【作者】 李伟；

【导师】 郑国钧； 尤敏；

【作者基本信息】 北京化工大学 ， 生物工程（专业学位）， 2015， 硕士

【摘要】 (-))γ-内酰胺是一种重要的化学中间体,主要可以用来制备阿巴卡韦和帕拉米韦等药物。本文主要研究内容为重组大肠杆菌发酵生产(+)γ-内酰胺酶的条件优化以及(+)γ-内酰胺酶的固定化方法研究。我们在15L发酵罐中通过对不同培养条件的实验比较,最终成功探索出一种适合于重组大肠杆菌生长产(+)γ-内酰胺酶的方法——双程培养双程诱导法,即在37℃下培养菌种至对数生长期开始时升温至42℃培养至放罐,在对数期结束后加入乳糖进行诱导约6h后加入等量乳糖再诱导一次,经过重复实验反复比较发现此法发酵所得(+)γ-内酰胺酶酶量最大。全细胞转化实验表明：0.16g湿菌体在50℃下反应1h催化2ml,5g/L的(±)γ-内酰胺ee值可达95%以上,此结果可以满足工业化生产需求。以离子交换树脂和氨基树脂(HA树脂)为固定化载体对酶的固定化进行了研究,结果表明：离子交换树脂在200g/L的高浓度底物反应下酶容易脱落,固定化酶重复使用次数较少且产物收率较低；HA树脂则呈现出较好的固定化效果,其酶活可达1200 U/g。对HA树脂为载体的固定化条件进行了研究,优化后的最佳条件为：交联温度30℃,戊二醛浓度4%,交联时间为2h,在此交联条件下树脂完成固定化反应后载酶量达到最大；最佳固定化条件为在30℃下固定化6h树脂载酶量可达最大。固定化后酶的Km值从3.93g/L升高到14.54g/L,Vmax从0.89g/L*min下降到0.63g/L*min;最适温度保持不变均为80℃,固定化后耐热性有所下降；最适pH保持不变均为8.0,固定化后pH耐受性大幅增强,在4-10的pH范围酶活几乎不受影响；进一步研究发现固定化后酶的表观活化能从40.03kJ/mol降低到33.61kJ/mol,实验说明固定化酶的反应不是受酶动力学的影响而是受到传质影响。重复实验显示50次反应后仍然有50%的酶活剩余,该结果可以满足工业化生产要求。本文还研究了新型固定化酶载体的制备及其运用。通过环氧氯丙烷与氧化石墨烯反应,对氧化石墨烯的羧基进行环氧化,研究结果表明修饰后的氧化石墨烯载体具有良好的应用前景。更多还原

【Abstract】 (-)γ-lactam product can be used for the preparation of carbon ring nucleoside analogues, such as abacavir and peramivir.In this paper, we mainly studied the (+)γ-lactamase fermentation conditions optimization as well as the (+)γ-lactamase enzyme immobilization methods.In the fermentation tank (15L), we successfully developed a method suitable for growth of E. coli after trying different culture conditions. Firstly, we cultivated the E. coli until the beginning of logarithmic phase at 37℃,then we raised the cultivated temperature to 42℃ until the end of fermentation, in the meantime, we added lactose to induce producing enzyme at the end of logarithmic phase and once more we added the same amount of lactose after 6h, finally we obtained the max enzyme activity. The experiment of whole cell demonstrated that e.e value can reach more than 95% under the condition of 50℃,1h, and the reaction system consist of 0.16g wet bacteria,2ml substrate (5g/L), the results can meet the demand of industrial production.Then we studied ion exchange resin and HA resin as the immobilized carrier. We found that ion exchange resin can not be used in high concentration(200g/L) of substrate, because the enzyme molecule released easily from the carrier in high concentration of substrate. HA resin demonstrated better effects in immobilization experiment, it is 1200U/g of the enzyme activity capacity. The best crosslinking temperature, crosslinking time and glutaraldehyde concentration were 30℃,6h and 4%, respectively. Immobilized enzyme kinetic experiments showed that the Km value increased from 3.93g/L to 14.54g/L, and the Vmax decreased from 0.89g/L to 0.63g/L The optimum temperature (80℃)and pH value (pH 8.0) of both the free and immobilized enzyme were the same. However, the pH tolerance range increased after immobilization from pH 7.0-9.0 to pH 4.0-10.0. Subsequently, the values of the apparent activation energy (Ea) of the free and immobilized (+)Y-lactamase were 40.03kJ/mol and 33.61 kJ/mol, respectively. Experimental studies demonstrated that the best operating temperature is 50℃ and the resin and substrate consumption ratio is about 2:1. A reusability assay demonstrated that 50% of the enzyme activity remained after 60 repeated batch experiments. Experimental studies showed that the industrial production prospects is considerable of the immobilized enzyme.In addition, we also studied the new type of enzyme carrier. We modified the carboxyl of graphene oxide with epoxy chloropropane in order to obtain a new enzyme immobilization material.The infrared spectrum scanning results showed that epoxy chloropropane was reacted successfully on graphene oxide. But it is difficult to put the new carrier into application, because the machining process of graphene oxide is complex and lead to a high cost.更多还原