大豆皮过氧化物酶提取、分离、纯化及其固定化研究

【作者】 高晶晶；

【导师】 高新；

【作者基本信息】 西北大学 ， 化学工艺， 2011， 硕士

【摘要】 目前我国大豆皮产量逐年增加,但是对大豆皮的利用却不够充分,因此需要合理的开发和利用大豆皮,提高其经济效益和社会效益。大豆皮过氧化物酶是一种活性很强的过氧化物酶,它具有广泛的催化氧化作用,因此对大豆皮过氧化物酶的研究具有重要意义。本课题在大豆皮过氧化物酶的提取、分离纯化以及固定化方面做了系统的研究。主要研究成果如下：(1)对大豆皮过氧化物酶采用PBS缓冲液(磷酸缓冲液,配制方法见2.1.3)提取和超声提取两种方法进行了对比研究,在单因素的基础上采用响应面优化分析得到了两种提取工艺的优化参数：PBS缓冲液提取工艺：提取温度38℃,提取时间6h,液料比20mL/g,提取液浓度0.02mol/L,提取次数3次,在此条件下1g大豆皮可提取出的酶活为2608.7U,酶的比活力为89.03U/mg；超声提取工艺：超声功率70W,提取温度38℃,提取时间41min,液料比20mL/g,提取液浓度0.02mol/L,提取次数3次,在此条件下1g大豆皮可提取出的酶活为3409.3U,酶的比活力89.96U/mg。实验数据表明超声提取法的提取时间短、提取酶活较高。(2)采用硫酸铵和丙酮分级沉淀法、硫酸锌除杂以及微孔滤膜过滤等方法对提取得到的粗酶进行纯化,得到的酶纯化倍数为29.01,纯酶的Rz值为1.89。(3)采用了氧化铝吸附-海藻酸钠包埋-戊二醛交联法和大孔树脂吸附-戊二醛交联法两种方法对大豆皮过氧化物酶进行固定化研究,并对树脂法的吸附过程进行了热力学和动力学研究。氧化铝吸附-海藻酸钠包埋-戊二醛交联法优化工艺条件为：纯酶10mg,氧化铝用量1.5g,吸附时间1h,海藻酸钠浓度4%,CaCl2浓度0.02 mol/L,戊二醛浓度0.3%,交联时间30min,此条件下酶的固定化率为77.3%；大孔树脂吸附-戊二醛交联法的优化工艺条件为：LX-42C型树脂1g(干重),震荡速度125r/min,温度35℃,吸附时间4h,给酶量30mg,戊二醛浓度0.4%,交联时间‘1h,此条件下酶的固定化率为89.47%。大孔树脂吸附过程的热力学和动力学研究表明吸附过程符合Langmuir吸附模型,且吸附过程是自发进行的吸热过程,其吸附速率符合准二级动力学方程。最后通过对固定化酶和游离酶的性质比较可知,固定化酶的热稳定性、酸碱稳定性和贮藏稳定性等均要高于游离酶,且固定化酶可以重复多次使用。更多还原

【Abstract】 The yield of soybean hull coats was arising year by year at present in our country,but its utilization was inadequate,so it was needed to develop and use of soybean hull coats reasonable to increase benefits of economic and social. Soybean hull peroxidase (SHP)was high activity and it was used broadly in oxidation,so there was significance to study of SHP.The thesis has done a systematic study on the extraction,isolation,purification and immobilization of peroxidase from the soybean hull coats.The mainly results as follows:(1) Soybean hull peroxidase was extracted by two methods which were using PBS buffer and ultrasonic wave,on the basis of single factor experiment, then compared of those methods using response surface analysis to get the optimal condition of the extraction.The optimal conditions of the PBS extraction were as follows:the temperature 38℃,the extraction time 6h,the ratio of solution and soybean hull at 20mL/g,PBS concentration 0.02mol/L,3 times.The enzyme activity was 2608.7U and enzyme specific activity was 89.03U/mg based on the above optimal conditions from 1g soybean hull coats.The optimal conditions of the ultrasonic wave extraction were as follows:ultrasonic power 70 W, the temperature 38℃, the extraction time 41min, the ratio of solution and soybean hull at 20mL/g,PBS concentration 0.02mol/L,3 times. The enzyme activity was 3409.3U and enzyme specific activity was 89.96U/mg based on the above optimal conditions from 1g soybean hull coats.The conclusion showed that the ultrasonic wave extraction was time-saving and high activity.(2)Using ammonium sulfate fractionated precipitation, acetone fractionated precipitation, zinc sulfate impurition and membrane filtration to isolate and purificat the crude SHP that was purified by 29.01,the value of Rz was 1.89.(3)The SHP was immobilized by using alumina adsorption-sodium alginate entrapping-glutaraldehyde crosslinking and macroporous resins adsorption-glutaraldehyde crosslinking, then studied the thermodynamics and dynamics for the adsorption process.The optimal conditions of the alumina adsorption-sodium alginate entrapping-glutaraldehyde crosslinking were as follows:pure enzyme 10mg, alumina 1.5g,the adsorption time lh, sodium alginate concentration 4%, calcium chloride concentration 0.02 mol/L, glutaraldehyde concentration 0.3%, crosslinking time 30min. The yield of immobilized was 77.3% based on the above optimal conditions.The optimal conditions of the macroporous resins adsorption-glutaraldehyde crosslinking were as follows:resin of LX-42C 1g(dry weight), the rate of oscillation 125r/min, the temperature 35℃, the adsorption time 4h, enzyme content given 30mg, glutaraldehyde concentration 0.4%, crosslinking time 1h. The yield of immobilized was 89.47% based on the above optimal conditions.The study of the thermodynamics and dynamics for adsorption process showed that was fit for Langmuir Model, the adsorption process was endothermic and spontaneous reaction, the rate of adsorption had well shown in agreement with quasisecondary kinetin equation.By comparing the characters of free SHP and immobilized SHP to find that the immobilized SHP was used many times and more stable in temperature, pH and storage.更多还原