【作者】 豆孝伟；

【导师】 梁智群；

【作者基本信息】 广西大学 ， 发酵工程， 2007， 硕士

【摘要】 豆豉溶栓酶是我国传统发酵食品豆豉在发酵过程中由蜡状芽孢杆菌产生的一种具有溶栓活性的丝氨酸蛋白酶。该酶具有安全性好，易被人体吸收，直接作用于血栓的主要基质——纤维蛋白，对顽固性血栓具有较强的分解能力，是一种很有潜力的新型溶栓药物。本论文以自主筛选的蜡状芽孢杆菌DC-101为出发菌株，经过紫外线和微波的联合诱变处理，筛选出一株能够稳定遗传的高产菌株DC-301。其酶活力由原来的2243U/mL提高到了5989U/mL，是出发菌株的2.67倍。通过单因素及正交试验分析，确定了该菌种产酶的最佳培养基和培养条件为：可溶性淀粉2％、豆浆5％、酵母浸膏0.3％、KH₂PO₄ 0.1％、K₂HPO₄ 0.3％、MgSO₄·7H₂O 0.05％、CaCl₂ 0.002％。初始pH 7.0、温度37℃，转速160 r/min、接种量2％。发酵液经过低温离心、活性炭脱色、蔗糖浓缩、硫酸铵盐析、透析、再次蔗糖浓缩、DEAE-C52阴离子交换柱层析处理，得到的粗酶经SDS-PAGE电泳分析显示达到了电泳纯，分子量约为27KD。酶学性质研究显示该酶最适酶促反应温度为40℃，最适pH为7.0，在最适温度和最适pH条件下性质较稳定。Mn²⁺有激活作用，Fe²⁺、Fe³⁺、Hg²⁺、Cu²⁺有不同程度的抑制作用。酶促反应V_m=0.87μg/L·min，K_m=0.002μg/L。PMSF能够完全抑制该酶活性，EDTA和EGTA对酶活性没有影响。更多还原

【Abstract】 Douchi Fibrinolytic Enzyme （DFE） is a fibrinolytic serineproteinase produced by Bacillus cereus screened from Douchi, a Chinesetraditional food. The enzyme is safer, easier to be absorbed by human andcan decompose fibrin directly. It also has a strong capability to resolutethe stubborn thrombus. DFE could be used to design fibrinolyticmedicine with lower cost by fermentation.A better mutagenesis DC-301 was obtained by a treatment ofradiation with microwave and ultraviolet. It is stable in subcultures withoutput of fibrinolytic enzyme from 2243U/ml to 5989U/ml, 2.67 times ofthat of the start Bacillus cereus DC-101. Single factor and orthogonaldesign were used to optimize the fementation conditions of DC-301 andgot a combination of medium and condition for DFE product: solublestarch 2%, soybean milk 5%, yeast extract 0.3%, KH₂PO₄ 0.1%, K₂HPO₄0.3%, MgSO₄·7H₂O 0.05%, CaCl₂ 0.002%; initial pH7.0, 37℃, 160 r/min,inoculation quantity 2%. DFE was purified from the broth by steps ofcentrifugation, activated charcoal decolorize, sucrose concentrate, ammonium sulfate fraction precipitation, dialysis, and DEAE-C52exchange chromatography. There is one protein brand on SDS-PAGEresult with molecular weight about 27KD.DFE was stable in its optimum temperature and pH as: 40℃, pH 7.0,V_m of 0.87μg/L·min, and K_m of 0.0026μg/L. Mn²⁺ can activates theability while Fe²⁺, Fe³⁺, Hg²⁺, and Cu²⁺ inhibit its ability with varyingdegree. The result that PMFS inhibited all activities while EDTA andEGTA no indicated that DFE was a kind of serine proteinase, not ametalloprotease.更多还原