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。
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.
引文
[1]中国科学院中国植物志编写委员会.中国植物志[M].北京：科学出版社,1989,41(1)：234
[2]Welinder K G, Larsen Y B. Covalent structure of soybean seed coat peroxidase[J]. Biochimica et Biophysica Acta,2004,1698:121-126
[3]王继强,张波.大豆皮的营养价值及在畜牧业中的应用[J].饲料博览,2004,2
[4]严群.大豆皮过氧化物酶的分离纯化研究[D].浙江：浙江大学,2005
[5]阙瑞琦.莲藕过氧化物酶的分离纯化、性质研究及固定化初探[D].重庆：西南大学,2008
[6]田国忠,李怀方,裘维蕃.植物过氧化物酶研究进展[J].武汉植物学研究进展,2001,19(4)：332-344
[7]Burel C, Berthe T, Mery J C,et al. Isoelectric focusing analysis of peroxidases in flax seedling hypocotyls grow in different light condition[J]. Plant Physiol Biochem,1994,32:853-860
[8]Lee M Y, Choi Y, Kim S S. Purification and immunological relationships of six radish isoperoxidases[J]. Plant Physiol Biochem,1994,32:259-265
[9]Asada K. Ascorbate peroxidase-a hydrogen peroxide-scavenging enzyme in plants[J]. Physiol Plant, 1992,85:235-241
[10]Buffard D, Breda C, Van Huystee, et al. Molecular cloning of complementary DNAs encoding two cationic peroxidases from cultivated peanut cells[J]. Proc Natl Acad Sci USA,1990,87:8874-8878
[11]刘稳.植物过氧化物酶超家族的分子结构[J].生命科学,2002,14(4)：212-214
[12]刘稳,李扬,高培基,等.过氧化物酶研究进展[J].纤维素科学与技术,2000,8(2)：50-64
[13]Intapruk C, Yamamoto K, Sekine M, et al. Regulatory sequences involved in the Peroxidase Gene expression in Arabidopsis Thaliana[J].Plan Cell Rep.1994,13:123-129
[14]Zin-Huang Liu, Mang-Jye Ger. Changes of Enzyme Activity. During Pollen Gemination in Maize, and possible Evidence of Lignin Synthesis[J]. Aust. J. Plant physiol.1997,24:329-335
[15]Meunier G, Meunier B. JAM Chem Soc,1985,107:2558
[16]Meunier G, Meunier B. J Biol Chem,1985,260:10567
[17]Jia J, Wong B, Wu A, et al. A method to construct a third-generation horseradish peroxidase biosensor:self-assembling gold nanoparticles to three-dimensional sol-gel network[J]. Anal.Chem. 2002,74:2217-2223
[18]严群,张建国,徐芝勇.大豆过氧化物酶研究进展[J].中国粮油学报,2005,20(3)：51-53
[19]Caza N, Bewtra J. K, Biswas N, et al. Removal of phenolic compounds from synthetic wasterwater using soybean peroxidase[J]. Water Research,1999,33(13):3012-3018
[20]Gijzen M. A deletion mutation at the eplocus causes low seed coat peroxidase activity in soybean[J]. Plant J.,1997,12(5):991-998
[21]Kamal J. K. A, Bhere D. V. Activity, stability and conformational flexibility of seed coat soybean peroxidase[J]. Journal of Inorganic Biochemistry,2003,94:236-242
[22]孙立水,高强.过氧化物酶的应用研究进展[J].化工技术与开发,2006,35(12)：13-17
[23]McEldoon J. P, Dordick J. S. Unusual Thermal Stability of Soybean Peroxidase[J]. Biotechno Prog., 1996,12(4):555-558
[24]Kamal J. K. A, Bhere D. V. Thermal and conformational stability of seed coat soybean peroxidase[J]. Biochemistry,2002,41(29):9034-9042
[25]Tsaprailis G, Chan D.W. S, English A.M. Conformational states in denaturants of cytochromec and horseradish peroxidases examined by fluorescence and circular Dichroism[J]. Biochemistry,1998,37(7):2004-2016
[26]SmishA.T, Sanders S.A,ThorneIey R.N.F, et al. Characterisation of a haem active-site mutant of horseradish peroxidase, Phe41-Val, with altered reactivity towards hydrogen peroxide and reducing substrates[J]. Eur. J. Biochem,1992,207:507-519
[27]Sessa D. J, Anderson R. L. Soybean peroxidases:purification and some propertiesfJ]. J. Agric. Food Chem.1981,29(5):960
