title: Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201.
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.
authors.affiliation: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Adeeba
authors.initials: A
authors.lastname: Hussain
authors.affiliation: Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Saito
authors.affiliation: Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Sekine
authors.affiliation: Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Misuzu
authors.initials: M
authors.lastname: Nameki
authors.affiliation: Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Matsushita
authors.affiliation: Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Katayama
mesh_terms: D020561:Alphaproteobacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D001483:Base Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D060106:Lakes / Q000382:microbiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013461:Sulfur Oxides / Q000378:metabolism; D013696:Temperature; D013855:Thiobacillus / Q000201:enzymology / Q000235:genetics; D013861:Thiocyanates / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D013461:Sulfur Oxides; D013861:Thiocyanates; D000641:Ammonia; C010063:carbonyl sulfide; D006867:Hydrolases; C074369:thiocyanate hydrolase; C031760:thiocyanate