Recombinant Thermoanaerobacter thermosulfurogenes Beta-galactosidase (lacZ), partial
Description
Enzyme Characteristics and Source Organism
Thermoanaerobacter thermosulfurogenes is a thermophilic, anaerobic bacterium thriving at high temperatures (60–75°C). Its β-galactosidase (lacZ) belongs to glycosyl hydrolase family 2 or 42, depending on structural homology . The term "partial" indicates that the recombinant enzyme may lack certain domains (e.g., signal peptides or non-catalytic regions) but retains functional activity.
Key features inferred from homologous enzymes:
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Thermostability: Retains activity at 65–85°C, with half-lives exceeding 16 hours at 80°C in related Thermotoga maritima β-galactosidase .
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Substrate Specificity: Hydrolyzes lactose, o-nitrophenyl-β-D-galactopyranoside (oNPG), and synthetic analogs like X-Gal .
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pH Optimum: Typically pH 5.5–7.0, aligning with neutral to mildly acidic environments .
Recombinant Expression and Purification
The lacZ gene is cloned into mesophilic hosts like Escherichia coli for overexpression. Example protocols include:
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Vector Systems: pIN-III-lpp or pACE vectors with inducible promoters (e.g., lac or groES) .
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Tagging: His-tag fusion for affinity chromatography (e.g., Ni-NTA) .
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Purification Steps: Heat treatment (denatures host proteins), ion-exchange chromatography, and gel filtration .
Table 1: Expression and Purification of Related β-Galactosidases
Biochemical Properties
Data from homologous enzymes suggest the following characteristics:
Table 2: Kinetic and Stability Parameters
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Inhibition: Glucose competitively inhibits activity, with K<sub>i</sub> values ~200 mM in Thermoanaerobacter brockii β-glucosidases .
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Metal Dependence: Mn<sup>2+</sup> enhances activity by 30–50% in Thermotoga maritima .
Industrial Applications
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Lactose Hydrolysis: Used in dairy industries to produce lactose-free products .
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Biosensors: Thermostable variants serve as reporters in anaerobic, high-temperature conditions (e.g., Geobacillus β-galactosidase with S-gal substrate) .
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Prebiotic Synthesis: Transglycosylation generates GOS, promoting gut microbiota growth .
Research Gaps and Future Directions
While Thermoanaerobacter thermosulfurogenes β-galactosidase shares features with well-studied homologs, specific data on its:
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Full-length vs. partial structure impacts on activity,
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Crystal structure for rational engineering,
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Performance in bioreactors under industrial conditions, remain uncharacterized in the literature reviewed.
Product Specs
Q&A
Frequently Asked Questions for Researchers Working with Recombinant Thermoanaerobacter thermosulfurogenes Beta-Galactosidase (lacZ), Partial
Advanced Research Questions
How can thermostability of recombinant T. thermosulfurogenes beta-galactosidase be optimized for industrial applications?
Approaches include:
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Site-directed mutagenesis: Target residues near the catalytic site (e.g., Glu389, Tyr429) or subunit interfaces to enhance stability.
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Directed evolution: Screen mutant libraries under high-temperature stress using robotic platforms.
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Additives: Test stabilizing agents (e.g., glycerol, Ca²⁺) during purification .
Data from comparative sequence analysis of thermophilic vs. mesophilic beta-galactosidases suggest that increased hydrophobic interactions and salt bridges contribute to thermostability .
What structural features underlie the enzyme’s activity and stability?
Key structural insights include:
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Catalytic residues: Glu389 and Tyr429 (homologous to Glu461 and Tyr503 in E. coli) , confirmed by mutagenesis to abolish activity .
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Quaternary structure: A homodimer (170 kDa) critical for activity, as disruption of subunit interactions reduces thermostability .
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Conserved regions: Five domains shared with E. coli beta-galactosidase, including the TIM barrel catalytic domain . Use X-ray crystallography or cryo-EM to resolve domain arrangements.
How can discrepancies in activity measurements between studies be resolved?
Common sources of variability and solutions:
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Substrate differences: Compare ONPG vs. lactose hydrolysis rates (Table 1) .
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Assay conditions: Standardize pH (7.0), temperature (60–70°C), and permeabilization methods (toluene vs. BugBuster) .
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Enzyme truncations: Partial sequences may lack regulatory domains; validate constructs via N-terminal sequencing .
Table 1: Beta-Galactosidase Activity Under Varying Conditions
| Condition | Activity (μmol/min/mg) | Source |
|---|---|---|
| 60°C, pH 7.0 (ONPG) | 450 ± 30 | |
| 70°C, pH 7.0 (ONPG) | 380 ± 25 | |
| Toluene-permeabilized | 220 ± 15 | |
| BugBuster-extracted | 310 ± 20 |
Which genetic tools are effective for engineering T. thermosulfurogenes beta-galactosidase?
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Knockout vectors: Use homologous recombination with 1.2 kb homology arms for precise editing .
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Broad-host-range plasmids: Employ vectors like pMTL85151 with terminators validated in Clostridium .
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Reporter systems: Dual enzymatic systems (e.g., GusA + LacZ) enable medium-throughput screening of terminator efficiency .
Methodological Considerations
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Troubleshooting low activity: Check for misfolding by comparing soluble vs. insoluble fractions . Use chaperone co-expression (e.g., GroEL/ES) in E. coli.
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Handling partial sequences: Verify truncation boundaries via alignment with full-length homologs (e.g., residues 1–716 in C. thermosulfurogenes) .
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Data contradiction: Reconcile mismatches in subunit stoichiometry (dimer vs. tetramer) using gel filtration under non-denaturing conditions .
