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Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus.

Leis B, Angelov A, Mientus M, Li H, Pham VT, Lauinger B, Bongen P, Pietruszka J, Gonçalves LG, Santos H, Liebl W - Front Microbiol (2015)

Bottom Line: We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli.In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only.Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Technische Universität München Freising, Germany.

ABSTRACT
Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

No MeSH data available.


Related in: MedlinePlus

Characterization of the purified esterase-active proteins EstA2 and EstB1. (A) Substrate specificity of various acyl chain length pNP-esters. (B) Temperature and (C) pH optimum. The assays were performed in 50 mM phosphate buffer (pH 6–7) or 50 mM Tris-HCl buffer (pH 7.2–8.8) under optimal activity parameters (80°C for EstA2 and 75°C for EstB1, respectively) against 1.25 mM pNP-butyrate. 0.065–1.3 μg of purified enzyme was used for the assays. Data represents average values and standard deviations (error bars).
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Figure 3: Characterization of the purified esterase-active proteins EstA2 and EstB1. (A) Substrate specificity of various acyl chain length pNP-esters. (B) Temperature and (C) pH optimum. The assays were performed in 50 mM phosphate buffer (pH 6–7) or 50 mM Tris-HCl buffer (pH 7.2–8.8) under optimal activity parameters (80°C for EstA2 and 75°C for EstB1, respectively) against 1.25 mM pNP-butyrate. 0.065–1.3 μg of purified enzyme was used for the assays. Data represents average values and standard deviations (error bars).

Mentions: The characterization of the recombinant enzymes revealed similar substrate preferences for short acyl chain substrates (pNP-C3 > C4 > C5 > C6 > C8) as the ones observed in T. thermophilus, while activities on long-chain substrates (over C10) were comparably low (Figure 3). Under the assay conditions used here, optimum activity of EstA2 was observed around 80°C and pH 8.0, while EstB1 was most active at 75°C and pH 8.0. The specific activities of both enzymes on para-nitrophenyl butyrate were 44.05 ± 2.06 U/mg protein for EstA2 and 4.13 ± 0.71 U/mg for EstB1. The influence of several additives, NaCl, KCl, CaCl2, MgCl2, EDTA, and PMSF at 1–10 mM final concentration was negligible. Our data suggests both proteins to be esterases rather than lipases due to their characteristic substrate specificities.


Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus.

Leis B, Angelov A, Mientus M, Li H, Pham VT, Lauinger B, Bongen P, Pietruszka J, Gonçalves LG, Santos H, Liebl W - Front Microbiol (2015)

Characterization of the purified esterase-active proteins EstA2 and EstB1. (A) Substrate specificity of various acyl chain length pNP-esters. (B) Temperature and (C) pH optimum. The assays were performed in 50 mM phosphate buffer (pH 6–7) or 50 mM Tris-HCl buffer (pH 7.2–8.8) under optimal activity parameters (80°C for EstA2 and 75°C for EstB1, respectively) against 1.25 mM pNP-butyrate. 0.065–1.3 μg of purified enzyme was used for the assays. Data represents average values and standard deviations (error bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389547&req=5

Figure 3: Characterization of the purified esterase-active proteins EstA2 and EstB1. (A) Substrate specificity of various acyl chain length pNP-esters. (B) Temperature and (C) pH optimum. The assays were performed in 50 mM phosphate buffer (pH 6–7) or 50 mM Tris-HCl buffer (pH 7.2–8.8) under optimal activity parameters (80°C for EstA2 and 75°C for EstB1, respectively) against 1.25 mM pNP-butyrate. 0.065–1.3 μg of purified enzyme was used for the assays. Data represents average values and standard deviations (error bars).
Mentions: The characterization of the recombinant enzymes revealed similar substrate preferences for short acyl chain substrates (pNP-C3 > C4 > C5 > C6 > C8) as the ones observed in T. thermophilus, while activities on long-chain substrates (over C10) were comparably low (Figure 3). Under the assay conditions used here, optimum activity of EstA2 was observed around 80°C and pH 8.0, while EstB1 was most active at 75°C and pH 8.0. The specific activities of both enzymes on para-nitrophenyl butyrate were 44.05 ± 2.06 U/mg protein for EstA2 and 4.13 ± 0.71 U/mg for EstB1. The influence of several additives, NaCl, KCl, CaCl2, MgCl2, EDTA, and PMSF at 1–10 mM final concentration was negligible. Our data suggests both proteins to be esterases rather than lipases due to their characteristic substrate specificities.

Bottom Line: We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli.In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only.Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Technische Universität München Freising, Germany.

ABSTRACT
Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

No MeSH data available.


Related in: MedlinePlus