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Improvement of the Stabilization and Activity of Protocatechuate 3,4-Dioxygenase Isolated from Rhizobium sp. LMB-1 and Immobilized on Fe3O4 Nanoparticles

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Abstract

Protocatechuate 3,4-dioxygenase (P34O), which is isolated from Rhizobium sp. LMB-1, catalyzes the ring cleavage step in the metabolism of aromatic compounds, and has great potential for environmental bioremediation. However, its structure is very sensitive to different environmental factors, which weaken its activity. Immobilization of the enzyme can improve its stability, allow reusability, and reduce operation costs. In this work, the relative molecular mass of the native P34O enzyme was determined to be 500 kDa by gel filtration chromatography on Sephadex G-200, and the enzyme was immobilized onto (3-aminopropyl) triethoxysilane-modified Fe3O4 nanoparticles (NPs) by the glutaraldehyde method. The optimum pH of immobilized and free P34O was unaffected, but the optimum temperature of immobilized P34O increased from 60 to 70 °C, and the thermal stability of immobilized P34O was better than that of the free enzyme and showed higher enzymatic activity at 60 and 70 °C. In addition, with the exception of Fe3+, most metal ions and organic chemicals could not improve the activity of free and immobilized P34O. The kinetic parameters of the immobilized P34O were higher than those of the free enzyme, and immobilized P34O on Fe3O4 NPs could be reused ten times without a remarkable decrease in enzymatic activity.

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Abbreviations

P34O:

Protocatechuate 3,4-dioxygenase

Fe3O4 NPs:

Fe3O4 nanoparticles

PAEs:

Phthalate esters

3-APTES:

(3-Aminopropyl) triethoxysilane

Ms:

Magnetization

VSM:

Vibrating sample magnetometer

XRD:

X-ray diffraction

TGA:

Thermogravimetric analysis

TSB:

Tryptone soya broth

MSM:

Minimum salt medium

PBS:

Phosphate-buffered saline

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

PCA:

Protocatechuic acid

JCPDS:

Joint Committee on Powder Diffraction Standards

bp:

Base pair

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Acknowledgements

This research was supported by National Natural Science Foundation of China (31401592).

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Authors and Affiliations

  1. Department of Food Science and Technology, School of Bioengineering, East China University of Science and Technology, Meilong RD 130, Shanghai, 200237, China

    Li-Shuang Zhang, Yue Fang & Ying Zhou

  2. Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong RD 130, Shanghai, 200237, China

    Bang-Ce Ye

  3. School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China

    Bang-Ce Ye

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  1. Li-Shuang Zhang
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Correspondence to Ying Zhou.

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Zhang, LS., Fang, Y., Zhou, Y. et al. Improvement of the Stabilization and Activity of Protocatechuate 3,4-Dioxygenase Isolated from Rhizobium sp. LMB-1 and Immobilized on Fe3O4 Nanoparticles. Appl Biochem Biotechnol 183, 1035–1048 (2017). https://doi.org/10.1007/s12010-017-2481-9

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