Recombinant Acinetobacter sp. PKHD-type hydroxylase ACIAD0531 (ACIAD0531)
Description
Introduction
Recombinant Acinetobacter sp. PKHD-type hydroxylase ACIAD0531 (ACIAD0531) refers to a hydroxylase enzyme, which is a type of oxygenase that catalyzes the incorporation of one or two atoms of oxygen into a substrate . Specifically, ACIAD0531 originates from Acinetobacter sp., a genus of bacteria known for its diverse metabolic capabilities, including the degradation of various organic compounds . The term "recombinant" indicates that the enzyme is produced through recombinant DNA technology, where the gene encoding the hydroxylase is cloned and expressed in a host organism, such as Escherichia coli, to produce large quantities of the enzyme for research and industrial applications . PKHD-type hydroxylases are characterized by their structural similarity to polycystic kidney and hepatic disease 1 (PKHD1) protein, although their precise function and substrates may vary . The enzyme ACIAD0531 is identified by the locus tag ACIAD0531, a unique identifier in the genome of Acinetobacter sp .
Alkane Hydroxylases in Acinetobacter sp.
Alkane hydroxylases are a class of enzymes that catalyze the initial step in the oxidation of alkanes, converting them into corresponding alcohols . In Acinetobacter sp. strain ADP1, the alkane hydroxylase is encoded by the alkM gene, which is essential for growth on alkanes as the sole carbon source . The alkM gene is located next to the alkR gene, which encodes a transcriptional regulator that controls the expression of alkM . AlkM shows sequence homologies with other bacterial integral-membrane hydrocarbon hydroxylases, suggesting it belongs to a novel protein family .
Cloning and Expression of Hydroxylases
To produce recombinant hydroxylases like ACIAD0531, researchers typically clone the gene encoding the enzyme into an expression vector, which is then introduced into a host organism such as E. coli . The expression vector contains regulatory elements that control the expression of the cloned gene, allowing for high-level production of the recombinant enzyme . The recombinant enzyme can then be purified using various biochemical techniques, such as affinity chromatography, for further study and application . For example, genes encoding p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii were cloned and expressed in E. coli, and the recombinant enzymes were purified and characterized .
Potential Applications
Recombinant hydroxylases like ACIAD0531 have a wide range of potential applications, including:
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Bioremediation: Hydroxylases can be used to degrade pollutants and clean up contaminated environments .
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Biocatalysis: Hydroxylases can be used as biocatalysts in industrial processes to produce valuable chemicals and pharmaceuticals .
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Drug Discovery: Hydroxylases can be used as targets for drug discovery, particularly in the development of new antibiotics to combat bacterial infections .
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Synthetic Biology: Hydroxylases can be engineered to create novel metabolic pathways and produce valuable products .
Table Summarizing Key Information
| Feature | Description |
|---|---|
| Enzyme Name | Recombinant Acinetobacter sp. PKHD-type hydroxylase ACIAD0531 |
| Origin | Acinetobacter sp. |
| Enzyme Type | Hydroxylase (Oxygenase) |
| Locus Tag | ACIAD0531 |
| Production Method | Recombinant DNA technology |
| Potential Applications | Bioremediation, biocatalysis, drug discovery, synthetic biology |
| Related Gene (Alkane example) | alkM (alkane hydroxylase-encoding gene in Acinetobacter sp. strain ADP1) |
| Regulatory Gene (Alkane) | alkR (transcriptional regulator of alkM in Acinetobacter sp. strain ADP1) |
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The specific tag will be determined during production. If you require a particular tag, please inform us, and we will prioritize its development.
Q&A
How should ACIAD0531 be stored and handled to maintain optimal stability?
The stability of ACIAD0531 depends on several factors including storage temperature, buffer composition, and exposure to freeze-thaw cycles. For optimal preservation:
| Storage Form | Recommended Temperature | Shelf Life | Notes |
|---|---|---|---|
| Lyophilized | -20°C to -80°C | 12 months | Preferred for long-term storage |
| Liquid | -20°C to -80°C | 6 months | Aliquot to avoid freeze-thaw cycles |
| Working solution | 4°C | Up to one week | For immediate experimental use |
Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol (final concentration) recommended for aliquoting and long-term storage. Repeated freezing and thawing should be avoided to prevent protein denaturation and activity loss .
What are the optimized conditions for recombinant expression of ACIAD0531 in E. coli?
