大孔树脂吸附–交联法固定脂肪酶
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摘要
【目的】为基于大孔树脂吸附结合环氧交联剂交联法固定脂肪酶等工业用酶奠定基础。【方法】使用大孔树脂吸附,而后环氧交联剂交联的方法进行脂肪酶的固定化,研究各因素对吸附–交联固定化的影响,并采用响应面法对固定化条件进行优化,制备固定化酶并考察其稳定性。【结果】筛选出大孔树脂HPD750为载体,聚乙二醇二缩水甘油醚为交联剂。最佳固定化条件为:吸附温度45℃,给酶量60 mg·g–1,交联温度30℃,交联时间12.5 h,pH6.36,交联剂体积分数为0.7%。由上述条件制备所得的固定化酶活力为565.31 U·g–1,酶活力回收率为32.16%。与游离酶相比,固定化酶的热稳定性和酸碱稳定性均有明显提升;连续操作10次,固定化酶活力仍保留34.86%,操作稳定性较好;4℃条件下储存30 d,固定化酶活力仍保留64.81%。【结论】大孔树脂HPD750为载体,聚乙二醇二缩水甘油醚为交联剂制备的固定化脂肪酶热稳定性、酸碱稳定性均得到显著提升,且具有良好的操作及储存稳定性。
【Objective】To provide a basis for immobilization of industrial enzymes such as lipase using macroporous resin for adsorption and epoxy crosslinker for crosslinking.【Method】The immobilization of lipase was performed through adsorption using macroporous resin as the carrier and epoxy crosslinker as the crosslinking agent. We investigated the effects of different factors on adsorption-crosslinking immobilization,and used response surface design to optimize the immobilization conditions. The immobilized enzyme was prepared and its stability was investigated.【Result】Macroporous resin HPD750 was selected as the carrier,and poly(ethylene glycol) diglycidyl ether was selected as the crosslinker. The optimal conditions of lipase immobilization were as follows: Adsorption temperature 45 ℃, enzyme addition amount 60 mg·g–1, crosslinking temperature 30 ℃, crosslinking time 12.5 h, pH6.36, and comcentration of crosslinking agent 0.7%. Under these optimized conditions, the immobilized lipase activity was 565.31 U·g–1 and the recycled rate of lipase activity was 32.16%. Compared with free lipase, the immobilized lipase exhibited obviously better thermal stability and pH stability. The immobilized lipase was of good operation stability and remained 34.86% of the original activity after repeated usage for 10 times. The immobilized lipase also exhibited good storage stability and remained 64.81% of the original activity after storage at 4 ℃ for 30 days.【Conclusion】Using macroporous resin HPD750 as the carrier and poly(ethylene glycol) diglycidyl ether as the crosslinker, the immobilized enzyme has significantly improved thermal stability and pH stability, and it also exhibits good operation stability and storage stability.
【Objective】To provide a basis for immobilization of industrial enzymes such as lipase using macroporous resin for adsorption and epoxy crosslinker for crosslinking.【Method】The immobilization of lipase was performed through adsorption using macroporous resin as the carrier and epoxy crosslinker as the crosslinking agent. We investigated the effects of different factors on adsorption-crosslinking immobilization,and used response surface design to optimize the immobilization conditions. The immobilized enzyme was prepared and its stability was investigated.【Result】Macroporous resin HPD750 was selected as the carrier,and poly(ethylene glycol) diglycidyl ether was selected as the crosslinker. The optimal conditions of lipase immobilization were as follows: Adsorption temperature 45 ℃, enzyme addition amount 60 mg·g–1, crosslinking temperature 30 ℃, crosslinking time 12.5 h, pH6.36, and comcentration of crosslinking agent 0.7%. Under these optimized conditions, the immobilized lipase activity was 565.31 U·g–1 and the recycled rate of lipase activity was 32.16%. Compared with free lipase, the immobilized lipase exhibited obviously better thermal stability and pH stability. The immobilized lipase was of good operation stability and remained 34.86% of the original activity after repeated usage for 10 times. The immobilized lipase also exhibited good storage stability and remained 64.81% of the original activity after storage at 4 ℃ for 30 days.【Conclusion】Using macroporous resin HPD750 as the carrier and poly(ethylene glycol) diglycidyl ether as the crosslinker, the immobilized enzyme has significantly improved thermal stability and pH stability, and it also exhibits good operation stability and storage stability.
