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High Efficiency Preparation and Characterization of Intact Poly(Vinyl Alcohol) Dehydrogenase from Sphingopyxis sp.113P3 in Escherichia coli by Inclusion Bodies Renaturation

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Abstract

Poly(vinyl alcohol) dehydrogenase (PVADH, EC 1.1.99.23) is an enzyme which has potential application in textile industry to degrade the poly(vinyl alcohol) (PVA) in waste water. Previously, a 1,965-bp fragment encoding a PVADH from Sphingopyxis sp. 113P3 was synthesized based on the replacement of the rare codons in Escherichia coli (E. coli). In this work, the deduced mature PVADH (mPVADH) gene of 1,887 bp was amplified by polymerase chain reaction (PCR) and inserted into the site between NcoI and HindIII in pET-32a(+). The constructed recombinant plasmid was transformed into E. coli Rosetta (DE3). In shake flask, the fusion protein of thioredoxin (Trx)-mPVADH was expressed precisely; however, Trx-mPVADH was found to accumulate mainly as inclusion bodies. After isolating, dissolving in buffer containing urea, purification, dialysis renaturation, and digesting with recombinant enterokinase/His (rEK/His), the bioactive mPVADH fragments were obtained with protein concentration of 0.56 g/L and enzymatic activity of 194 U/mL. The K m and V max values for PVA 1799 were 2.33 mg/mL and 15.7 nmol/(min·mg protein), respectively. 1H-NMR and infrared (IR) spectrum demonstrated that its biological function was oxidizing hydroxyl groups of PVA 1799 to form diketone, and PVA 1799 could be degraded completely by successive treatment with mPVADH and oxidized PVA hydrolase (OPH).

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Abbreviations

PVA:

Poly(vinyl alcohol)

PVADH:

Poly(vinyl alcohol) dehydrogenase

mPVADH:

Mature poly(vinyl alcohol) dehydrogenase

rEK/His:

Recombinant enterokinase/His

tPVADH:

Truncated poly(vinyl alcohol) dehydrogenase

oxiPVA:

Oxidized PVA

Mv:

Viscosity weight

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Acknowledgments

This project was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (111-2-06), the National High Technology Research and Development Program of China (863 Program, 2011AA100905), the Priority Academic Program Development of Jiangsu Higher Education Institution, the PhD Research Fund of Jiangnan University, and the 973 Program (2012CB720802 and 2012CB720806).

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Dongxu Jia, Yu Yang, Zhengcong Peng, Dongxu Zhang, Jianghua Li, Long Liu, Guocheng Du & Jian Chen

  2. Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China

    Dongxu Jia

  3. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China

    Yu Yang, Zhengcong Peng, Dongxu Zhang, Jianghua Li, Long Liu, Guocheng Du & Jian Chen

  4. School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

    Guocheng Du

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  1. Dongxu Jia
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  2. Yu Yang
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  3. Zhengcong Peng
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  4. Dongxu Zhang
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  5. Jianghua Li
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  8. Jian Chen
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Correspondence to Guocheng Du.

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Jia, D., Yang, Y., Peng, Z. et al. High Efficiency Preparation and Characterization of Intact Poly(Vinyl Alcohol) Dehydrogenase from Sphingopyxis sp.113P3 in Escherichia coli by Inclusion Bodies Renaturation. Appl Biochem Biotechnol 172, 2540–2551 (2014). https://doi.org/10.1007/s12010-013-0703-3

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  • DOI: https://doi.org/10.1007/s12010-013-0703-3

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