While specific optimization studies for ACIAD0531 expression are not directly documented in the search results, evidence from similar recombinant protein expression studies suggests the following parameters may be effective starting points:
| Parameter | Optimized Condition | Rationale |
|---|---|---|
| Expression vector | pET28a or similar | Common for high-level expression of recombinant proteins |
| Host strain | E. coli BL21(DE3) | Deficient in proteases, suitable for recombinant protein expression |
| Media | Terrific Broth (TB) | Yields higher specific activity compared to LB media |
| IPTG concentration | 0.5 mM | Balance between protein yield and specific activity |
| Induction temperature | 25°C | Lower temperatures often improve protein solubility |
| Shaking speed | 150 rpm | Moderate aeration for optimal enzyme activity |
| Induction period | 18 hours | Extended period for maximum protein production |
This optimization framework is based on studies with similar recombinant enzymes where the highest enzyme specific activity was achieved at lower temperatures (25°C) despite higher protein expression levels at 37°C . The specific activity of recombinant enzymes was demonstrated to be higher in TB media (1.65±0.1 μmol·min⁻¹·mg⁻¹) compared to LB media (1.35±0.1 μmol·min⁻¹·mg⁻¹) .
What experimental design approach is most effective for optimizing multiple parameters in ACIAD0531 expression?
Split-plot experimental design provides an efficient framework for optimizing ACIAD0531 expression parameters, particularly when dealing with both hard-to-change and easy-to-change factors:
| Design Element | Application to ACIAD0531 Expression | Statistical Consideration |
|---|---|---|
| Whole plots | Host strain selection, vector construction, media type | Requires fewer level changes, analyzed at the whole-plot level |
| Sub-plots | IPTG concentration, temperature, induction time, shaking speed | Can be varied within whole plots, analyzed at the sub-plot level |
| Randomization | Restricted randomization at whole-plot level, complete randomization at sub-plot level | Accounts for different experimental unit sizes |
| Analysis | Mixed-model ANOVA | Addresses the two error terms from different randomization levels |
The split-plot design is particularly valuable when optimizing recombinant protein expression due to the practical constraints of changing certain experimental factors. This approach enhances efficiency by optimizing the allocation of resources while maintaining statistical validity .
What are the established methods for measuring ACIAD0531 enzymatic activity?
As a PKHD-type hydroxylase (EC 1.14.11.-), ACIAD0531 activity measurement requires detection of hydroxylation reactions. Standard methodological approaches include:
| Assay Type | Principle | Advantages | Limitations |
|---|---|---|---|
| Spectrophotometric | Measures changes in absorbance associated with substrate consumption or product formation | Real-time monitoring, relatively simple equipment requirements | May lack specificity, potential interference from sample components |
| HPLC-based | Separates and quantifies substrates and products | High specificity, quantitative analysis of multiple reaction components | Requires specialized equipment, not real-time |
| Oxygen consumption | Measures O₂ uptake during catalysis using oxygen electrode | Direct measurement of co-substrate utilization, real-time analysis | Requires specialized equipment, potential interference from other O₂-consuming reactions |
| Coupled enzyme assays | Links hydroxylase activity to secondary enzymatic reactions with measurable outputs | Can amplify signal for low-activity enzymes | Complexity in optimization, potential for false results due to effects on coupled enzymes |
For optimal results, method selection should consider the specific substrate preferences, reaction mechanisms, and experimental contexts relevant to ACIAD0531.
How does ACIAD0531 function in the context of aromatic compound degradation pathways?
While direct evidence for ACIAD0531's specific role is limited in the provided search results, analysis of related Acinetobacter enzymes suggests potential involvement in aromatic compound degradation pathways:
| Pathway Component | Potential Role of ACIAD0531 | Related Enzymes in Acinetobacter |
|---|---|---|
| Toluene degradation | Hydroxylation steps leading to benzoate formation | Hydroxylases involved in ring modification |
| Xylene metabolism | Conversion of methyl groups or ring hydroxylation | Enzymes catalyzing formation of methylbenzoate intermediates |
| Aromatic ring cleavage | Introduction of hydroxyl groups to facilitate ring opening | Dioxygenases that incorporate oxygen into aromatic structures |
Research with Acinetobacter baumannii has demonstrated involvement in degradation pathways where hydroxylases play critical roles in transforming aromatic compounds . The specific substrates and reaction mechanisms of ACIAD0531 require further characterization to precisely position this enzyme within these metabolic networks.
How can protein engineering approaches be applied to enhance ACIAD0531 catalytic properties?
