Chauhan Shikha, Kanwar Kriti, Sharma Deepika, Singh Harjodh, Sharma Deepak, Ahuja Vishal, Azmi Wamik
Department of Biotechnology, Himachal Pradesh University, Summerhill Shimla, Shimla, Himachal Pradesh, 171005, India.
University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, 140413, India.
Biotechnol Lett. 2025 Jul 22;47(4):79. doi: 10.1007/s10529-025-03615-2.
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.
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 K and V 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.
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.
本研究采用响应面法（RSM）优化革兰氏阴性菌污染金色杆菌KU665299在深层发酵条件下生产胞外胶原酶的理化变量。同时还揭示了胶原酶降解人血液中主要结构蛋白胶原蛋白的能力。
本研究通过响应面法（RSM）成功将胶原酶活性提高了1.2倍，并通过硫酸铵沉淀和DEAE-琼脂糖层析法纯化酶，使其活性提高了5.33倍（比活性为538.0 U/mg）。SDS-PAGE分析确定其分子量为32 kDa。该酶活性的最佳条件为pH 7.5和40℃。对胶原酶KU665299的动力学研究表明其对胶原蛋白具有特异性，K值和V值分别为0.059 mg/l和588.24 μmol/min/mg。锌离子和钙离子增强了酶的活性，而EDTA和DTT则强烈抑制其活性。纯化后的胶原酶在消化血凝块方面表现出显著的效率，在37℃下4（此处原文有误，应为40）分钟内可完全溶解1 ml血凝块，显示出显著的溶栓潜力。
本研究成功优化并表征了一种来自污染金色杆菌KU665299的新型胶原酶，揭示了其在降解胶原蛋白方面的高特异性、稳定性和效率，以及其快速消化血凝块的潜在溶栓特性的良好能力。
