Properties of a newly identified esterase from Bacillus sp. K91 and its novel function in diisobutyl phthalate degradation.
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.
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.
Junmei
J
Ding
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.
Chaofan
C
Wang
Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, Yunnan, China.
Zhenrong
Z
Xie
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.
Junjun
J
Li
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.
Yunjuan
Y
Yang
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.
Yuelin
Y
Mu
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.
Xianghua
X
Tang
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.
Bo
B
Xu
Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, Yunnan, China.
Junpei
J
Zhou
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.
Zunxi
Z
Huang
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003993:Dibutyl Phthalate / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013696:Temperature
D003993:Dibutyl Phthalate; D004950:Esterases; C025605:diisobutyl phthalate