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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

Analysis of the protein expressed in E. coli BL21 cells following purification on a 12% SDS-PAGE.Lane M, protein molecular marker; Lane 1, before induction with 0.7 mM IPTG; Lane 2, after induction with IPTG and grown at 20°C for 20 h; and Lane 3, purified recombinant CarEW (∼53.76 kDa).
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pone.0119216.g003: Analysis of the protein expressed in E. coli BL21 cells following purification on a 12% SDS-PAGE.Lane M, protein molecular marker; Lane 1, before induction with 0.7 mM IPTG; Lane 2, after induction with IPTG and grown at 20°C for 20 h; and Lane 3, purified recombinant CarEW (∼53.76 kDa).

Mentions: To investigate the biochemical properties of the enzyme, the CarEW gene was expressed in the pEASY-E2 vector as a 6 × His tagged fusion protein and induced with 0.7 mM IPTG at 20°C for 20 h. The crude enzyme extracted from recombinant E. coli BL21 cells was purified using Ni2+–NTA metal–chelating affinity chromatography and analyzed by SDS–PAGE. As shown in Fig. 3 (lane 3), one band corresponded in size to the calculated molecular mass of CarEW was detected (∼53.76 kDa). The band was absent in the control lane from the E. coli BL21 cells carrying only the non-recombinant pEASY-E2 vector (Fig. 3, lane 1), which was cultured and induced under the same conditions as that for the E. coli BL21 (pEASY-E2-CarEW) cells. The isoelectric point (pI) was 4.88. The kinetic parameters of different substrates were determined at pH 7.5 and 45°C using the purified recombinant CarEW. ρ-NPC4 was the best substrate for CarEW according to the highest maximum initial velocity Kcat/Km values (Table 1). Besides, CarEW also displayed quite high enzyme activity towards ρ-NPC2, ρ-NPC6, ρ-NPC8, ρ-NPC10 according to the initial velocity Kcat/Km values in Table 1. The catalytic efficiency toward ρ-NPC4 was approximately 1.5-fold higher than toward ρ-NPC2. No significant esterase activity was observed for the substrates with a chain length ≥ C12. Compared with the ρ-NP substrates, the catalytic efficiency of DiBP and MiBP were not high. The Kcat/Km values for DiBP and MiBP were 0.109 and 0.031 (s−1 mM−1), respectively (Table 1).


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)

Analysis of the protein expressed in E. coli BL21 cells following purification on a 12% SDS-PAGE.Lane M, protein molecular marker; Lane 1, before induction with 0.7 mM IPTG; Lane 2, after induction with IPTG and grown at 20°C for 20 h; and Lane 3, purified recombinant CarEW (∼53.76 kDa).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119216.g003: Analysis of the protein expressed in E. coli BL21 cells following purification on a 12% SDS-PAGE.Lane M, protein molecular marker; Lane 1, before induction with 0.7 mM IPTG; Lane 2, after induction with IPTG and grown at 20°C for 20 h; and Lane 3, purified recombinant CarEW (∼53.76 kDa).
Mentions: To investigate the biochemical properties of the enzyme, the CarEW gene was expressed in the pEASY-E2 vector as a 6 × His tagged fusion protein and induced with 0.7 mM IPTG at 20°C for 20 h. The crude enzyme extracted from recombinant E. coli BL21 cells was purified using Ni2+–NTA metal–chelating affinity chromatography and analyzed by SDS–PAGE. As shown in Fig. 3 (lane 3), one band corresponded in size to the calculated molecular mass of CarEW was detected (∼53.76 kDa). The band was absent in the control lane from the E. coli BL21 cells carrying only the non-recombinant pEASY-E2 vector (Fig. 3, lane 1), which was cultured and induced under the same conditions as that for the E. coli BL21 (pEASY-E2-CarEW) cells. The isoelectric point (pI) was 4.88. The kinetic parameters of different substrates were determined at pH 7.5 and 45°C using the purified recombinant CarEW. ρ-NPC4 was the best substrate for CarEW according to the highest maximum initial velocity Kcat/Km values (Table 1). Besides, CarEW also displayed quite high enzyme activity towards ρ-NPC2, ρ-NPC6, ρ-NPC8, ρ-NPC10 according to the initial velocity Kcat/Km values in Table 1. The catalytic efficiency toward ρ-NPC4 was approximately 1.5-fold higher than toward ρ-NPC2. No significant esterase activity was observed for the substrates with a chain length ≥ C12. Compared with the ρ-NP substrates, the catalytic efficiency of DiBP and MiBP were not high. The Kcat/Km values for DiBP and MiBP were 0.109 and 0.031 (s−1 mM−1), respectively (Table 1).

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