氨基化Fe_3O_4-SiO_2固定化磷脂酶A_1
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摘要
以磁性Fe_3O_4-SiO_2纳米颗粒为载体,研究固定化条件对磷脂酶活力的影响,通过响应面试验得到最优固定化条件为:固定化pH 6.7、固定化温度30℃、固定化时间7.9 h、戊二醛质量分数8.3%、加酶量8.2 mL/50 mg,在此条件下酶活力回收率能达到63.6%,蛋白固载率68%。并对制备的固定化磷脂酶的化学组分、形态结构和粒径进行分析,结果表明磷脂酶固定化效果较好,粒径均一,载体平均粒径为200 nm左右。固定化酶热稳定性、pH值稳定性和贮藏稳定性增强,最适反应温度为50℃,最适pH 6.0,重复操作10次后保留60%以上的初始酶活力。
In this investigation, Fe_3O_4-SiO_2 magnetic nanoparticles were used as a carrier to study the effects of different operating conditions on the immobilization of phospholipase A1. The optimal conditions determined using response surface methodology were as follows: pH 6.7, temperature 30 ℃, immobilization time 7.9 h, glutaraldehyde concentration 8.3%, and amount of enzyme 8.2 mL/50 mg. Under these conditions, the recovery of enzymatic activity was as high as 63.6% and the percentage of protein immobilization was 68%. The chemical composition, morphological structure and particle size of the immobilized phospholipase were characterized. The results showed that the ef?ciency of phospholipase immobilization was good; uniform particle size distribution was observed with a mean particle size of about 200 nm. The thermal, pH and storage stability of the immobilized enzyme were enhanced relative to the free one. The optimum reaction temperature for the immobilized enzyme was 50 ℃ and optimum pH 6.0. Over 60% of the enzymatic activity remained after tenth repeated use.
In this investigation, Fe_3O_4-SiO_2 magnetic nanoparticles were used as a carrier to study the effects of different operating conditions on the immobilization of phospholipase A1. The optimal conditions determined using response surface methodology were as follows: pH 6.7, temperature 30 ℃, immobilization time 7.9 h, glutaraldehyde concentration 8.3%, and amount of enzyme 8.2 mL/50 mg. Under these conditions, the recovery of enzymatic activity was as high as 63.6% and the percentage of protein immobilization was 68%. The chemical composition, morphological structure and particle size of the immobilized phospholipase were characterized. The results showed that the ef?ciency of phospholipase immobilization was good; uniform particle size distribution was observed with a mean particle size of about 200 nm. The thermal, pH and storage stability of the immobilized enzyme were enhanced relative to the free one. The optimum reaction temperature for the immobilized enzyme was 50 ℃ and optimum pH 6.0. Over 60% of the enzymatic activity remained after tenth repeated use.
引文
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[2]段书平.大豆油酶法脱胶的生产应用[J].中国油脂,2013,38(8):11-13.DOI:10.3969/j.issn.1003-7969.2013.08.004.
[3]LIU L,WANG J,TAO H,et al.Study on enzymatic degumming technology for peanut oil with lecitase ultra[J].Shandong Agricultural Sciences,2016,48(2):108-111.DOI:10.14083/j.issn.1001-4942.2016.02.026.
[4]余榛榛,常明,刘睿杰,等.磷脂酶C在酶法脱胶中的研究进展[J].中国油脂,2013,38(7):19-22.DOI:10.3969/j.issn.1003-7969.2013.07.006.
[5]YU D,MA Y,JIANG L,et al.Stability of soybean oil degumming using immobilized phospholipase A(2)[J].Journal of Oleo Science,2014,63(1):25-30.DOI:10.5650/jos.ess13105.
[6]毛程鑫,李桂华,薛武军,等.菜籽油酶法脱胶的研究[J].中国粮油学报,2016,31(8):75-79.DOI:10.3969/j.issn.1003-0174.2016.08.013.
[7]徐赢华,王国敬,李春,等.酶法脱胶在植物油脂精炼中的应用进展[J].农业工程学报,2015,31(23):269-276.DOI:10.11975/j.issn.1002-6819.2015.23.036.
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[9]QU Y,SUN L,LI X,et al.Enzymatic degumming of soybean oil with magnetic immobilized phospholipase A2[J].LWT-Food Science and Technology,2016,73:290-295.DOI:10.1016/j.lwt.2016.06.026.
[10]水龙龙,操丽丽,庞敏,等.大孔树脂固定化磷脂酶A1及其用于菜籽油脱胶工艺的优化[J].食品科学,2018,39(4):241-247.DOI:10.7506/spkx1002-6630-201804036.
[11]刘倩倩,邱永乾,刘炯钦,等.大孔树脂固定化磷脂酶D[J].工业微生物,2016,46(3):25-30.DOI:10.3969/j.issn.1001-6678.2016.03.005.
[12]陈丽萍,宋鹏,解桂东,等.海藻酸钠固定化磷脂酶用于大豆油脱胶[J].农业机械,2013(17):53-56.DOI:10.16167/j.cnki.1000-9868.2013.17.005.
[13]占剑峰,胡孝明,王蔚新,等.聚乙烯醇-海藻酸钠固定化磷脂酶A1的研究[J].食品科技,2016,41(7):8-13.DOI:10.13684/j.cnki.spkj.2016.07.002.
