【作者】 李品周；

【导师】 施巧琴； 施碧红；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 酶一般都不稳定，易失活，霉菌脂肪酶稳定性显得更差。目前国内外改善酶的稳定性包括2个方面：1．改变酶分子周围环境条件来提高酶分子的稳定性。2．从分子水平改造酶分子结构。本文按第1种研究路线对扩展青霉产生的脂肪酶的稳定性的研究，结果总结如下：（1）发酵培养基以黄豆饼粉5％、玉米淀粉1.2％有利于保持酶的稳定性：K₂SO₄0.03％、FeSO₄0.03％、ZnSO₄0％既有利于脂肪酶的合成，也有利于保持酶的稳定性；发酵培养基中添加透性剂对该菌生长、合成代谢均不利，因此也难以提高脂肪酶的稳定性。（2）在20h内，能较稳定维持发酵液和过滤液中脂肪酶活性的最佳PH为8.5：菌丝体中脂肪酶活性的最佳PH为6.0；但随着温度升高和贮存的时间延长，酶稳定性越来越低。（3）在扩展青霉（Penicillium cxpansum）FS1884生长、合成代谢过程中，蛋白酶的合成在20h左右达到高峰，而脂肪酶在20-40 h左右，因此对脂肪酶合成影响不大，但发酵后蛋白酶的量即使是少量，也会使脂肪酶部分水解失活。该蛋白酶受EDTA抑制，且EDTA对脂肪酶的活性有一定的促进作用。分离纯化后的蛋白酶，通过分析测量得分子量为52.5kda，为实际工作中选择相关分子筛提供参考数据。（4）从粗酶中分离出来的淀粉和粗纤维按同等量再加入纯化后液体酶中，使酶稳定性有所下降；从粗酶中分离出来的总糖和金属离子（金属离子以金属化合物形式）按同等量再加入纯化后液体酶中，均对不影响脂肪酶稳定性。（5）外加一定浓度的金属离子、糖、醇、盐到粗酶液中，分别放置在35℃、25℃、15℃下，于2、4、9d，取样进行残余酶活测定，结果为：KCl、Cacl₂、四硼酸钠、丁二酸钠、山梨糖、丙三醇均可提高粗酶热稳定性的半衰期，对以上因子进行正交试验结果：液体脂肪酶稳定剂的最佳组合配方为4mM的Ca²⁺、0.0313M的四硼酸钠和4M的丙三醇，在40℃下可提高脂肪酶热稳定性半衰期84小时，35℃下提高酶半衰期为790 h。而固体脂肪酶稳定剂的最佳组合配方为2mM的Ca²⁺、0.0313 M四硼酸钠和0.5％山梨糖，在40℃下可提高脂肪酶热稳定性半衰期30小时，35℃下提高酶半衰期为166 h。更多还原

【Abstract】 Stable enzymes are generally not susceptible to deactivation fungal lipase stability is even worse. Improve enzyme stability at home and abroad, including two areas : 1. Changes to improve environmental conditions around the enzyme molecule enzyme molecules stability. 2. From the level of molecules to transform the structure of enzyme molecules. This kind of study in accordance with paragraph 1 of the extension of pro-line mycophenolate lipase stability of the study, results are summarized as follows :1. Fermentation medium containing 5% of soybean meal, 1.2% of corn starch may help enzymes stability; 0.03% of K₂SO₄ and 0.03% of FeSO₄ were conducive to both the synthesis and the stability of lipase. Addition of permeable agent to the fermentation medium was harmful to both the growth and metabolic of the species, and it would be unfavorable to the stability of lipase.2. The optimum pH to maintain the lipase activity in the fermentation broth and filtration within 20 hours was 8.5. and pH 6.0 for the lipase activity in the mycelium; the lipase stability was decreased with the increasing of the temperature and storage time.3. A protease was synthesized and reached peak in 20 hours during the growth of Penicillium expansum FS1884. Whereas the lipase was synthesized in 20 to 40 hr, hence the protease have little effect on the synthesis of lipase. However, even a small amount of protease in the late of fermentation may lead to partial hydrolyzing of lipase. The study showed that the protease activity was inhibited by addition of EDTA, and the lipase activity can be enhanced by EDTA. The molecule weight of the protease was assayed as 52.5 kDa after purification of the protein, which provided a reference for selection of suitable molecule screen.4. The stability of lipase was declined after addition of equate amount of the starch and fiber separated from the crude enzyme; the lipase activity had little changed after addition equate amount of total sugar and metal ions （in the style of metal compounds） isolated from the crude enzyme.5. The half life of the crude lipase thermal stability was increased by adding a certain concentration of KCI, CaCl₂, sodium tetraborate, sodium succinic acid, sorbitol, glycerol to the crude enzyme and incubating at temperatures of 35℃, 25℃, 15℃for 2, 4 and 9 days, respectively. Results of orthogonal test showed that the optimum stable agent for liquid lipase was composted of 4 mM Ca²⁺, 0.0313 M of sodium tetraborate and 4 M of glycerol, which would increase the half life of lipase thermal stability for 84 hrs at a temperature of 40℃, and 790 hrs at 37℃. The optimum stable agent for solid lipase was made of 2 mM of Ca²⁺, 0.0313 M of sodium tetraborate and 0.5% of sorbitol, which would enhance the half life of lipase thermal stability for 30 hrs at a temperature of 40℃, and 166 hr at 35℃.更多还原