Recombinant Mesoplasma florum Enolase (eno)
Description
Introduction to Recombinant Mesoplasma florum Enolase (eno)
Enolase is a 47 kDa enzyme involved in glycolysis and gluconeogenesis, catalyzing the reversible conversion of D-2-phosphoglycerate . In Mesoplasma florum, a near-minimal bacterium, enolase is part of its core metabolic machinery . M. florum is an emerging model organism in systems and synthetic biology because it has a small genome, grows quickly, and isn't harmful . Recombinant M. florum enolase refers to enolase produced through recombinant DNA technology, allowing for detailed studies of its structure, function, and interactions .
Characteristics of Mesoplasma florum
M. florum is characterized by its minimal genome (~800 kb) and rapid growth rate, with a doubling time of approximately 32 minutes at an optimal growth temperature of 34°C . It exhibits four typical bacterial growth phases in rich medium . The bacterium's small size and well-defined growth kinetics make it suitable for comprehensive analyses, including transcriptome and proteome profiling .
Expression and Purification of Recombinant Enolase
Recombinant expression and purification are crucial steps in studying enolase. For example, recombinant Fasciola hepatica enolase (rFhENO) has been expressed in the BL21 ClearColi system, with purification achieved through NiNTA-Agarose affinity chromatography . Such procedures yield highly purified enzyme suitable for structural and functional studies .
Functional Roles and Metabolic Significance
Enolase plays a vital role in the glycolytic pathway of M. florum, with high expression levels observed for metabolic genes involved in glycolysis .
Highly expressed genes in M. florum :
-
L-lactate dehydrogenase (mfl596)
-
Glyceraldehyde-3-phosphate dehydrogenase (mfl578)
-
Phosphoglycerate kinase (mfl577)
These enzymes are essential for energy production and metabolic balance within the bacterium .
Genome-Scale Modeling
Genome-scale metabolic modeling has been used to identify metabolic functions encoded in the M. florum genome . These models integrate experimental data, such as substrate uptake rates and biomass composition, to create functional models like the iJL208 genome-scale model . Such models help predict and explain metabolic behaviors and are validated using growth data on various carbohydrates .
Potential Applications and Further Research
Characterizing recombinant M. florum enolase provides a foundation for synthetic genomics and genome engineering . Understanding its structure-function relationship may lead to biotechnological applications or drug targets . Further research could explore enolase's role in stress response, metabolic regulation, and interactions with other cellular components .
florum Growth Kinetics Analysis
| Parameter | Value |
|---|---|
| Optimal Temperature | 34°C |
| Doubling Time (FCM) | 30.8 ± 2.9 min |
| Doubling Time (CFUs) | 32.7 ± 0.9 min |
| Stationary Phase Cell Concentration | ~1 × 10^10 cells/ml |
Transcriptome Profiling
| Category | Details |
|---|---|
| Average Pearson Correlation | 0.91 (between replicates) |
| CDS with Detectable Expression | 660 (FPKM > 0) |
| mRNA Molecules | Approximately 420 molecules in exponential phase |
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during ordering for customized preparation.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires advance notice and incurs additional charges.
The specific tag type is determined during production. Please inform us of any tag type preferences for prioritized development.
Target Background
Function: Enolase catalyzes the reversible interconversion of 2-phosphoglycerate and phosphoenolpyruvate, playing a crucial role in carbohydrate catabolism via glycolysis.
KEGG: mfl:Mfl468
STRING: 265311.Mfl468
Q&A
Basic Research Questions
-
What is Mesoplasma florum Enolase and what is its primary function in cellular metabolism?
Mesoplasma florum Enolase (eno) is a key glycolytic enzyme found in the near-minimal bacterium M. florum, catalyzing the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP). This bacterium is particularly interesting for systems and synthetic biology due to its small genome (~800 kb), fast growth rate (~32 minutes doubling time at optimal temperature of 34°C), and lack of pathogenic potential . The enolase protein in M. florum is encoded by the gene with Uniprot accession number Q6F0Z7 . Like other enolases, it plays a critical role in energy metabolism, particularly in the glycolytic pathway. The enzyme requires metal ions (typically Mg²⁺) as cofactors for optimal catalytic activity. Methodologically, enolase activity can be assessed by measuring the conversion rate of 2-PGA to PEP using spectrophotometric assays that track changes in absorbance at specific wavelengths.
-
What experimental methods are used to produce and purify recombinant Mesoplasma florum Enolase?
Production of recombinant M. florum Enolase typically employs heterologous expression systems, most commonly E. coli strains optimized for protein expression (e.g., BL21). The methodological approach involves:
-
Cloning the M. florum eno gene (often codon-optimized for the expression host) into an appropriate expression vector with a suitable affinity tag (His6-tag, GST-tag, etc.)
-
Transforming the construct into competent E. coli cells
-
Inducing protein expression using IPTG or auto-induction media
-
Cell lysis using sonication, French press, or chemical methods
-
Purification via affinity chromatography, followed by additional purification steps like ion exchange and/or size exclusion chromatography
-
Validation of purity using SDS-PAGE and Western blotting
Quality control typically includes assessment of enzymatic activity and structural integrity. The FreeGenes resource has created E. coli codon-optimized versions of M. florum genes, including enolase, which are standardized for MoClo assembly , facilitating recombinant expression.
-
-
How is enzymatic activity of Mesoplasma florum Enolase measured in laboratory settings?
The enzymatic activity of M. florum Enolase can be measured using approaches similar to those employed for other bacterial enolases, such as Mycoplasma synoviae enolase . The standard methodology includes:
-
Spectrophotometric assays that track the conversion of 2-PGA to PEP at 240 nm
-
Coupled enzyme assays where PEP production is linked to NADH oxidation via pyruvate kinase and lactate dehydrogenase
-
Determination of kinetic parameters such as Km and Vmax under different conditions
For example, in studies of M. synoviae enolase, researchers determined Km and Vmax values of 1.1 × 10⁻³ M and 0.739 μmol/L/min, respectively . Similar kinetic analysis can be applied to M. florum Enolase to characterize its catalytic efficiency. These experiments typically require optimization of buffer conditions, including pH, ionic strength, and metal ion concentration for maximum enzyme activity.
-