[14]于殿宇,马莺,刘晶,等.高碘酸钠氧化法固定化磷脂酶A1的研究[J].食品工业科技,2012,33(7):188-190.DOI:10.13386/j.issn1002-0306.2012.07.006.
[15]于殿宇,马莺,张欢,等.磁性固定化PLA1的制备及其在脱胶应用中的研究[J].食品工业科技,2014,35(19):128-131.DOI:10.13386/j.issn1002-0306.2014.19.019.
[16]WU X C,ZHANG Y,WU C Y,et al.Preparation and characterization of magnetic Fe3O4/CRGO nanocomposites for enzyme immobilization[J].Transactions of Nonferrous Metals Society of China,2012,22(10):s162-s168.DOI:10.1016/S1003-6326(12)61703-8.
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[18]杨兆壬.表面羧基化Fe3O4磁性微球固定化CA酶的制备与研究[D].泉州:华侨大学,2014.
[19]谈昭君.磁性载体的制备及其固定化纤维素酶的研究[D].兰州:兰州理工大学,2015.
[20]李晔,张恒建,廖明霞,等.氨基酸分子改性的SiO2杂化材料用于胰蛋白酶固定化[J].高等学校化学学报,2011,32(5):1100-1105.DOI:10.3969/j.issn.1674-9057.2012.04.022.
[21]阮贵华,刘玉花,陈正毅,等.Fe3O4磁性纳米粒子固定化胰蛋白酶[J].桂林理工大学学报,2012,32(4):569-574.DOI:10.3969/j.issn.1674-9057.2012.04.022.
[22]XIE W,ZANG X.Covalent immobilization of lipase onto aminopropyl-functionalized hydroxyapatite-encapsulated-γ-Fe2O3,nanoparticles:a magnetic biocatalyst for interesterification of soybean oil[J].Food Chemistry,2017,227:397-403.DOI:10.1016/j.foodchem.2017.01.082.
[23]唐玲利,黄东萍,侯路琦,等.磁性纳米粒子的制备及对α-淀粉酶的固定化研究[J].中国卫生检验杂志,2014(10):1369-1372.
[24]MA’MANI L,SHEYKHAN M,HEYDARI A.Nanosilver embedded on hydroxyapatite-encapsulatedγ-Fe2O3:superparamagnetic catalyst for chemoselective oxidation of primary amines to N-monoalkylated hydroxylamines[J].Applied Catalysis A:General,2011,395(1):34-38.DOI:10.1016/j.apcata.2011.01.015.
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[27]易笑生,胡铁,冯超,等.Fe3O4-SiO2载体制备和脂肪酶固定化条件优化[J].食品工业科技,2015,36(1):235-238.DOI:10.13386/j.issn1002-0306.2015.01.040.
[28]WANG F,GUO C,YANG L R,et al.Magnetic mesoporous silica nanoparticles:fabrication and their laccase immobilization performance[J].Bioresource Technology,2010,101(23):8931-8935.DOI:10.1016/j.biortech.2010.06.115.
[29]NAWANI N,SINGH R,KAUR J.Immobilization and stability studies of a lipase from thermophilic Bacillus sp:the effect of process parameters on immobilization of enzyme[J].Electronic Journal of Biotechnology,2007,9(5):559-565.DOI:10.2225/vol9-issue5-fulltext-9.
[30]朱珊珊,李铁,王永华,等.磷脂酶A1的固定化及应用研究[J].中国油脂,2010,35(12):33-37.
[31]吕雯姣,余兆硕,唐柳欢,等.响应面优化超声波辅助水酶法提取荸荠多糖[J].农产品加工,2016(23):1-5.DOI:10.16693/j.cnki.1671-9646(X).2016.12.001.
[32]罗磊,张冰洁,朱文学,等.响应面试验优化超声辅助提取金银花叶黄酮工艺及其抗氧化活性[J].食品科学,2016,37(6):13-19.DOI:10.7506/spkx1002-6630-201606003.
[33]施瑛,裴斐,周玲玉,等.响应面法优化复合酶法提取紫菜藻红蛋白工艺[J].食品科学,2015,36(6):51-57.DOI:10.7506/spkx1002-6630-201506010.
[34]占剑峰.磷脂酶A1的发酵制备、分离纯化及催化特性的研究[D].合肥:合肥工业大学,2013.
[35]张晓闻,刘彦琳.柠檬酸修饰Fe3O4磁性粒子的制备与表征[J].辽宁工业大学学报(自然科学版),2016,36(4):277-280.DOI:10.15916/j.issn1674-3261.2016.04.17.
[36]ZHAO J,LUQUE R,QI W,et al.Facile surfactant-free synthesis and characterization of Fe3O4@3-aminophenol-formaldehyde core-shell magnetic microspheres[J].Journal of Materials Chemistry A,2014,3(2):519-524.DOI:10.1039/C4TA03821E.
[37]李涛,张龙,陈颖,等.微米聚苯胺/Fe3O4空心球的制备及吸波性能[J].高等学校化学学报,2015,36(4):772-780.DOI:10.7503/cjcu20141000.
[38]LIU F,NIU F,PENG N,et al.Synthesis,characterization,and application of Fe3O4@SiO2-NH2 nanoparticles[J].RSC Advances,2015,5(23):18128-18136.DOI:10.1039/C4RA15968C.