【作者】 张勇；

【导师】 王忠彦；

【作者基本信息】 四川大学 ， 微生物学， 2002， 硕士

【摘要】 以实验室保存菌株BL.PY为出发菌株，该菌为地衣芽孢杆菌（Bacillus licheniformis），可以产生血纤维蛋白溶解酶（Plasmin）。该菌株经过复壮后，酶活大约为800U。用低能N离子注入纤溶酶产生菌，以20kev,剂量为1×10¹⁴至4×10¹⁵ions／cm²·s的N离子为诱变剂，产生可遗传的变异，一定范围内纤溶酶产生菌的正向突变率，致死率与离子注入剂量成正相关，离子注入后纤溶酶产生菌的菌体形态发生一定程度上的改变。从大约120株突变株中筛选到一株编号为BL.Z高产菌，摇瓶发酵表明该突变菌株酶产量可提高51.13％，达到1210U。通过单因素实验以及正交分析法探索该菌株的最佳发酵条件为（％）：蔗糖 3，黄豆粉 0.2，酵母膏0.1， MgSO₄·7H₂O 0.1， CaCl₂ 0.03，K₂HPO₄ 0.3， KH₂PO₄ 0.04，通气量为1：0.5—1：1，转速为480rpm，37℃发酵4天，其纤溶酶活力达到1320U，较出发菌株提高了65％。 发酵液通过硫酸铵分级盐析沉淀，CM-Sephadex C-25阳离子交换层析，DEAE-Sephadex A-50阴离子交换层析和Sephadex G-75凝胶过滤，获得一种具有纤溶酶活性的蛋白酶BK，其分子量大约为23KD。以苯胺-硫酸法测定中性糖含量为7.55％，以3，5-二硝基水杨酸法（DNS法）测总还原糖含量分别为7.45％。以硅胶G薄层层析法鉴定糖的种类，该酶所含糖蛋白主要为中性戊糖，同时研究了一些金属离子的添加对纤溶酶的活性的影响，EDTA对酶活影响不大，而PMSF对酶活有较强的抑制作用，说明该酶的活性中心有丝氨酸参与，属丝氨酸酶系，而其作用中心无Ca²⁺，Mg²⁺等金属离子参与。脲和SDS变性剂对酶有一定 的抑制作用，但较弱，可以看出酶的抗变性剂能力较强。更多还原

【Abstract】 A novel plasmin-producing bacterial strain PY,which had been identified as Bacillus LicheniformaoUsing a dosage of about 1 X 1014 to 4X 1015 N ions/cm2 -s and 20kev, the strain B L.PY. was treated by N ion implantation and a stable mutant strain with high yield of plasmin is obtained .In general, the rate of mutation and death grows as the increasing of ion dosage. The shape of strains changed. Shaking fermentation indicates that compared with that of normal strain about 120 strians, the enzymetic production of the mutant one increases 51.13%, reaching 1210U. Fermentation conditions for enzyme production have been established. Composition of the medium (%) is edible sucrose 3, soybean poder 1.2, yeast extract 0.2, MgSO4 7H2O 0.1, CaCl2 0.03, K2HPO4 0.3, KH2PO40.04, pH 7.2, After fermentation in 1.5 liter of fermentorat 37"C with an aeration rate of 1: 0.5 - 1:1 for 4 days, the average fibrinolytic activity was about 1320U/ml culture broth,increase by 65% over the mutant.The purification of enzyme BK include saline extraction,, ion-exchange chromatography on CM-Sephadex C-25 and DEAE-Sephadex A-50, and gel-filtration on Sephadex G-75 ,with a molecular weight of 23 KD. The protocols used were phenols.sulfuric acid method,3,5-dinitrosalicylic acid method and thin layer chromatography of salic gel G. Reducing sugar ang the tatol sugar weremeasured with 3,5-2nitro-salcylic acid.We found that their contents were 7.45% and 7.49% respectnely.The categories of sugar were obtained using silica gel sheet chromatography .The glyco proteins that this enzyme contains mortly were hetrosamine .In the same time,a research was performed to find out how the additionnal metal hydronium would affect the activity of the fibrinolytic enzyme. We could draw an inclusion that EDTA did not have an obvipns inhibitoryaction.These showed that serine was one of the conponents of the active center while metal hydromium wasn’t Urea and denaturant SDS inhibited the activity of the enzyme to a degree,but we could still find that the enzyme’s ability of anti-denaturant was strong.更多还原