Journal
ENZYME AND MICROBIAL TECHNOLOGY
Volume 42, Issue 5, Pages 381-388Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2008.01.009
Keywords
Streptomyces coelicolor; lipases; over-expression
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The sequencing of the genome of Streptomyces coelicolor A3(2) identified more than 50 putative esterase genes and two of them encode secreted putative hydrolases showing sequence similarity to the lipase from Streptomyces rimosus [Vujaklija D, Schroder W, Abramic M, Zou P Lescic I, Franke P, et al. A novel steptomycete lipase: cloning, sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene. Arch Microbiol 2002;178:124-301. In this report, we present the cloning, expression and purification, as well as the characterization of these two hydrolases. Spectrophotometric analysis of the purified enzymes with chromogenic substrate p-nitrophenyl (p-NP)-palmitate showed that the enzyme SCO1725 (SCI11.14c) was active in a pH range from 7.5 to 9, whereas SCO7513 (SC25F8.05) activity was higher at more alkaline pH (8.5-10). The activity of the former enzyme was highest between 20 and 30 degrees C, while the second showed maximum activity at temperatures ranging from 45 to 55 degrees C. Enzymatic assays with various esters of p-NP showed a preference mid-length acyl chain (C-10-C-12) for SCO1725, and long chain fatty acids (C-16-C-18) for SC07513. The impact of various divalent metal ions, PMSF, and DTT on lipase activity indicated that Ag2+ strongly inhibited both enzymes. The cations Ca2+, Mg2+, Co2+ and Cu2+ did not significantly reduce the lipolytic activity of SCO1725 and SCO7513, except for Co2+ and Cu2+ which partially inhibited SCO7513 at a concentration of 10 mM. DTF inhibited activity of SCO1725 but not that of SCO7513. Both hydrolases were resistant to PMSF inhibition, suggesting the presence of a hydrophobic lid hindering access to the catalytic site. However, when calcium was combined with Triton X-100 in the incubation mixture, inhibition occurred, which suggests that lid opening may be induced in the absence of a substrate. Finally, these results based on genome mining of Streptomyces chromosome provide substantial information for the classification of both putative hydrolases as true lipases (EC 3.1.1.3). (C)d 2008 Elsevier Inc. All rights reserved.
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