Nelson-Somogyi assay

• Chromogenic substrate assay: Utilize specific chromogenic substrates that release measurable products upon enzymatic hydrolysis

Viscometric method:

• Measure the decrease in viscosity of a β-1,3-glucan solution as the enzyme hydrolyzes the polysaccharide, shortening the chain length

HPLC analysis:

• Analyze the oligosaccharide products released by enzymatic action using HPLC with appropriate detectors

• This method allows for qualitative and quantitative assessment of the enzyme's mode of action

Activity gel electrophoresis:

• Incorporate β-1,3-glucan substrates into polyacrylamide gels

• After electrophoresis and appropriate incubation, stain for enzyme activity zones

For recombinant enzymes specifically, it's essential to include proper controls such as heat-inactivated enzyme samples and substrate-only reactions to account for background activity .

How can researchers optimize the expression and purification of recombinant Pinus halepensis Glucan endo-1,3-beta-glucosidase?

Optimizing expression and purification of recombinant Pinus halepensis Glucan endo-1,3-beta-glucosidase requires attention to several critical factors:

Expression optimization:

• Vector selection: Choose appropriate vectors with strong promoters compatible with the host system (baculovirus system is commonly used for this enzyme)

• Codon optimization: Adapt the coding sequence to the codon usage bias of the expression host to improve translation efficiency

• Expression conditions: Optimize temperature, induction time, and inducer concentration for maximum protein yield

• Host cell selection: The baculovirus expression system utilizing insect cells is preferred for complex eukaryotic proteins requiring post-translational modifications

Purification strategy:

• Affinity tag selection: The tag type should be determined during the manufacturing process based on the specific requirements of the protein

• Chromatography methods:

  • Initial capture using affinity chromatography if tags are present

  • Ion exchange chromatography based on the protein's isoelectric point

  • Size exclusion chromatography as a polishing step

• Quality assessment: SDS-PAGE analysis to confirm purity (target >85%)

Optimizing enzyme activity:

• Buffer composition: Test various buffer systems to identify optimal pH and ionic strength

• Additives: Evaluate the effects of stabilizers such as glycerol, BSA, or specific ions

• Storage conditions: Determine optimal formulation for maintaining stability and activity

How is Glucan endo-1,3-beta-glucosidase involved in plant stress responses?

Glucan endo-1,3-beta-glucosidase plays significant roles in plant stress responses, particularly in defense against pathogens and adaptation to abiotic stresses:

Pathogen defense mechanisms:

• The enzyme hydrolyzes β-1,3-glucans, which are major structural components of fungal cell walls, thus directly inhibiting fungal growth

• It releases oligosaccharide fragments that can act as elicitors, triggering broader defense responses in plants

Abiotic stress response:
Research on Pinus halepensis under heat stress conditions has revealed that proteins involved in stress response mechanisms, including metabolic enzymes, show altered expression patterns. While glucan endo-1,3-beta-glucosidase specifically was not highlighted, the study identified 27 proteins related to heat stress response with significant changes in abundance .

Under heat stress conditions (40°C for 4h, 50°C for 30 min, and 60°C for 5 min), proteins with increased abundance were primarily involved in:

• Regulation of metabolism (glycolysis, TCA cycle)

• Amino acid biosynthesis

• Flavonoid formation

• DNA binding and cell division

• Transcription regulation

• Protein lifecycle management

These findings suggest that enzymes like glucan endo-1,3-beta-glucosidase may be part of an integrated stress response system in plants, potentially contributing to acquired thermotolerance and oxidative stress management .

What is the role of Glucan endo-1,3-beta-glucosidase in plant-pathogen interactions?

Glucan endo-1,3-beta-glucosidase serves as a critical component in plant-pathogen interactions through several mechanisms:

Direct antifungal activity:

• The enzyme hydrolyzes β-1,3-glucans in fungal cell walls, weakening their structural integrity and inhibiting fungal growth

• This enzymatic activity is particularly important for delaying pathogenic fungi growth and decreasing disease-related damage in plants

Defense response activation:

• The enzyme releases oligosaccharide fragments from fungal cell walls that can function as elicitors

• These elicitors can trigger broader plant defense responses, including the production of other pathogenesis-related proteins and phytoalexins

Pathogenesis-related protein expression:

• Glucan endo-1,3-beta-glucosidase genes are classified as pathogenesis-related (PR) genes, specifically belonging to the PR-2 family

• The expression of these genes is typically induced upon pathogen attack, wounding, or treatment with elicitors

• Promoter analysis of glucan endo-1,3-beta-glucosidase genes (though in potato rather than pine) has revealed multiple regulatory elements involved in stress responses

Transcriptional regulation:
The regulation of glucan endo-1,3-beta-glucosidase genes involves complex mechanisms:

• Multiple transcription start sites (TSSs) have been identified in glucan endo-1,3-beta-glucosidase genes

• Common motifs shared among promoter regions suggest coordinated regulation of these genes

• These motifs are concentrated between +1 and -500 bp of the TSSs and are distributed on both positive and negative strands

Understanding these regulatory mechanisms provides potential targets for enhancing plant disease resistance through genetic engineering approaches .

