Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala

Chen, Jie; Luo, Xiao-Jing; Chen, Qi; Pan, Jiang; Zhou, Jiahai; Xu, Jian

doi:10.1186/s40643-015-0067-3

Cited by 12 publications

(6 citation statements)

References 29 publications

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“…Cells were cultured as described previously (Qian et al 2011). Vector pET-28a (+) for heterogeneous expression studies (Chen et al 2015) was obtained from Novagen (Shanghai, China).…”

Section: Bacterial Strains and Vectorsmentioning

confidence: 99%

Identification of a new thermostable and alkali-tolerant α-carbonic anhydrase from Lactobacillus delbrueckii as a biocatalyst for CO2 biomineralization

Jiang

Yongjuan

et al. 2015

Bioresour. Bioprocess.

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Background: Carbonic anhydrase (CA, EC 4.2.1.1), an ancient enzyme and the fastest among many enzymes, is a useful biocatalyst for carbon capture use and storage (CCUS). The use of alkaline buffers and high temperatures are favorable for biomineralization. Hence, the stability of CA under such harsh conditions is extremely important for its practical application. Methods and results:Herein, we report a new thermostable and alkaline-tolerant α-CA (designated as LdCA), with only 26 % identity to bovine CA (BCA), which was identified by genome mining from Lactobacillus delbrueckii CGMCC 8137. It was overexpressed in Escherichia coli in a soluble form and purified to electrophoretic homogeneity by HisTrap affinity chromatography. The dimer protein had a subunit molecular weight of 23.8 kDa and showed extremely high stability at pH 6.0-11.0 and 30-60 °C. Its activity was maintained even after incubation at 90 °C for 15 min. The half-lives of the enzyme measured at 30, 40, and 50 °C were 630, 370, and 177 h, respectively. At pH 9.0, 10.0, and 11.0, its half-lives were 105, 65, and 41 min, respectively. LdCA was applied at 50 °C to accelerate the formation of calcium carbonate in a vaterite phase. Conclusions:In summary, a new CA with high thermal and alkaline stability was identified from a general bacterium, demonstrating an effective strategy for discovering new and useful biocatalysts.

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“…Cells were cultured as described previously (Qian et al 2011). Vector pET-28a (+) for heterogeneous expression studies (Chen et al 2015) was obtained from Novagen (Shanghai, China).…”

Section: Bacterial Strains and Vectorsmentioning

confidence: 99%

Identification of a new thermostable and alkali-tolerant α-carbonic anhydrase from Lactobacillus delbrueckii as a biocatalyst for CO2 biomineralization

Jiang

Yongjuan

et al. 2015

Bioresour. Bioprocess.

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“…Crystallization and Diffraction Data Collection. Crystals were grown at 18 °C using the sitting-drop vapor diffusion method 46 by mixing the protein solution (2 μL, 14 mg/mL) with the reservoir solution (2 μL) containing 25% (w/v) PEG 3350, 0.1 M Tris-HCl (pH 8.5), and 0.2 M NaCl. Crystals were cryoprotected with reservoir solution plus 15% glycerol.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Engineering of Cyclohexanone Monooxygenase for the Enantioselective Synthesis of (S)-Omeprazole

Zhang

et al. 2019

ACS Sustainable Chem. Eng.

