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Purification and Characterization of an Endo-d-arabinase Produced by Cellulomonas

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Abstract

An extracellular endo-d-arabinase enzyme produced by the bacterial strain of Cellulomonas was purified 77.1-fold with 0.20% recovery for protein by DEAE Sepharose anion exchange, Sephacryl S-300 gel filtration and blue Sepharose affinity chromatography, and designated as CEDAase. The apparent molecular mass of CEDAase was 45 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CEDAase is an endoenzyme for arabinogalactan with the main and specific product of hexa-arabinofuranoside. It reacts optimally with its substrate, arabinogalactan, at approximately pH 8.0 and at 40 °C. CEDAase shows stability in the pH range of 6.0–9.0 and at the temperature below 50 °C. The Km measured for the CEDAase was 55.6 μM, with an apparent Vmax of 0.083 μmol/min. To our knowledge, for the first time, the current work obtains an extracellular Cellulomonas endo-d-arabinase enzyme that might be potentially served as a tool enzyme for hydrolyzing specific cell wall such as Mycobacterium cell. It is purified as an important potential initial material basis for mass spectrometric sequencing and chemical gene synthesis. It may make it possible to clone and express this valuable endo-d-arabinase and make it available to the mycobacteria scientific community.

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Abbreviations

AG:

Arabinogalactan

CEDAase:

Cellulomonas endo-d-arabinase

DEAE Sepharose:

Diethylaminoethyl Sepharose

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TB:

Tuberculosis

MDR:

Multiple drugs resistant (or Multiple drugs resistance)

PBS:

Phosphate buffer solution

HAC:

Acetic acid

TLC:

Thin layer chromatography

HPLC:

High performance liquid chromatography

PAD:

Pulsed amperometric detector

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Acknowledgments

This work was supported by a grant from National Natural Science Foundation of China (306570416).

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Authors and Affiliations

  1. Department of Biotechnology, Dalian Medical University, 9 West Lvshun Southern Road, 116044, Dalian, China

    Ming-Zhong Sun, Xiao-Ying Zhang & Yi Xin

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  1. Ming-Zhong Sun
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  2. Xiao-Ying Zhang
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  3. Yi Xin
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Correspondence to Yi Xin.

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Ming-Zhong Sun and Xiao-Ying Zhang have contributed equally as the first authors.

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Sun, MZ., Zhang, XY. & Xin, Y. Purification and Characterization of an Endo-d-arabinase Produced by Cellulomonas . Protein J 31, 51–58 (2012). https://doi.org/10.1007/s10930-011-9374-5

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