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

IF 2.1 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2025-07-22 DOI:10.1007/s10529-025-03615-2

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

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引用次数: 0

Abstract

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 K_m and V_max 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.

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胶原酶KU665299的优化、表征及其在体外凝块消化中的应用

目的：采用响应面法（RSM）优化革兰氏阴性菌株污染黄杆菌KU665299深层发酵产胞外胶原酶的理化参数。它还揭示了胶原酶降解胶原蛋白的能力，胶原蛋白是人体血液中的主要结构蛋白。结果：通过响应面法（Response Surface Methodology， RSM）和DEAE-Sepharose色谱法分别将胶原酶活性提高了1.2倍和5.33倍，比活性为538.0 U/mg。SDS-PAGE分析鉴定其分子量为32 kDa。酶活性的最佳条件为pH 7.5和40℃。胶原酶KU665299对胶原蛋白具有特异性，Km和Vmax值分别为0.059 mg/l和588.24µmol/min/mg。锌离子和钙离子增强了其活性，EDTA和DTT则对其有较强的抑制作用。纯化的胶原酶在消化血凝块方面表现出显著的效率，在37°C下40分钟内完全溶解1 ml血凝块，显示出显著的溶栓潜力。结论：本研究成功地对一种新型胶原酶进行了优化和表征，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血栓特性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.