How do promoter regions and regulatory elements influence Glucan endo-1,3-beta-glucosidase expression in response to different stressors?

The expression of Glucan endo-1,3-beta-glucosidase genes is regulated by complex promoter regions with multiple regulatory elements that respond to various stressors. While specific information about Pinus halepensis promoters is limited in the search results, studies on homologous genes in Solanum tuberosum provide valuable insights:

Promoter structure and organization:

• Transcription start sites (TSSs) have been identified in glucan endo-1,3-beta-glucosidase genes, with numbers ranging from 1 to 3 per gene

• The majority (84.2%) of promoter regions showed high predictive scores (≥0.90)

• Common motifs shared among glucan endo-1,3-beta-glucosidase gene promoters are concentrated between +1 and −500 bp of the TSSs

• These motifs are distributed on both positive (30 instances) and negative (25 instances) strands

Regulatory elements and their functions:
Five candidate motifs shared by glucan endo-1,3-beta-glucosidase gene promoter sequences have been discovered, potentially binding specific transcription factors that regulate gene expression in response to:

• Pathogen attack

• Wounding

• Temperature stress

• Hormonal signals (like ethylene, jasmonic acid, or salicylic acid)

Stress-responsive expression patterns:
Under heat stress conditions in Pinus halepensis, significant changes in protein expression profiles were observed, including proteins involved in:

• Metabolism regulation (glycolysis, TCA cycle)

• Amino acid biosynthesis

• Flavonoid formation

• DNA binding and cell division

• Transcription regulation

• Protein lifecycle

What comparative genomic approaches can help identify conserved functional domains in Glucan endo-1,3-beta-glucosidase across different species?

Comparative genomic approaches offer powerful tools for identifying conserved functional domains in Glucan endo-1,3-beta-glucosidase across species. Based on methodologies described in the search results, researchers can employ the following approaches:

Phylogenetic analysis:

• Use the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method to construct phylogenetic trees from multiple glucan endo-1,3-beta-glucosidase sequences

• Include sequences from diverse species (e.g., Pinus halepensis, Solanum tuberosum, Nicotiana tabacum, Solanum lycopersicum, and Arabidopsis thaliana)

• Calculate genetic distances using the p-distance method, considering all codon positions (1st+2nd+3rd+Noncoding)

• Use tools like Molecular Evolution Genetic Analysis X32 (MEGA X32) for comprehensive analysis of genetic distances, conserved sites, variable sites, and base composition

Sequence alignment and domain identification:

• Perform multiple sequence alignments to identify conserved regions across species

• Use domain prediction tools to annotate functional domains (catalytic domains, carbohydrate-binding modules, etc.)

• Identify critical amino acid residues involved in catalysis or substrate binding

Promoter region analysis:

• Compare promoter regions of glucan endo-1,3-beta-glucosidase genes across species

• Identify conserved regulatory elements using tools like Neural Network Promoter Prediction (NNPP) and MEME (Multiple EM for Motif Elicitation)

• Map the spatial distribution of common motifs and analyze their potential role in gene regulation

CpG island analysis:

• Identify CpG islands in promoter regions using parameters such as GC content ≥55% and observed to expected CpG ratio ≥0.65

• Compare methylation patterns across species to understand epigenetic regulation

SSR (Simple Sequence Repeat) analysis:

• Screen sequences for di-, tri-, tetra-, penta-, and hexanucleotide SSR motifs using tools like SSRIT

• Analyze the distribution and conservation of these repeats across species

These comparative approaches can reveal evolutionarily conserved features of glucan endo-1,3-beta-glucosidase, providing insights into critical functional domains and potential targets for protein engineering .

How can structural biology techniques be applied to elucidate the catalytic mechanism of Pinus halepensis Glucan endo-1,3-beta-glucosidase?

