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Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium.

Guo G, Fang T, Wang C, Huang Y, Tian F, Cui Q, Wang H - Sci Rep (2015)

Bottom Line: The enzymes activity both increased in the presence of Fe(3+), Fe(2+), Cu(2+) and Al(3+) and showed no significant inhibition by other tested metal ions.The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively.As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation.

View Article: PubMed Central - PubMed

Affiliation: State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, 100084, China.

ABSTRACT
Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0-30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe(3+), Fe(2+), Cu(2+) and Al(3+) and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation.

No MeSH data available.


Related in: MedlinePlus

Detection of C23O1 and C23O2 overproduced in pET28a/C23O1 and pMAL-c5X/C23O2.High amounts of 35 kDa were mainly soluble. M: maker;1: pET28a in the precipitation (major amount of insoluble protein); 2: pET28a in supernatant (minor amount of protein); 3: pET28a/C23O1; 4: purified pET28a/C23O1; 5: purified C23O1 after digested; 6: pMAL-c5X; 7: pMAL-c5X/C23O2; 8: purified pMAL-c5X/C23O2; 9: purified C23O2 after digested.
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f2: Detection of C23O1 and C23O2 overproduced in pET28a/C23O1 and pMAL-c5X/C23O2.High amounts of 35 kDa were mainly soluble. M: maker;1: pET28a in the precipitation (major amount of insoluble protein); 2: pET28a in supernatant (minor amount of protein); 3: pET28a/C23O1; 4: purified pET28a/C23O1; 5: purified C23O1 after digested; 6: pMAL-c5X; 7: pMAL-c5X/C23O2; 8: purified pMAL-c5X/C23O2; 9: purified C23O2 after digested.

Mentions: The C23O1 and C23O2 genes were subcloned into pET-28a (+) and pMAL-c5X respectively, and overexpressed in E.coli BL21 (DE3) with 0.5 mM IPTG induction. The recombinant C23Os were purified by chromatography and the purification process was analyzed by SDS-PAGE (Fig. 2). The results indicated that both proteins were correctly synthesized into E. coli host cells and the molecular mass of the two purified proteins were approximately 35 kDa. After digestion with protease, the two purified enzyme showed the activity values up to 136.7 U and 52.3 U respectively. No C23O activity was detected in the control strain BL21 (ED3)-pET-28a (+) and BL21 (ED3)-pMAL-c5X.


Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium.

Guo G, Fang T, Wang C, Huang Y, Tian F, Cui Q, Wang H - Sci Rep (2015)

Detection of C23O1 and C23O2 overproduced in pET28a/C23O1 and pMAL-c5X/C23O2.High amounts of 35 kDa were mainly soluble. M: maker;1: pET28a in the precipitation (major amount of insoluble protein); 2: pET28a in supernatant (minor amount of protein); 3: pET28a/C23O1; 4: purified pET28a/C23O1; 5: purified C23O1 after digested; 6: pMAL-c5X; 7: pMAL-c5X/C23O2; 8: purified pMAL-c5X/C23O2; 9: purified C23O2 after digested.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664950&req=5

f2: Detection of C23O1 and C23O2 overproduced in pET28a/C23O1 and pMAL-c5X/C23O2.High amounts of 35 kDa were mainly soluble. M: maker;1: pET28a in the precipitation (major amount of insoluble protein); 2: pET28a in supernatant (minor amount of protein); 3: pET28a/C23O1; 4: purified pET28a/C23O1; 5: purified C23O1 after digested; 6: pMAL-c5X; 7: pMAL-c5X/C23O2; 8: purified pMAL-c5X/C23O2; 9: purified C23O2 after digested.
Mentions: The C23O1 and C23O2 genes were subcloned into pET-28a (+) and pMAL-c5X respectively, and overexpressed in E.coli BL21 (DE3) with 0.5 mM IPTG induction. The recombinant C23Os were purified by chromatography and the purification process was analyzed by SDS-PAGE (Fig. 2). The results indicated that both proteins were correctly synthesized into E. coli host cells and the molecular mass of the two purified proteins were approximately 35 kDa. After digestion with protease, the two purified enzyme showed the activity values up to 136.7 U and 52.3 U respectively. No C23O activity was detected in the control strain BL21 (ED3)-pET-28a (+) and BL21 (ED3)-pMAL-c5X.

Bottom Line: The enzymes activity both increased in the presence of Fe(3+), Fe(2+), Cu(2+) and Al(3+) and showed no significant inhibition by other tested metal ions.The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively.As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation.

View Article: PubMed Central - PubMed

Affiliation: State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, 100084, China.

ABSTRACT
Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0-30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe(3+), Fe(2+), Cu(2+) and Al(3+) and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation.

No MeSH data available.


Related in: MedlinePlus