Characterization of a novel thermostable esterase from Thermus scotoductus SA-01: evidence of a new family of lipolytic esterases.
An esterase, designated EstTs1, was identified and characterized from a genomic library of Thermus scotoductus SA-01 (ATCC 700910). The library was screened in Escherichia coli for lipolytic activity on tributyrin agar plates. A 1.7-kb DNA fragment from a lipolytic positive clone was sequenced and two open reading frames (ORFs) were identified. A 774-bp ORF, designated EstTs1 with an estimated molecular mass of 28.6 kDa, and a 693-bp ORF, designated EstTs2 with an estimated molecular mass of 25.6 kDa, were identified. These two ORFs appear to form part of an operon. Sequence analysis showed that both proteins contained the G-X-S-X-G signature sequence motif present in most esterases and lipases. The deduced amino sequence of EstTs1 was found to display significant sequence identity with putative hydrolase proteins from both Thermus aquaticus Y51MC23 and Thermus thermophilus HB27. Similarly, EstTs2, also displayed significant homology to a second putative hydrolase protein present in the same two organisms. The cloning and characterization of these two ORFs from T. aquaticus Y51MC23 and T. thermophilus strain HB27 encoding putative hydrolase genes have not been reported. E. coli cells harbouring EstTs1 on a multicopy vector produced a clearing zone on tributyrin agar plates, whereas no enzymatic activity was observed for E. coli harbouring EstTs2 on a multicopy vector. EstTs1 displayed optimum activity at pH 7 and 80 degrees C with a half life of 48 h at 70 degrees C.
du Plessis
Erika M
EM
CSIR Biosciences, Box 395, Pretoria, 0001, Gauteng, South Africa. emdplessis@csir.co.za
Berger
Eldie
E
Stark
Therese
T
Louw
Maureen E
ME
Visser
Daniel
D
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006207:Half-Life; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008066:Lipolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004950:Esterases