Advanced structural biology techniques can provide crucial insights into the catalytic mechanism of Pinus halepensis Glucan endo-1,3-beta-glucosidase. While the search results don't provide specific structural information for this enzyme, standard methodological approaches include:

X-ray crystallography:

• Purify recombinant enzyme to high homogeneity (>95%)

• Optimize crystallization conditions through screening of various precipitants, buffers, and additives

• Collect diffraction data and solve the structure through molecular replacement using homologous structures or experimental phasing methods

• Analyze the active site architecture to identify catalytic residues and substrate-binding regions

Cryo-electron microscopy (Cryo-EM):

• Prepare enzyme samples for grid preparation and vitrification

• Collect high-resolution image data

• Perform image processing and 3D reconstruction

• Analyze the resulting structures to understand conformational states

Computational approaches:

• Homology modeling: Create structural models based on homologous enzymes with known structures

• Molecular dynamics simulations: Simulate enzyme-substrate interactions and catalytic mechanisms

• Quantum mechanics/molecular mechanics (QM/MM): Model the electronic changes during catalysis

Functional analysis techniques:

• Site-directed mutagenesis: Modify specific amino acid residues predicted to be involved in catalysis or substrate binding

• Enzyme kinetics: Measure the effects of mutations on catalytic parameters (kcat, KM)

• Substrate specificity analysis: Test activity on various β-1,3-glucan substrates of different lengths and structures

These structural studies would aim to answer key questions about:

• The identity and roles of catalytic residues in the active site

• The structural basis for substrate recognition and binding

• Conformational changes during catalysis

• The mechanism of glycosidic bond hydrolysis

Understanding these structural features would provide a foundation for enzyme engineering efforts aimed at enhancing catalytic efficiency, altering substrate specificity, or improving stability under various conditions.

What are the common challenges in working with recombinant Pinus halepensis Glucan endo-1,3-beta-glucosidase and how can they be addressed?

Table 1: Common Challenges and Troubleshooting Strategies for Recombinant Pinus halepensis Glucan endo-1,3-beta-glucosidase

To ensure reliable results when working with this enzyme, researchers should:

• Validate each new batch by SDS-PAGE to confirm purity (>85%)

• Establish standardized protocols for reconstitution and storage

• Include appropriate controls in all enzymatic assays

• Carefully document storage conditions and freeze-thaw cycles

• Consider the shelf life limitations (6 months for liquid form, 12 months for lyophilized form at -20°C/-80°C)

How can researchers design experiments to investigate the ecological role of Glucan endo-1,3-beta-glucosidase in Pinus halepensis?

Designing experiments to investigate the ecological role of Glucan endo-1,3-beta-glucosidase in Pinus halepensis requires multidisciplinary approaches spanning molecular, physiological, and field-based methods:

Field-based ecological studies:

• Comparative analysis across ecological gradients:

  • Sample Pinus halepensis populations across Mediterranean climate gradients

  • Measure enzyme activity in tissues (needles, bark, roots) and correlate with environmental factors

  • Compare populations with different pathogen pressure or fire history

• Response to natural disturbances:

  • Monitor enzyme expression and activity before and after fire events

  • Assess relationship between enzyme levels and post-fire regeneration capacity

  • Pinus halepensis is known to regenerate vigorously after fire via serotinous cones

Controlled environment experiments:

• Pathogen challenge experiments:

  • Expose seedlings to common fungal pathogens

  • Measure temporal and spatial patterns of enzyme induction

  • Correlate enzyme activity with disease resistance

• Abiotic stress experiments:

  • Apply heat stress treatments (e.g., 40°C for 4h, 50°C for 30 min, and 60°C for 5 min) as described in previous studies

  • Measure enzyme activity alongside other stress-responsive proteins

  • Analyze metabolite changes (particularly sucrose and amino acids like glutamine, glycine, and cysteine)

• Transgenic approaches:

  • Create lines with altered expression of the enzyme (RNAi knockdown or overexpression)

  • Assess consequences for growth, development, and stress tolerance

  • Use somatic embryogenesis as a model system, which has been established for Pinus halepensis

Molecular ecology approaches:

• Population genetics:

  • Analyze genetic variation in glucan endo-1,3-beta-glucosidase genes across populations

  • Use SSR (Simple Sequence Repeat) markers identified in the gene

  • Correlate genetic variants with ecological factors or phenotypic traits

• Transcriptomic analysis:

  • Compare gene expression profiles under different ecological conditions

  • Analyze co-expression networks to identify ecological functions

  • Study promoter activity using reporter gene constructs

These multifaceted approaches would provide comprehensive insights into how this enzyme contributes to the ecological success of Pinus halepensis in Mediterranean ecosystems, particularly in relation to pathogen defense and adaptation to abiotic stresses like fire and heat .

What are promising applications of Pinus halepensis Glucan endo-1,3-beta-glucosidase in biotechnology and plant breeding?