Protein engineering strategies offer significant potential for enhancing ACIAD0531's properties for research applications:
| Engineering Approach | Methodology | Expected Outcomes | Experimental Considerations |
|---|---|---|---|
| Rational design | Site-directed mutagenesis targeting active site residues based on structural predictions | Altered substrate specificity, improved catalytic efficiency | Requires structural knowledge or reliable homology models |
| Directed evolution | Random mutagenesis followed by screening/selection for improved variants | Novel properties not predictable by rational approaches | Requires high-throughput screening capability |
| Semi-rational design | Targeting specific regions (loops, domains) with focused mutagenesis libraries | Combined benefits of rational and random approaches | Balances exploration with guided design |
| Domain swapping | Replacement of functional domains with those from related enzymes | Hybrid enzymes with combined properties | May cause structural instability if domains are incompatible |
Similar approaches have successfully enhanced stability and catalytic properties in other therapeutic enzymes, as demonstrated with the Anabaena variabilis phenylalanine ammonia lyase (AvPAL) where mutations improved stability properties while maintaining catalytic function .
What analytical techniques should be employed for comprehensive characterization of ACIAD0531 structure-function relationships?
Understanding the relationship between ACIAD0531 structure and function requires integration of multiple analytical approaches:
| Analytical Technique | Information Provided | Application to ACIAD0531 Research |
|---|---|---|
| X-ray crystallography | High-resolution static structure | Identification of active site architecture, substrate binding pockets |
| Circular dichroism (CD) | Secondary structure content and stability | Monitoring structural changes under varying conditions (pH, temperature) |
| Site-directed mutagenesis | Functional importance of specific residues | Systematic probing of potential catalytic and substrate-binding residues |
| Hydrogen-deuterium exchange mass spectrometry (HDX-MS) | Protein dynamics and conformational changes | Investigating structural flexibility and solvent accessibility |
| Molecular dynamics simulations | Time-dependent structural behavior | Predicting conformational changes during substrate binding and catalysis |
| Substrate specificity profiling | Enzyme preference for different substrates | Defining the substrate scope and potential biological functions |
A comprehensive characterization would integrate these methodologies to develop a complete understanding of how ACIAD0531's structure determines its catalytic behavior and substrate preferences.
What are common challenges in recombinant ACIAD0531 expression and how can they be addressed?
Researchers working with recombinant ACIAD0531 may encounter several technical challenges:
| Challenge | Possible Causes | Recommended Solutions |
|---|---|---|
| Low expression yield | Codon bias, protein toxicity, transcriptional/translational inefficiency | Codon optimization, use of specialized host strains, lower induction temperature (25°C instead of 37°C), optimization of IPTG concentration (0.5 mM) |
| Inclusion body formation | Rapid overexpression, improper folding, hydrophobic interactions | Reduced induction temperature, co-expression with chaperones, addition of solubility enhancers, use of solubility tags |
| Loss of enzymatic activity | Improper folding, missing cofactors, oxidation of critical residues | Addition of cofactors (Fe²⁺), reducing agents, optimization of buffer conditions |
| Protein aggregation during storage | Freeze-thaw cycles, improper buffer conditions | Addition of glycerol (5-50%), storage as aliquots, optimization of buffer composition |
| Batch-to-batch variability | Inconsistent expression conditions, purification procedures | Implementation of standardized protocols, detailed documentation of conditions, quality control measures |
How can experimental design address contradictory optimization parameters for ACIAD0531?
When optimizing ACIAD0531 expression and activity, researchers often encounter trade-offs between different parameters:
| Parameter Conflict | Experimental Observation | Resolution Strategy |
|---|---|---|
| Protein yield vs. specific activity | Higher IPTG concentration (1 mM) maximizes yield but moderate concentration (0.5 mM) optimizes activity | Prioritize based on research goals; consider two-stage optimization |
| Expression temperature effect | 37°C maximizes expression level but 25°C yields higher specific activity | Use lower temperature for functional studies, higher temperature when protein quantity is critical |
| Induction duration | Longer induction (18h) increases yield but may reduce specific activity at higher temperatures | Combine longer induction with lower temperature to balance yield and activity |
| Aeration level (shaking speed) | Higher speeds increase growth rate but moderate speeds (150 rpm) optimize enzyme activity | Use moderate shaking speed during induction phase |
A split-plot experimental design approach is particularly valuable for resolving these conflicts by allowing systematic investigation of interaction effects between different parameters while accounting for the practical constraints of changing certain factors . This approach enables researchers to develop optimized protocols that balance competing objectives based on specific research requirements.