引文
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[12]侯爱军,徐冰斌,梁亮,等.改进铜皂-分光光度法测定脂肪酶活力[J].皮革科学与工程,2011,21(1):22-27.
[13]LEI L,BAI Y,LI Y,et al.Study on immobilization of lipase onto magnetic microspheres with epoxy groups[J].J Magn Magn Mater,2009,321(4):252-258.
[14]HASAN F,SHAH A A,HAMEED A.Industrial applications of microbial lipases[J].Enzyme Microb Technol,2006,39(2):235-251.
[15]JE H H,NOH S,HONG S G,et al.Cellulose nanofibers for magnetically-separable and highly loaded enzyme immobilization[J].Chem Eng J,2017,323(1):425-433.
[16]徐珊,李任强,郑振华,等.脂肪酶的包埋和交联固定化研究[J].云南农业大学学报(自然科学版),2017,32(6):1-9.
[17]钱明华.一种经济、高效环氧树脂及交联剂固定化脂肪酶的研究[D].广州:暨南大学,2018.
[2]WU Z,QI W,WANG M,et al.Lipase immobilized on novel ceramic supporter with Ni activation for efficient cinnamyl acetate synthesis[J].J Mol Catal B:Enzym,2014,110:32-38.
[3]LI Y,WANG W,HAN P.Immobilization of Candida,sp.99-125 lipase onto silanized SBA-15 mesoporous materials by physical adsorption[J].Korean J Chem Eng,2014,31(1):98-103.
[4]ARAVINDAN R,ANBUMATHI P,VIRUTHAGIRI T.Lipase applications in food industry[J].Indian J Biotechnol,2007,6(2):141-158.
[5]SHELDON R A.Enzyme immobilization:The quest for optimum performance[J].Adv Synth Catal,2007,349(8/9):1289-1307.
[6]贾存勤,李阳春,屠鹏飞,等.HPD系列大孔吸附树脂预处理方法研究[J].中国中药杂志,2005,30(18):1425-1427.
[7]LI C,ZHANG G,LIU N,et al.Preparation and properties of rhizopus oryzae lipase immobilized using an adsorption-crosslinking method[J].Int J Food Prop,2016,19(8):1776-1785.
[8]KILINC A,TEKE M,ONAL S,et al.Immobilization of pancreatic lipase on chitin and chitosan[J].Prep Biochem Biotechnol,2006,36(2):153-163.
[9]DOSANJH N S,KAUR J.Immobilization,stability and esterification studies of a lipase from a Bacillus sp.[J].Biotechnol Appl Biochem,2002,36:7-12.
[10]SCHOEVAART R,WOLBERS M W,GOLUBOVIC M,et al.Preparation,optimization,and structures of crosslinked enzyme aggregates(CLEAs)[J].Biotechnol Bioeng,2004,87(6):754-762.
[11]ALNOCH R,RODRIGUES DE MELO R,PALOMO J,et al.New tailor-made alkyl-aldehyde bifunctional supports for lipase immobilization[J].Catalysts,2016,6(12):191.
[12]侯爱军,徐冰斌,梁亮,等.改进铜皂-分光光度法测定脂肪酶活力[J].皮革科学与工程,2011,21(1):22-27.
[13]LEI L,BAI Y,LI Y,et al.Study on immobilization of lipase onto magnetic microspheres with epoxy groups[J].J Magn Magn Mater,2009,321(4):252-258.
[14]HASAN F,SHAH A A,HAMEED A.Industrial applications of microbial lipases[J].Enzyme Microb Technol,2006,39(2):235-251.
[15]JE H H,NOH S,HONG S G,et al.Cellulose nanofibers for magnetically-separable and highly loaded enzyme immobilization[J].Chem Eng J,2017,323(1):425-433.
[16]徐珊,李任强,郑振华,等.脂肪酶的包埋和交联固定化研究[J].云南农业大学学报(自然科学版),2017,32(6):1-9.
[17]钱明华.一种经济、高效环氧树脂及交联剂固定化脂肪酶的研究[D].广州:暨南大学,2018.