【作者】 吴昊；

【导师】 张洪斌；

【作者基本信息】 合肥工业大学 ， 制药工程， 2020， 硕士

【摘要】 右旋糖酐是葡萄糖经过脱水缩合形成的一种生物多糖,广泛应用于食品、医药、化工、污水处理等多个领域,在这些领域中发挥很重要的作用。右旋糖酐的衍生物右旋糖酐铁是一种临床应用价值较高的补铁剂。本课题在用酶法制备并获得低分子量右旋糖酐T5的基础上,通过氧化反应获得氧化型右旋糖酐,随后选用合适的氧化型右旋糖酐制备右旋糖酐铁并优化合成工艺,同时,对氧化型右旋糖酐和右旋糖酐铁的理化性质进行研究和分析。研究内容及分析结果如下:1、本课题对重组毕赤酵母产右旋糖酐酶的工艺进行进一步优化,随后对酶的热稳定性和酶法制备低分子量右旋糖酐进行研究。其中产右旋糖酐酶的最适工艺条件是每24h添加0.25%山梨醇和0.62%甲醇,pH值为5.08。在探究孵育温度对右旋糖酐酶活力的半衰期的影响实验中发现,30℃温度条件是比较适合右旋糖酐酶的长时间催化反应。通过研究底物浓度、酶浓度和反应时间对右旋糖酐酶催化降解高分子量右旋糖酐的影响实验,发现底物浓度过大不利于此酶催化反应,酶浓度的增加会影响底物降解速率,反应时间的延长虽然会使底物降解得更多,但是降解速率会随之减小。右旋糖酐酶对右旋糖酐的亲和力随其分子量的减小而降低。5%底物溶液加7.5U/mL右旋糖酐酶反应7h,此反应体系能够在较短时间内制备右旋糖酐T5,其回收率较高。2、用NaClO/NaBr和H2O2分别氧化右旋糖酐生成氧化型右旋糖酐DOB和DOP,研究结果表明随着投入氧化剂浓度的增加,DOB的羧基含量增长幅度要大于DOP的,DOB和DOP均发生略微降解,但DOB的降解程度要轻于DOP的。在一定氧化剂浓度范围内,DOP(H2O24g/100g)与铁(Ⅲ)离子的络合能力要弱于DOB(活性氯2g/100g)的。氧化反应会使右旋糖酐的结晶区的x结构向无定形转化,DOB的氧化主要发生在C-1位上的活泼羟基,DOP的氧化会在C-1位的羟基被氧化成羧基后,使C-1和C-2之间的连接键断裂,再继续将C-2位上的羟基氧化成羧基。3、通过单因素试验对右旋糖酐铁(DOBIC)的制备工艺进行条件优化,并对DOBIC进行理化性质分析。研究结果表明,DOBIC的络合反应最优条件是三氯化铁投入质量20g、pH9、反应温度为90℃、反应时间为2h。研究发现DOBIC的比原始右旋糖酐和DOB的表面更光滑紧实。DOBIC和DOB的热稳定性要优于原始右旋糖酐,DOBIC在30℃-203℃温度范围内具有较好的热稳定性。DOB与铁(Ⅲ)离子络合形成了铁核β-FeOOH结构,DOB中的羟基和羧基参与络合反应,DOBIC结晶性较差,呈无定形。DOBIC的分子量为70kDa,其易溶于水,且其游离铁的含量小于0.2%。更多还原

【Abstract】 Dextran is a kind of biological polysaccharide formed by the dehydration condensation of glucose.It is widely used in many fields such as food,medicine,chemical industry,sewage treatment and other fields,and plays an important role in these fields.One of the derivatives of dextran,the iron dextran is a kind of iron supplement with high clinical value.Based on the enzymatic preparation and obtaining low molecular weight dextran T5,this subject obtained oxidized dextran through oxidation reaction,and then selected the appropriate oxidized dextran to prepare iron dextran and optimizes the synthesis process.At the same time,the physical and chemical properties of oxidized dextran and iron dextran were studied and analyzed.The research content and analysis results were as follows:1.This subject further optimized the process of producing dextranase from recombinant Pichia pastoris.Then,studied the thermal stability of the enzyme and the process of preparing low molecular weight dextran by enzyme method.The optimal process conditions for producing dextranase were to add 0.25%sorbitol and 0.62%methanol every 24h,and the pH value is 5.08.In the experiment of exploring the influence of incubation temperature on the half-life of dextranase activity,it was found that the temperature condition of 30℃ is more suitable for the long-term catalytic reaction of dextranase.In the experiment to explore the effect of incubation temperature on the half-life of dextranase activity,it was found that the temperature condition of 30℃ was more suitable for the long-term catalytic reaction of dextranase.By studying the effect of substrate concentration,enzyme concentration and reaction time on the catalysis of dextranase to degrade high molecular weight dextran,it was found that excessive substrate concentration was not conducive to this enzyme-catalyzed reaction.The increase of the enzyme concentration accelerated the substrate degradation rate.Although the extension of the reaction time caused the substrate to degrade more,the degradation rate decreased accordingly.The affinity of dextranase for dextran decreased as its molecular weight decreased.The 5%substrate solution plus 7.5U/mL dextranase was reacted for 7h.This reaction system prepared dextran T5 in a relatively short time,and its recovery rate was high.2.NaClO/NaBr and H2O2 were used to oxidize dextran to generate oxidized dextran DOB and DOP,respectively.The research results showed that with the increase of the concentration of the input oxidant,the increase of DOB carboxyl content was greater than that of DOP.Both DOB and DOP degraded slightly,but the degradation degree of DOB was lighter than that of DOP.In a certain oxidant concentration range,the iron(Ⅲ)chelating capacity of DOP(H2O2 4g/100g)was weaker than that of DOB(active chlorine 2g/100g).The oxidation reaction would transform the structure of the crystalline region of dextran to amorphous.The oxidation of DOB mainly occurred at the active hydroxyl group at the C-1 position.The oxidation of DOP would break the bond between C-1 and C-2 after the hydroxyl group at C-1 position was oxidized to carboxyl,and then oxidized the hydroxyl group at the C-2 position to a carboxyl group.3.Through the single factor test,the preparation process of iron dextran(DOBIC)was optimized,and analyzed the physical and chemical properties of DOBIC.The results showed that the optimal conditions for DOBIC complexation reaction was ferric chloride input 20g,pH 9,reaction temperature 90℃,and reaction time 2h.The surface of DOBIC was smoother and firmer than that of the original dextran and DOB.The thermal stability of DOBIC and DOB was better than that of the original dextran.DOBIC had better thermal stability in the temperature range of 30℃-203℃.The complex of DOB and iron(Ⅲ)ions formed an iron core β-FeOOH structure.The hydroxyl and carboxyl groups in DOB participated in the complexation reaction.DOBIC had poor crystallinity and was amorphous.The molecular weight of DOBIC was 70kDa,it was easily soluble in water,and its free iron content was less than 0.2%.更多还原