Article

Characterization of a novel thermostable esterase from Thermus scotoductus SA-01: evidence of a new family of lipolytic esterases.

CSIR Biosciences, Box 395, Pretoria, 0001, Gauteng, South Africa.
Current Microbiology (impact factor: 1.82). 12/2009; 60(4):248-53. DOI:10.1007/s00284-009-9533-5 pp.248-53
Source: PubMed

ABSTRACT 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.

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Keywords

1.7-kb DNA fragment
 
deduced amino sequence
 
display significant sequence identity
 
E. coli cells harbouring EstTs1
 
E. coli harbouring EstTs2
 
Escherichia coli
 
estimated molecular mass
 
form part
 
G-X-S-X-G signature sequence motif present
 
lipolytic positive clone
 
open reading frames
 
proteins
 
putative hydrolase proteins
 
second putative hydrolase protein present
 
Sequence analysis
 
T. thermophilus strain HB27 encoding putative hydrolase genes
 
Thermus aquaticus Y51MC23
 
Thermus scotoductus SA-01
 
tributyrin agar plates
 
two ORFs