[28]Miranda M. V, Ferandez-LahoreH. M, Dobrecky J, et al. The extractive purification of peroxidase from plant raw materials in aqueous two-phase systems[J]. Acta Biotechnol,1998,18(3):179
[29]刘金磊,苏涛,李典鹏,等.苦瓜过氧化物酶的提取分离及性质测定[J].广西科学,2007,14(4)：407-410
[30]范军,李纯,稽跃琴,等.辣根过氧化物酶的分离制备和纯化[J].安徽农业大学学报,1998,25(3)：313-316
[31]陈守文.酶工程[M].北京：科学出版社,2008,41-43
[32]季钟煜,费锦鑫.中性辣根过氧化物酶制法新进展[J].生命的化学,1996,16(3)：36
[33]王志兵,解丛林,张凤清,等.豆壳中过氧化物酶的提纯[J].食品研究与开发,2010,31(8)：159-162
[34]郭勇.酶工程[M].北京：科学出版社,2004,96-106
[35]刘稳,方靖,高培基.豆壳过氧化物酶的分离纯化及其性质研究[J].中国生物化学与分子生物学报,1998,14(5)：577-582
[36]刘均洪,邱龙辉,李俊峰,等.豆壳过氧化物酶的分离纯化新工艺研究[J].化学工程,2005,33(6)：31-34
[37]郭孝武.一种提取中草药化学成分的力法-超声提取法[J].天然产物研究与开发,1998,11(3)：37-40
[38]罗贵民.酶工程[M].北京：化学工业出版社,2003,56
[39]金锋.微孔淀粉-海藻酸钠固定化木瓜蛋白酶的研究[J].中国酿造,2009,7,83-85
[40]张伟,杨秀山.酶的固定化技术及其应用[J].自然杂志,2000,22(5)：282-286
[41]屈建莹,陈文静.明胶固定辣根过氧化物酶制备H202传感器[J].化学学报,2010,68(3)：257-262
[42]杨玉玲.酶固定化技术及载体材料研究新进展[J].粮油食品科技,2001,9(5)：22-25
[43]袁勤生,赵健,王维育.应用酶学[M].上海：华东理工大学出版社,1994,229-235
[44]李鸿玉,厉重先,李祖明.海藻酸钠固定化果胶酶的研究[J].食品科技,2010,35(4)：21-24
[45]左鹏,于少明,杨杰茹,等.辣根过氧化物酶固定化载体材料的研究进展[J].材料导报,2007,21(11)：46-48
[46]洪伟杰,张朝晖,芦国营.辣根过氧化物酶固定化新进展[J].现代食品科技,2006,22(1)：177-180
[47]程俊.层柱黏土固定辣根过氧化物酶及其在含酚废水处理中的应用[D].安徽：合肥工业大学,2006
[48]陈姗姗,仇农学.阴离子交换树脂固定化果胶酶研究[J].陕西师范大学学报(自然科学版),2008,36(2)：102-105
[49]袁新跃.树脂固定化多酚氧化酶及其催化茶多酚形成茶黄素的研究[D].北京：中国农业科学院.2009
[50]吴叶明.木素过氧化物酶的生产、固定化及其在染料降解中应用[D].浙江：浙江工业大学,2008
[51]程晓滨,荚荣,李平生,等.锰过氧化物酶的耦合固定化及其性质研究[J].生物工程学报,2007,23(1)：90-95
[52]张丽华.氯过氧化物酶的固定化及其应用研究[D].陕西：陕西师范大学,2008
[53]袁新跃,江和源,张建勇,等.不同树脂载体固定化多酚氧化酶的研究[J].安徽农业科学, 2009,37(7)：2832-2834
[54]王燕,张凤宝,朱怀工,等.大孔阴离子树脂DEAE-E/H固定化氨基酰化酶的研究[J].离子交换与吸附,2005,21(3)：248-254
[55]印明善,马秀贞,叶乃芝,等.氧化铝为载体的固定化葡萄糖异构酶某些性质的研究[J].生物工程学报,1986,2(1)：52-56
[56]王娜,朱启忠,任延刚,等.海藻酸钠固定化木聚糖酶的研究[J].北方园艺,2009,5：92-94
[57]简-路易斯,雷蒙.酶活检测[M].北京：化学工业出版社,2008,1-6
[58]魏永锋,闫宏涛.催化荧光光度法测定辣根过氧化物酶及甲胎蛋白[J].分析化学研究简报,2000,28(1)：99-101
[59]李忠光,龚明.愈创木酚法测定植物过氧化物酶活性的改进[J].植物生理学通讯,2008,44(2)：323-324
[60]于彤,薛淑云,段春生.以对苯二酚为底物伏安法测定辣根过氧化物酶[J].精细石油化工,2005,6：61-64
[61]马莺,工静,牛天骄.功能性食品活性成分测定[M].北京：化学工业出版社,2005
[62]黄文华.应用Phenyl Bio-Sephar FF进行辣根过氧化物酶精分离的研究[J].生命科学仪器,2005,3(4)：49-51
[63]葛宇,许时婴,王璋.响应面法优化冰淇淋复配稳定剂配方研究[J].食品科学,1995,191(11)：5-9
[64]慕运动.响应面方法及其在食品工业中的应用[J].郑州工程学院院报,2002,22(3)：91-94
[65]唐璇,李稳宏,李新生,等.黄姜黄色素在大孔树脂上的吸附特性及热力学研究[J].西北大学学报(自然科学版),2008,38(4)：579-582
[66]Maliyekkal S M, Shukla S, Philip L, et al. Enhanced Fluoride Removal from Drinking Water by Magnesia-amended Activated Alumina Granules[J]. Chem.Eng.J.,2008,140(1/3):183-192
[67]Diban N, Ruiz G, Urtiaga A, et al. Recovery of the Main Pear AromaCompound by Adsorption/Desorption onto Commercial Granular Activated Carbon:Equilibrium and Kinetics[J]. J.Food Eng.,2008,84(1):82-91
[68]何炳林,黄文强.离子交换与吸附树脂[M].上海：上海科技教育出版社,1995：174
[69]天津大学物理化学教研室.物理化学(第四版上册)[M].北京：高等教育出版社,2001,224
[70]Sari A, Tuzen M. Biosorption of Total Chromium from Aqueous Solution by Red Algae(Ceramium virgatum):Equilibrium, Kinetic and Thermodynamic Studies[J]. J.Hazard.Mater.,2008,160(2/3): 349-355
[71]刘步云,姚忠,周治,等.螫合树脂对铜离子的吸附动力学和热力学[J].过程工程学报,2009.9(5)：865-870
[72]Kennedy L J, Vijaya J J, Sekaran G, et al. Equilibrium, Kinetic and Thermodynamic Studies on the Adsorption of m-Cresol onto Micro and Mesoporous Carbon[J]. J.Hazard.Mater.,2007,149(1): 134-143