Several promising research directions exist for applications of Pinus halepensis Glucan endo-1,3-beta-glucosidase in biotechnology and plant breeding:

Enhanced disease resistance:

• Development of transgenic plants with optimized expression of glucan endo-1,3-beta-glucosidase genes to enhance fungal pathogen resistance

• Engineering of the promoter regions to achieve precise spatial and temporal expression patterns in response to pathogen attack

• Stacking with other defense-related genes for durable and broad-spectrum resistance

Abiotic stress tolerance:

• Exploration of the enzyme's role in heat stress responses, building on findings that showed altered protein expression patterns under high temperature conditions

• Development of "primed" plants through controlled stress exposure during development, potentially involving glucan endo-1,3-beta-glucosidase regulation

• Engineering of stress-responsive promoter elements to optimize enzyme expression under adverse conditions

Forest management applications:

• Development of molecular markers based on glucan endo-1,3-beta-glucosidase genes for selecting pathogen-resistant Pinus halepensis genotypes

• Application in reforestation programs in Mediterranean regions where the species is native

• Integration with fire management strategies, given the species' ability to regenerate after fire via serotinous cones

Enzyme biotechnology:

• Production of recombinant enzyme for applications in agricultural fungal disease management

• Enzyme engineering to enhance catalytic efficiency, stability, or substrate specificity

• Development of novel detection systems for fungal pathogens based on enzyme-substrate interactions

Fundamental research:

• Further characterization of the enzyme's role in plant-microbe interactions, including potential involvement in symbiotic relationships

• Comparative genomic studies to understand evolutionary adaptations of the enzyme in Mediterranean conifers

• Investigation of potential roles beyond pathogen defense, such as involvement in growth and development processes

These applications would benefit from the continued refinement of production methods for the recombinant enzyme and deeper understanding of its regulatory mechanisms , potentially leading to innovative solutions for sustainable forestry and agriculture.

How might climate change impact the expression and function of Glucan endo-1,3-beta-glucosidase in Mediterranean forest ecosystems?

Climate change is expected to significantly impact Mediterranean forest ecosystems, potentially affecting the expression and function of Glucan endo-1,3-beta-glucosidase in Pinus halepensis through several mechanisms:

Increased temperature stress:

Changes in pathogen pressure:

• Climate change is expected to alter the geographic range and virulence of forest pathogens

• As a key defense enzyme against fungal pathogens, glucan endo-1,3-beta-glucosidase expression and activity may need to adapt to new pathogen challenges

• Changes in precipitation patterns may influence fungal disease incidence, potentially requiring adjusted expression patterns of defense enzymes

Altered fire regimes:

• Mediterranean regions are predicted to experience more frequent and intense wildfires

• Pinus halepensis is adapted to regenerate vigorously after fire via serotinous cones

• Post-fire recovery likely involves complex gene expression changes, potentially including defense enzymes like glucan endo-1,3-beta-glucosidase

• Understanding the enzyme's role in post-disturbance recovery could inform forest management strategies

Potential research approaches:

Understanding these climate change impacts would provide valuable insights for forest management and conservation strategies in Mediterranean ecosystems where Pinus halepensis is a key species .

What are the optimal assay conditions for measuring Pinus halepensis Glucan endo-1,3-beta-glucosidase activity?

Establishing optimal assay conditions is critical for accurately measuring Pinus halepensis Glucan endo-1,3-beta-glucosidase activity. While specific assay conditions for this particular enzyme are not explicitly detailed in the search results, a methodological framework based on similar enzymes can be proposed:

Buffer and pH optimization:

• Test a range of buffer systems (sodium acetate, sodium phosphate, citrate-phosphate) across pH range 4.0-7.0

• Typical optimum pH for plant β-1,3-glucanases is often between 4.5-5.5

• Buffer concentration is typically 50-100 mM

Temperature optimization:

• Assess activity across temperature range 25-60°C

• Consider native habitat of Pinus halepensis (Mediterranean region) when determining physiologically relevant temperature ranges

• Include controls for enzyme stability at each temperature point

Substrate selection and concentration:

• Laminarin (β-1,3-glucan from Laminaria digitata) is commonly used at 0.5-1% (w/v)

• Other potential substrates include pachyman, curdlan, or synthetic chromogenic/fluorogenic substrates

• Determine Km and Vmax by testing substrate concentration ranges (typically 0.1-10 mg/mL)

Assay development considerations:

• Reducing sugar assay:

  • After enzyme reaction, measure released reducing sugars using DNS or Nelson-Somogyi methods

  • Calibrate with glucose standard curve (0-500 μg/mL)

  • Typical reaction times: 15-60 minutes

• Viscometric assay:

  • Measure decrease in viscosity of substrate solution over time

  • Requires specialized viscometer equipment

• Chromogenic/fluorogenic substrate assays:

  • More sensitive but may not perfectly mimic natural substrate interactions

  • Simpler quantification via spectrophotometer or fluorometer

Table 2: Standard Assay Components for Glucan endo-1,3-beta-glucosidase Activity

These parameters should be systematically optimized for the specific recombinant Pinus halepensis enzyme to ensure reproducible and physiologically relevant activity measurements.