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Enzymatic asymmetric sulfoxidation using molecular oxygen as the oxidant is a promising green chemistry approach to chiral sulfoxide production. Despite the broad substrate spectrum of cyclohexanone monooxygenases (CHMOs), some unnatural substrates with bulky functional groups, such as the pharmaceutically relevant omeprazole sulfide, cannot be effectively accepted by CHMOs. Herein, we describe a set of variants derived from an Acinetobacter calcoaceticus CHMO (AcCHMO), whose active sites adjacent to the substrate tunnel were altered to shift the substrate specificity from cyclohexanone monooxygenation toward omeprazole sulfide sulfoxidation. We performed homologous modeling and molecular docking to identify key residues that might affect the substrate specificity. Two libraries of residues lining the active center of AcCHMO were then constructed and screened by an effective halo-based selection method using the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole). Functional evaluation of the resultant variants showed that the substrate specificity of AcCHMO was markedly altered from the small natural substrate (cyclohexanone) toward the desired bulky substrate (omeprazole sulfide) despite the extremely poor activity detected even for the best variant, M2 (0.61 U/ g prot ). The crystal structure of M2 complexed with a flavin adenine dinucleotide (FAD) prosthetic group was determined, which provided insight into the altered substrate specificity. To improve the activity of enzyme M2 toward pharmaceutical precursor omeprazole sulfide, we performed both local and global protein engineering among the two CASTing libraries surrounding FAD + and NADP + prosthetic groups and an error-prone PCR library of the full-length AcCHMO. As a result, variant M6 was obtained, giving a 50-fold higher activity compared to M2. This structure-guided protein engineering of AcCHMO provided a promising candidate for converting omeprazole sulfide into (S)-omeprazole using a green biocatalytic method.

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“…OPH based biodegradation, which can efficiently hydrolyze OPs to benign molecules, is one of the most attractive technologies for removing OPs (Schenk et al 2016;Kim et al 2014a, b;Ramalho et al 2016;Theriot and Grunden 2011). Many naturally occurring or engineered OPHs have been developed so far (Cherny et al 2013;Khare et al 2012;Luo et al 2014;Abe et al 2014;Bigley et al 2016;Chen et al 2015;Jackson et al 2009). However, in spite of high activity, free OPHs are difficult to be recovered from aqueous solution, and are often denatured quickly under harsh environments such as high temperatures and high pHs that are usually used in industrial washing and water treatment, hindering its usage in practical applications (Giudice et al 2016;Bai et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation

Cheng

Zhao

Luo

et al. 2018

Bioresour. Bioprocess.

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Background: Enzymatic biodegradation of organophosphate pesticides (OPs) is a promising technology to remove these toxic compounds. However, its application in industrial washing was restricted by the lack of efficient immobilized enzymes that can work at high temperatures and high pHs in the presence of various detergents. Therefore, it is necessary to develop a simple method to prepare a robust immobilized enzyme for efficient degradation of OPs. Results: An organophosphate hydrolase (OPH), PoOPH M9 , was conjugated and immobilized with a commercially available polymer, Pluronic F127. The prepared cross-linked enzyme-polymer conjugate (CLEPC) displayed higher pH stability in the range from 7.0 to 11.0 and a higher optimal temperature (50 °C) than that of free PoOPH M9 (30 °C). Its half-life and apparent k cat /K M reached 12.8 h at 50 °C and 390.3 ± 7.8 mM −1 s −1 , respectively, which were even better than that of the traditional cross-linked enzyme aggregates (CLEA, 7.2 h and 10.9 ± 1.7 mM −1 s −1). The activity of PoOPH M9 CLEPC was further enhanced up to 2.5-fold by the anionic, nonionic and biocompatible detergents, which was first observed. 0.15 mM Malathion was degraded completely by PoOPH M9 CLEPC after activation within 10 min in the presence of 0.1% (w/w) detergents of all types at pH 9.0 and 25 °C, demonstrating its capability in degrading OPs at practically relevant conditions. Conclusion: The conjugation of Pluronic F127 in enzyme immobilization could effectively reduce the activity loss of immobilized enzymes and enhance their stability and activity at high temperatures and high pHs. In addition, the activity of CLEPC can be even enhanced in the presence of various detergents. This technology can be extended easily to produce other immobilized polymer-enzyme conjugates due to its simplicity.

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Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala

Cited by 12 publications

References 29 publications

Identification of a new thermostable and alkali-tolerant α-carbonic anhydrase from Lactobacillus delbrueckii as a biocatalyst for CO2 biomineralization

Identification of a new thermostable and alkali-tolerant α-carbonic anhydrase from Lactobacillus delbrueckii as a biocatalyst for CO2 biomineralization

Engineering of Cyclohexanone Monooxygenase for the Enantioselective Synthesis of (S)-Omeprazole

Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation

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