Characterization of a Recombinant Thermostable Dehalogenase Isolated from the Hot Spring Thermophile Sulfolobus tokodaii

Bachas-Daunert, Philip G; Law, Stacy A.; Wei, Yinan

doi:10.1007/s12010-009-8589-9

Cited by 8 publications

(10 citation statements)

References 31 publications

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“…Characterisation of this dehalogenase suggested that it is maximally active at 60 °C (Rye et al 2009 ). L-HAD tolerates pH environments between 4 and 10, and remains fully active after incubation at 70 °C for 4 h (Bachas-Daunert et al 2009 ).…”

Section: Introductionmentioning

confidence: 99%

l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1

2016

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l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1 is a stereospecific enzyme that acts exclusively on l-isomers of 2-chloropropionate and dichloroacetate. The amino acid sequence of this enzyme is substantially different from those of other l-specific dehalogenases produced by other organisms. DehL has not been crystallised, and hence its three-dimensional structure is unavailable. Herein, we review what is known concerning DehL and tentatively identify the amino acid residues important for catalysis based on a comparative structural and sequence analysis with well-characterised l-specific dehalogenases.

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Section: Introductionmentioning

confidence: 99%

l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1

2016

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“…This enzyme belongs to the L -2-halogen acid (L-2-HAD) superfamily and is vital for industrial applications because of its efficient catalytic activity, stereoselectivity, thermal stability and tolerance to a wide pH range. As a monomer globin, ST2570 was a suitable model enzyme for testing site-specific covalent immobilization [19].…”

Section: Introductionmentioning

confidence: 99%

Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

et al. 2016

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Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

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“…Previously published studies also suggest that the K m values at these temperatures are comparable. 32,36 Unfortunately, global heating of the system, which includes the hydrogel and surrounding environment, is highly energy inefficient and can result in unwanted side effects. This is especially true when applying the materials for selective functionality in a complex matrix.…”

Section: Resultsmentioning

confidence: 99%

“…The reaction results in the stereospecific dehalogenation of CPA and formation free chloride, which can be measured by a chloride ion-selective electrode (ISE). L-2-HAD ST has optimal activity at pH 9.5 and 70 °C and maintains its activity when immobilized on a solid matrix . The demonstration of the long-term activity of the enzyme in solution, as well as its ability to work under extreme conditions and retain activity upon immobilization, allowed us to formulate the hypothesis that L-2-HAD ST can maintain its enzymatic activity after chemical conjugation to acrylic acid monomers and polymerization into a hydrogel network.…”

Section: Resultsmentioning

confidence: 99%

Nanoparticle-Mediated Remote Control of Enzymatic Activity

Knecht

Ali²,

Wei

et al. 2012

ACS Nano

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Nanomaterials have found numerous applications as tunable, remotely controlled platforms for drug delivery, hyperthermia cancer treatment, and various other biomedical applications. The basis for the interest lies in their unique properties achieved at the nanoscale that can be accessed via remote stimuli. These properties could then be exploited to simultaneously activate secondary systems that are not remotely actuatable. In this work, iron oxide nanoparticles are encapsulated in a bisacrylamide-crosslinked polyacrylamide hydrogel network along with a model dehalogenase enzyme, L-2-HADST. This thermophilic enzyme is activated at elevated temperatures and has been shown to have optimal activity at 70 °C. By exposing the Fe3O4 nanoparticles to a remote stimulus, an alternating magnetic field (AMF), enhanced system heating can be achieved, thus remotely activating the enzyme. The internal heating of the nanocomposite hydrogel network in the AMF results in a 2-fold increase in enzymatic activity as compared to the same hydrogel heated externally in a water bath, suggesting that the internal heating of the nanoparticles is more efficient than the diffusion limited heating of the water bath. This system may prove useful for remote actuation of biomedical and environmentally relevant enzymes and find applications in a variety of fields.

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Characterization of a Recombinant Thermostable Dehalogenase Isolated from the Hot Spring Thermophile Sulfolobus tokodaii

Cited by 8 publications

References 31 publications

l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1

l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1

Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

Nanoparticle-Mediated Remote Control of Enzymatic Activity

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