基于DoseResp模型的角蛋白酶发酵策略研究及其在血栓降解中的应用

Keratinase: fermentation optimization based on DoseResp model and application in thrombolysis

摘要:

【目的】角蛋白酶是一类具有高效降解角蛋白纤维特性的丝氨酸蛋白酶，角蛋白酶的高效工业化生产有利于促进其在制革、纺织、饲料、肥料、日化、医疗等领域的广泛应用。本研究以课题组前期自主构建的重组角蛋白酶工程菌枯草芽孢杆菌(Bacillus subtilis) WB600-pMA5-KerBv为研究对象，通过系统的发酵优化提升工程菌的产酶能力，并创新探究角蛋白酶在降解血栓纤维蛋白中的应用潜力。【方法】通过单因素试验确定发酵培养基成分，随后借助响应面分析方法优化产角蛋白酶的发酵培养基，确定对菌体生长和产酶具有显著影响的因素及最优浓度。基于DoseResp模型通过预测菌种最佳生长点指导其在5 L发酵罐水平的扩大产酶。最后通过血栓以及纤维蛋白原降解试验探究角蛋白酶对血栓纤维蛋白的降解能力。【结果】经优化确定重组菌产角蛋白酶的最佳发酵培养基为(g/L)：葡萄糖25.0，酵母粉25.0，豆粕15.0，磷酸氢二钾14.04，磷酸二氢钾2.58，氯化镁0.3；基于DoseResp模型通过预测菌种最佳生长点指导5 L发酵罐水平扩大生产，在优化条件下，细菌浓度OD₆₀₀从摇瓶的2.45提高至77.80，酶活从摇瓶的4 471 U/mL升至21 301.67 U/mL，提高约4.76倍。该角蛋白酶对纤维蛋白原以及血液凝块均表现出显著的降解能力。【结论】本研究通过系统的发酵优化以及基于模型预测的罐上产酶研究，有效提高了角蛋白酶在枯草芽孢杆菌中的发酵产量，并为该酶在血栓降解领域的应用提供了研究基础，同时拓展了角蛋白酶的应用价值。

关键词:角蛋白酶;培养基;发酵条件;优化;纤维蛋白原降解

Abstract:

[Objective] Keratinases, a class of serine proteases capable of degrading keratin, have important application potential and research value in the utilization of keratin resources. The efficient industrial production of keratinase is helpful to promoting its application in leather, textiles, feed, chemical fertilizers, daily chemicals, and medicine. In this study, we optimized the fermentation conditions of Bacillus subtilis WB600-pMA5-KerBv, a recombinant keratinase-producing strain constructed in our laboratory, to improve the enzyme production. Furthermore, we explored the potential application of keratinase in the degradation of fibrin. [Methods] First, the composition of the fermentation medium was determined by single factor experiments. Then, response surface methodology was employed to optimize the medium formula for producing keratinase, and the factors significantly affecting the growth and enzyme production of bacteria and the optimum concentrations were determined. Subsequently, the DoseResp model was adopted to predict the optimal growth point of the strain and thus guide the expansion of enzyme production in a 5 L fermenter. Finally, the blood clot and fibrinogen degradation experiments were carried out to evaluate the degradation performance of the keratinase. [Results] The formula of the fermentation medium for producing keratinase by the recombinant strain was optimized as follows (g/L): glucose 25.0, yeast powder 25.0, soybean meal 15.0, dipotassium phosphate 14.04, potassium dihydrogen phosphate 2.58, and magnesium chloride 0.3. The optimal growth point of the strain was predicted based on the DoseResp model to guide the expansion of production in a 5 L fermenter. Under the optimized conditions, the OD₆₀₀ (bacterial biomass) increased from 2.45 in a shake flask to 77.80, and the enzyme activity increased by about 4.76 times from 4 471 U/mL in a shake flask to 21 301.67 U/mL. In addition, the keratinase showcased remarkable degradation ability on fibrinogen and blood clots. [Conclusion] The systematic fermentation optimization and model-based prediction of enzyme production in fermenters improved the production of keratinase in Bacillus subtilis. The findings provided a research basis for the application of keratinase in thrombolysis.

Key words:keratinase;medium;fermentation conditions;optimization;fibrinogen degradation