Limits...
Properties of a newly identified esterase from Bacillus sp. K91 and its novel function in diisobutyl phthalate degradation.

Ding J, Wang C, Xie Z, Li J, Yang Y, Mu Y, Tang X, Xu B, Zhou J, Huang Z - PLoS ONE (2015)

Bottom Line: In particular, genes involved in the hydrolysis of these compounds were not conclusively identified.Two intermediate products were identified, and a potential biodegradation pathway was proposed.Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

View Article: PubMed Central - PubMed

Affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, Yunnan, China; Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming 650500, Yunnan, China.

ABSTRACT
The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ρ-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45 °C, with ρ-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

No MeSH data available.


Related in: MedlinePlus

The results of HPLC analyses of catabolic intermediates.(A) DiBP, MiBP and PTH mixture for the standard sample. (B) The results of DiBP degradation after incubation with CarEW. (C) Reaction of MiBP with 10 mM citric acid-Na2HPO4 solution (pH 7.5) buffer instead of CarEW. (D) The results of MiBP degradation after incubation with CarEW.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352063&req=5

pone.0119216.g006: The results of HPLC analyses of catabolic intermediates.(A) DiBP, MiBP and PTH mixture for the standard sample. (B) The results of DiBP degradation after incubation with CarEW. (C) Reaction of MiBP with 10 mM citric acid-Na2HPO4 solution (pH 7.5) buffer instead of CarEW. (D) The results of MiBP degradation after incubation with CarEW.

Mentions: The products of the reaction catalyzed by CarEW were identified by comparison to the mass spectra (m/z) of reaction with no enzyme using ESI-MS. Firstly, a full ion scan using the DiBP mixture without the enzyme CarEW as the control (Fig. 5) was performed. In Fig. 5B, we detected DiBP (m/z 301) and two other possible products PTH and MiBP which exhibited [M+Na]+ molecule m/z of 189 and 245. However, only DiBP (m/z 301) was detected in the control in Fig. 5A. To further confirm the two possible products were PTH and MiBP, a daughter ion scan (ESI-MS-MS) was used. Two corresponded products phthalic acid (PTH; m/z 165) and its mono-derivative monoisobutyl phthalate (MiBP; m/z 221) were identified. The ion polarities of the two compounds were both negative ([M-H]¯) (Fig. 5C and D). On the basis of the potential biases for detecting compounds among assays, the main intermediate products were further identified by HPLC using the mixture of DiBP, MiBP, and PTH for the standard (Fig. 6A). The PTH and MiBP were identified in the reaction of DiBP with the enzyme CarEW at the retention time 4.488 and 9.262, which were close to the standard sample mixture (Fig. 6B). In order to further test whether MiBP can be hydrolyzed to PTH by CarEW, the reaction of MiBP with CarEW was performed used the same conditions as DiBP. The result showed that MiBP can also be hydrolyzed to PTH by CarEW (Fig. 6D). No PTH was detected in the MiBP reaction without the enzyme CarEW (Fig. 6C). The combined results of ESI-MS and HPLC led to the identification of two products, namely, PTH and MiBP. And the main degradation process of DiBP is supposed to be the stepwise hydrolysis of the ester bonds. On the basis of the products, a pathway for incomplete DiBP degradation by CarEW was proposed (Fig. 7).


Properties of a newly identified esterase from Bacillus sp. K91 and its novel function in diisobutyl phthalate degradation.

Ding J, Wang C, Xie Z, Li J, Yang Y, Mu Y, Tang X, Xu B, Zhou J, Huang Z - PLoS ONE (2015)

The results of HPLC analyses of catabolic intermediates.(A) DiBP, MiBP and PTH mixture for the standard sample. (B) The results of DiBP degradation after incubation with CarEW. (C) Reaction of MiBP with 10 mM citric acid-Na2HPO4 solution (pH 7.5) buffer instead of CarEW. (D) The results of MiBP degradation after incubation with CarEW.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352063&req=5

pone.0119216.g006: The results of HPLC analyses of catabolic intermediates.(A) DiBP, MiBP and PTH mixture for the standard sample. (B) The results of DiBP degradation after incubation with CarEW. (C) Reaction of MiBP with 10 mM citric acid-Na2HPO4 solution (pH 7.5) buffer instead of CarEW. (D) The results of MiBP degradation after incubation with CarEW.
Mentions: The products of the reaction catalyzed by CarEW were identified by comparison to the mass spectra (m/z) of reaction with no enzyme using ESI-MS. Firstly, a full ion scan using the DiBP mixture without the enzyme CarEW as the control (Fig. 5) was performed. In Fig. 5B, we detected DiBP (m/z 301) and two other possible products PTH and MiBP which exhibited [M+Na]+ molecule m/z of 189 and 245. However, only DiBP (m/z 301) was detected in the control in Fig. 5A. To further confirm the two possible products were PTH and MiBP, a daughter ion scan (ESI-MS-MS) was used. Two corresponded products phthalic acid (PTH; m/z 165) and its mono-derivative monoisobutyl phthalate (MiBP; m/z 221) were identified. The ion polarities of the two compounds were both negative ([M-H]¯) (Fig. 5C and D). On the basis of the potential biases for detecting compounds among assays, the main intermediate products were further identified by HPLC using the mixture of DiBP, MiBP, and PTH for the standard (Fig. 6A). The PTH and MiBP were identified in the reaction of DiBP with the enzyme CarEW at the retention time 4.488 and 9.262, which were close to the standard sample mixture (Fig. 6B). In order to further test whether MiBP can be hydrolyzed to PTH by CarEW, the reaction of MiBP with CarEW was performed used the same conditions as DiBP. The result showed that MiBP can also be hydrolyzed to PTH by CarEW (Fig. 6D). No PTH was detected in the MiBP reaction without the enzyme CarEW (Fig. 6C). The combined results of ESI-MS and HPLC led to the identification of two products, namely, PTH and MiBP. And the main degradation process of DiBP is supposed to be the stepwise hydrolysis of the ester bonds. On the basis of the products, a pathway for incomplete DiBP degradation by CarEW was proposed (Fig. 7).

Bottom Line: In particular, genes involved in the hydrolysis of these compounds were not conclusively identified.Two intermediate products were identified, and a potential biodegradation pathway was proposed.Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

View Article: PubMed Central - PubMed

Affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, Yunnan, China; Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming 650500, Yunnan, China.

ABSTRACT
The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ρ-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45 °C, with ρ-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

No MeSH data available.


Related in: MedlinePlus