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Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201
- Source :
- Microbiology (Reading, England). 159(Pt 11)
- Publication Year :
- 2013
-
Abstract
- Strain THI201, a member of the alphaproteobacteria, is a novel thiocyanate (SCN(-))-degrading bacterium isolated from lake water enriched with potassium thiocyanate (KSCN). This bacterium carries the enzyme thiocyanate hydrolase (SCNase) that hydrolyses thiocyanate to carbonyl sulfide and ammonia. Characterization of both native and recombinant SCNase revealed properties different from known SCNases regarding subunit structure and thermostability: SCNase of strain THI201 was composed of a single protein and thermostable. We cloned and sequenced the corresponding gene and determined a protein of 457 amino acids of molecular mass 50 267 Da. Presence of a twin-arginine (Tat) signal sequence of 32 amino acids was found upstream of SCNase. The deduced amino acid sequence of SCNase showed 83% identity to that of a putative uncharacterized protein of Thiobacillus denitrificans ATCC 25259, but no significant identity to those of three subunits of SCNase from Thiobacillus thioparus strain THI115. The specific activities of native and recombinant enzyme were 0.32 and 4-15 µmol min(-1) (mg protein)(-1), respectively. The maximum activity of SCNase was found in the temperature range 30-70 °C. The thiocyanate-hydrolysing activity in both enzymes was decreased by freeze-thawing, although 25-100% of the activity of recombinant protein could be retrieved by treating the enzyme at 60 °C for 15 min. Furthermore, both native and recombinant enzymes retained the activity after pre-treatment of the protein solution at temperatures up to 70 °C.
- Subjects :
- DNA, Bacterial
Thiocyanate hydrolase
Hydrolases
Protein subunit
ved/biology.organism_classification_rank.species
Molecular Sequence Data
Sulfur Oxides
Gene Expression
Biology
Protein Sorting Signals
Microbiology
Thiobacillus
chemistry.chemical_compound
Ammonia
Potassium thiocyanate
Enzyme Stability
Amino Acid Sequence
Cloning, Molecular
Peptide sequence
Biotransformation
Thermostability
Alphaproteobacteria
chemistry.chemical_classification
Thiocyanate
Base Sequence
Sequence Homology, Amino Acid
ved/biology
Temperature
Sequence Analysis, DNA
Molecular biology
Recombinant Proteins
Amino acid
Molecular Weight
Lakes
Biochemistry
chemistry
Thiocyanates
Subjects
Details
- ISSN :
- 14652080
- Volume :
- 159
- Issue :
- Pt 11
- Database :
- OpenAIRE
- Journal :
- Microbiology (Reading, England)
- Accession number :
- edsair.doi.dedup.....9bb4d950986a554c68e85af50b089d66