Recombinant Kluyveromyces lactis Beta-galactosidase (LAC4), partial
Description
2.1. Escherichia coli Expression
The LAC4 gene has been successfully expressed in E. coli as a soluble His-tagged enzyme :
| Parameter | Detail |
|---|---|
| Subunit molecular mass | 118 kDa |
| Oligomerization state | Multimeric (dimer predominant) |
| Metal ion requirement | Mn²⁺ |
| Imidazole sensitivity | Inactivated above 50 mM |
2.2. K. lactis Promoter Engineering
The native LAC4 promoter (P<sub>LAC4</sub>) was modified to eliminate unintended expression in E. coli while maintaining functionality in yeast :
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Pribnow box mutations:
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Integrative vector pKLAC1: Enables cloning of toxic genes (e.g., bovine enterokinase) in E. coli without leakage .
3.1. Kinetic Parameters2
| Substrate | K<sub>m</sub> (mM) | V<sub>max</sub> (μmol·min⁻¹·mg⁻¹) |
|---|---|---|
| oNPG | 1.5 | 560 |
| Lactose | 20 | 570 |
3.2. Optimal Conditions23
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Temperature: 37–40°C
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pH: 7.0
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Thermostability: Reduced in suppressor-revertant strains (e.g., 50% activity loss at 45°C) .
Secretion Strategies
Partial secretion (≤30% extracellular activity) was achieved using:
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Signal peptides: K. lactis killer toxin or S. cerevisiae α-factor pre-pro domains .
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N-terminal mutagenesis: Enhanced secretion efficiency by modifying hydrophobic regions .
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Challenges: High molecular weight (∼470 kDa tetramer) and oligomerization limit full secretion .
5.1. Strain Optimization5
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Chromosomal integration: LAC4 promoter-driven systems enable stable expression without antibiotic selection.
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β-Galactosidase as a biosensor: Enzyme activity correlates with heterologous gene transcription rates .
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High-density fermentation: Engineered strains (e.g., VAK367-D4) resist lysis, enabling prolonged protein accumulation .
5.2. Case Studies
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Human serum albumin (HSA): Secreted at 150 mg/L using α-mating factor fusions .
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Bovine enterokinase: Active enzyme produced in K. lactis via pKLAC1, circumventing E. coli toxicity .
Research Implications
Product Specs
Q&A
Experimental Design for Studying Recombinant Beta-galactosidase Activity
Q: How can I design an experiment to study the activity of recombinant beta-galactosidase from Kluyveromyces lactis in different conditions? A: To study the activity of recombinant beta-galactosidase, you should consider varying substrate concentrations (e.g., lactose), temperatures, and pH levels. Additionally, assess the effects of inhibitors like galactose and activators such as glucose. Use kinetic models like the Michaelis-Menten equation as a baseline and adjust based on experimental data .
Genetic Basis of Beta-galactosidase in Kluyveromyces lactis
Q: What genetic locus is responsible for encoding beta-galactosidase in Kluyveromyces lactis, and how does it affect enzyme activity? A: The LAC4 locus encodes beta-galactosidase in Kluyveromyces lactis. Mutations in this locus significantly reduce enzyme activity, and the level of activity is directly proportional to the number of LAC4 gene copies .
Optimization of Reaction Conditions for Beta-galactosidase
Q: What are the optimal conditions for maximizing the hydrolytic and transgalactosylation activities of beta-galactosidase from Kluyveromyces lactis? A: Optimal conditions typically involve a pH range of 4.4 to 7.0 and temperatures between 37°C to 50°C. The presence of ions like Mn²⁺ can enhance activity. For transgalactosylation reactions, higher temperatures may favor product yield .
Data Contradiction Analysis in Kinetic Studies
Q: How can I address discrepancies between experimental data and predicted kinetic models for beta-galactosidase activity? A: Discrepancies often arise from oversimplification of kinetic models. Consider incorporating additional factors such as substrate inhibition (e.g., galactose at low concentrations) and activation effects (e.g., glucose at low concentrations). Develop new models that account for these complexities to better fit experimental data .
Immobilization Techniques for Beta-galactosidase
Q: What immobilization methods can enhance the stability and reusability of beta-galactosidase from Kluyveromyces lactis? A: Techniques such as calcium alginate immobilization and enzyme attachment to supports like chitosan-glutaraldehyde can improve enzyme stability and allow for repeated use in biocatalytic processes .
Advanced Research Questions: Genetic Engineering
Q: How can genetic engineering be used to improve the thermal stability and catalytic efficiency of beta-galactosidase from Kluyveromyces lactis? A: Genetic engineering strategies involve modifying the LAC4 gene to enhance thermal resistance and catalytic activity. This can be achieved by expressing the enzyme in thermally stable hosts or through site-directed mutagenesis to alter key amino acids involved in enzyme stability and activity .
Comparative Analysis with Other Beta-galactosidases
Q: How does the beta-galactosidase from Kluyveromyces lactis compare to those from other sources in terms of hydrolytic and transgalactosylation activities? A: Kluyveromyces lactis beta-galactosidase exhibits higher hydrolytic activity compared to some other sources like Aspergillus oryzae and Bacillus circulans, but lower transgalactosylation activity. This makes it suitable for applications requiring efficient lactose hydrolysis .
Biotechnological Applications
Q: What are some potential biotechnological applications of recombinant beta-galactosidase from Kluyveromyces lactis? A: Applications include lactose hydrolysis in dairy products, production of galacto-oligosaccharides (GOS) as prebiotics, and synthesis of other valuable oligosaccharides through transgalactosylation reactions .
Analytical Techniques for Beta-galactosidase Activity
Q: What analytical methods can be used to assess the activity and stability of beta-galactosidase from Kluyveromyces lactis? A: Techniques such as spectrophotometry (using substrates like o-nitrophenyl-β-D-galactoside), two-dimensional acrylamide gel electrophoresis, and Western blotting can be employed to analyze enzyme activity and stability .
