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Objective <p>The study employed response surface methodology (RSM) to optimize physicochemical variables for extracellular collagenase production by gram negative bacterial strain <i>Chryseobacterium contaminans</i> KU665299 under submerged fermentation. It is also revealing the ability of collagenase to degrade collagen, main structural protein in human blood.</p> Result <p>The study successfully enhanced collagenase activity by 1.2 folds through Response Surface Methodology (RSM) and 5.33 folds through purification of enzyme using ammonium sulfate precipitation and DEAE-Sepharose chromatography (specific activity with 538.0 U/mg). SDS-PAGE analysis identified its molecular weight as 32&#xa0;kDa. Optimal conditions for the enzyme’s activity were pH 7.5 and 40&#xa0;°C. Kinetic studies of collagenase KU665299 revealed specificity for collagen, with <i>K</i><sub>m</sub> and <i>V</i><sub>max</sub> values of 0.059&#xa0;mg/l and 588.24&#xa0;µmol/min/mg, respectively. Zinc and calcium ions enhanced activity, while EDTA and DTT strongly inhibited it. The purified collagenase demonstrated remarkable efficiency in digesting blood clots, fully dissolving 1&#xa0;ml clots within 40&#xa0;min at 37&#xa0;°C, showcasing significant thrombolytic potential.</p> Conclusion <p>The study successfully optimized and characterized a novel collagenase from <i>C. contaminans</i> KU665299, revealing its high specificity, stability, and efficiency in degrading collagen and its promising ability to rapidly digest blood clots for potential thrombolytic properties.</p>

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Optimization and characterization of collagenase KU665299 and its application in effective in-vitro clot digestion

  • 期刊论文

  • 发表日期：

    2025年7月22日

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  • Shikha Chauhan,
  • Kriti Kanwar,
  • Deepika Sharma,
  • Harjodh Singh,
  • Deepak Sharma,
  • Vishal Ahuja,
  • Wamik Azmi

摘要

Objective

The study employed response surface methodology (RSM) to optimize physicochemical variables for extracellular collagenase production by gram negative bacterial strain Chryseobacterium contaminans KU665299 under submerged fermentation. It is also revealing the ability of collagenase to degrade collagen, main structural protein in human blood.

Result

The study successfully enhanced collagenase activity by 1.2 folds through Response Surface Methodology (RSM) and 5.33 folds through purification of enzyme using ammonium sulfate precipitation and DEAE-Sepharose chromatography (specific activity with 538.0 U/mg). SDS-PAGE analysis identified its molecular weight as 32 kDa. Optimal conditions for the enzyme’s activity were pH 7.5 and 40 °C. Kinetic studies of collagenase KU665299 revealed specificity for collagen, with Km and Vmax values of 0.059 mg/l and 588.24 µmol/min/mg, respectively. Zinc and calcium ions enhanced activity, while EDTA and DTT strongly inhibited it. The purified collagenase demonstrated remarkable efficiency in digesting blood clots, fully dissolving 1 ml clots within 40 min at 37 °C, showcasing significant thrombolytic potential.

Conclusion

The study successfully optimized and characterized a novel collagenase from C. contaminans KU665299, revealing its high specificity, stability, and efficiency in degrading collagen and its promising ability to rapidly digest blood clots for potential thrombolytic properties.