【作者】 李赟高；

【导师】 江波；

【作者基本信息】 江南大学 ， 食品科学， 2008， 硕士

【摘要】 谷氨酰胺转胺酶(TGase)是一种催化酰基转移反应的转移酶,它可催化蛋白质分子内和分子间ε-(γ-谷酰胺)-赖氨酸的异型肽键的形成,改善食品的品质,目前已广泛应用于食品工业中,但是成本过高的问题严重制约了TGase的使用和推广。本文以轮枝链霉菌SK4.001为出发菌株,研究了以豆饼粉酶解液作为培养基氮源对TGase酶活的影响。实验发现,胰蛋白酶是水解豆饼粉较好的酶。发酵培养基中酶解液添加量为100%。在胰蛋白酶水解豆饼粉单因素实验的基础上,采用正交实验进行优化。优化后的最佳酶解条件为温度35℃, pH 8.5,豆饼粉浓度10%,加酶量1000U/g底物。按此条件进行酶解,所获得的酶解物作为轮枝链霉菌产TGase发酵培养基的氮源,发酵42h后,酶活达到最高为5.04U/mL。因此,豆饼粉酶解物可以替代鱼粉蛋白胨作为轮枝链霉菌产TGase发酵培养基的氮源。对轮枝链霉菌SK4.001生物合成TGase的酶学性质进行了研究。研究发现,TGase的最适反应温度为35℃,最适反应pH为7.0。20℃到40℃之间,温度稳定性较好。TGase在pH 6.0-7.0之间比较稳定。添加各种常见的金属离子均会使TGase酶活有一定的损失,添加乙二胺四乙酸并不会使TGase失活。以苯甲基氧化碳酰-L-谷氨酰胺甘氨酸(CBZ-Gln-Gly)为底物,得到TGase对底物(CBZ-Gln-Gly)的Km值为53.88 mmol/L,对底物(CBZ-Gln-Gly)的Vmax为0.98μmol/mL·min。对轮枝链霉菌SK4.001生物合成TGase的机理进行研究。研究发现TGase先以酶原的形式表达,随后酶原经发酵液中的一些水解蛋白酶的作用下水解成具有活性的成熟酶。离子交换层析法(填料:Fractogel EMD SO3-)可以分离纯化酶原。中性蛋白酶酶解酶原,发现酶原在一定条件下可以水解成成熟酶,但随着加入的蛋白酶浓度增大,酶解时间增长,酶原被酶解的程度也增高,同时非特异性切割也得到加强,成熟酶也随即被水解。用CTAB处理发酵液可以提高酶活,但CTAB并不是直接作用于酶原,其提高酶活的机理还有待于进一步研究。利用神经网络理论对轮枝链霉菌SK4.001产TGase过程进行建模。研究发现,BP神经网络能够较好地对整个发酵过程进行模拟和预测,网络模拟的最大相对误差为4.76%,网络预测的最大相对误差在13.99%,为轮枝链霉菌SK4.001发酵产TGase在线测量和控制提供了参考。更多还原

【Abstract】 Transglutaminase (TGase) is a kind of thiol enzyme that catalyzes acryl-transfer reaction, which introduces inter- or intra-molecular covalent crosslinks in many proteins, changing the quality of food products. TGase has been widely used in food industry.The effects of soybean cake powder hydrolyzates as nitrogen source on TGase biosynthesis of Streptoviticillium SK4.001 were studied. The experiment results showed that trypsin could hydrolyze soybean cake powder effectively. The amount of hydrolyzates added into fermented culture was 100%. The best hydrolysis conditions were that: temperature 35℃, pH 8.5, the concentration of soybean cake 10%, and the amount of enzyme 1000U per gram of substrate. After 42h’s fermentation, TGase’s activity could reach 5.04U/mL when soybean cake powder hydrolyzates were used as nitrogen source. So it could replace fish peptone as well.TGase from Streptoviticillium SK4.001 exhibited optimum activity at 35℃, pH 7.0 for CBZ-Gln-Gly. TGase was stable at 20-40℃, pH 6.0-7.0. TGase was inhibited by some common metal ions, but was not inhibited by EDTA. Kinetic parameters were determined in the reaction mixtures containing variable mounts of the substrate N-CBZ-Gln-Gly (0-30 mmol/L). The value of Michaelis constant (Km) and maximum velocity (Vmax) determined from Lineweaver-Burk plots were 53.88 mmol/L and 0.98μmol/mL·min, respectively.The zymogen of TGase (pro-TGase) was found during cultivation of Streptoviticillium SK4.001. The pro-TGase was purified by ion-exchange chromatography (Fractogel EMD SO3-). The pro-TGase was hydrolyzed by dispase under suitable condition. The pro-TGase and TGase were both hydrolyzed by dispase when the concentration of protease or the time of enzymolysis increased. TGase’s activity increased when CTAB was added into zymotic fluid fermented for 30h, but CTAB did not act on pro-TGase and the mechanism was not clear.The models of Streptoviticillium SK4.001’s fermention were constructed using neural network to train experimental data,and the predicted value of the models was compared with the measured value.The result showed that theses models could accurately predict the time course of cell growth,glycerol consumption,and TGase’s activity well during the fermentation.The maximum relative error of netwok’s simulation reached 4.76% compared with 13.99% of netwok’s prediction, and the models constructed could be put into practical use.更多还原