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Purification and Characterization of a Novel Collagenase from Bacillus pumilus Col-J

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Abstract

The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery. The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature for the enzyme reaction was 45 °C. More than 50% of the original activity still remained after 5 min of incubation at 70 °C or 10 min at 60 °C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable over a pH range of 6.5–8.0. The collagenase activity was strongly inhibited by Mn2+, Pb2+, ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and β-mercaptoethanol. However, Ca2+ and Mg2+ greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K m and V max of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively.

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Acknowledgments

This research was supported by the Technology Innovation Fund for Young Scholars of Sichuan Agricultural University. The authors thank Dr. Zou Likou for kindly supplying the purification columns used in this study. They also acknowledged Ni Yan and Yin Huaqi for their help in the preparation of the manuscript.

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Authors and Affiliations

  1. College of Life Science, Sichuan Agricultural University, Ya’an, 625014, China

    Qi Wu, Chen Li, Chenglei Li & Hui Chen

  2. College of Food Science, Sichuan Agricultural University, Ya’an, 625014, China

    Liu Shuliang

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  1. Qi Wu
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  2. Chen Li
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  3. Chenglei Li
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  4. Hui Chen
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  5. Liu Shuliang
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Correspondence to Qi Wu.

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Wu, Q., Li, C., Li, C. et al. Purification and Characterization of a Novel Collagenase from Bacillus pumilus Col-J. Appl Biochem Biotechnol 160, 129–139 (2010). https://doi.org/10.1007/s12010-009-8673-1

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