Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2020, Vol. 40 Issue (5): 57-68    DOI: 10.13523/j.cb.1912027
    
Immobilization of Marine Candida Lipase Using Novel Epoxy Cross-linker and Amino Carrier
ZHU Heng1,2,3,ZHANG Ji-fu4,ZHANG Yun1,HU Yun-feng1,3,**()
1 CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences,Guangzhou 510301,China
2 University of Chinese Academy of Sciences, Beijing 100049, China
3 Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou),Guangzhou 511458,China
4 Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,China
Download: HTML   PDF(1854KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

After immobilization, the stability and environmental tolerance of the free enzyme were optimized, which can improve the utilization rate of enzymes and reduce the production costs in diverse fields such as food, medicine, chemical industry, environment and leather, and thus possesses great application potentials. The utilization of novel crosslinking agents in immobilized enzyme process has greatly advanced the study of immobilized enzymes.The optimized crosslinking and immobilization conditions were obtained by using the new crosslinking agent polyethylene glycol diglycidyl ether (PEGDGE), which was immobilized with the amino carrier LX-1000HA, and by using single factor and orthogonal experiment optimization: crosslinking temperature 30℃, crosslinking time 2h, crosslinking agent concentration 0.75%, pH 7.0, enzyme amount 800U, carrier volume 0.5g, immobilized time 2h, immobilized temperature 45℃. Using the optimized immobilization process, the immobilized enzyme LX-1000HA-PEGDGE-CRL was prepared to achieve 160.81U/g which was about twice of the activities of immobilized enzyme LX-1000HA-GA-CRL(obtained by LX-1000HA and glutaraldehyde crosslinked lipase) and LX-1000EA-PEGDGE-CRL(obtained by short-chain amino carrier LX-1000EA and PEGDGE crosslinked lipase). The optimal reaction temperature of immobilized enzyme LX-1000HA-PEGDGE-CRL was found to be 15℃ higher than that of free enzyme; the immobilized enzyme could still remain 70% of its original activity after incubation at 70℃ for 3h; the immobilized enzyme could remain about 65% of its original activity after recycling for 6 times; the immobilized enzyme also behaved good tolerance to acid and alkali condition and good storage stability, the immobilized enzyme could remain about 70% of the initial enzyme activity after storage at 4℃ for 30 days. After the comparation of immobilized enzyme LX-1000HA-PEGDGE-CRL with free enzyme, immobilized enzyme LX-1000HA-GA-CRL and immobilized enzyme LX-1000EA-PEGDGE-CRL, the immobilized enzyme LX-1000HA-PEGDGE-CRL behaved better application effect in thermo-tolerance and reusability.

Key words： Polyethylene glycol diglycidyl ether      Amino-carrier      Lipase      Immobilization      Enzymatic property     
Received: 18 December 2019      Published: 02 June 2020
ZTFLH:  Q814.2  
Corresponding Authors: Yun-feng HU     E-mail: yunfeng.hu@scsio.ac.cn
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
Heng ZHU
Ji-fu ZHANG
Yun ZHANG
Yun-feng HU
Cite this article:

ZHU Heng,ZHANG Ji-fu,ZHANG Yun,HU Yun-feng. Immobilization of Marine Candida Lipase Using Novel Epoxy Cross-linker and Amino Carrier. China Biotechnology, 2020, 40(5): 57-68.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1912027     OR     https://manu60.magtech.com.cn/biotech/Y2020/V40/I5/57

Fig.1 Schematic diagram of the immobilization of lipase using LX-1000HA
水平 因素
A(载体量)(g) B(pH) C(温度)(℃) D(时间)(h)
1 1.00 7 50 1
2 1.25 8 55 2
3 1.50 9 60 3
Table 1 Orthogonal experimental factor level table
Fig.2 Cross-linking and immobilization conditions were determined by single-factor experiments (a) Screening crosslinking agent (b) The effect of pH on the activity of immobilized enzymes (c) The effect of the concentration of the crosslinking agent on the activity of the immobilized enzyme (d) The effect of cross-linking temperature on the activity of the immobilized enzyme (e) The effect of carrier volume on the enzyme activity of the immobilized enzyme (f) The effect of immobilization temperature on the enzyme activity of the immobilized enzyme (g)The effect of crosslinking time on the activity of the immobilized enzyme (h) The effect of immobilization time on the activity of immobilized enzyme
序号 载体量(g) pH 温度(℃) 时间(h) 绝对酶活(U/g) 相对酶活(%) 酶活回收率(%)
1 0.50 6 40 1 52.80 50.12 5.40
2 0.50 7 45 2 105.38 100.00 11.83
3 0.50 8 50 3 74.27 70.51 4.87
4 0.75 6 45 3 43.68 41.47 4.66
5 0.75 7 50 1 60.24 57.19 8.83
6 0.75 8 40 2 40.30 38.25 4.45
7 1.00 6 50 2 47.63 45.22 6.60
8 1.00 7 40 3 46.26 43.93 8.33
9 1.00 8 45 1 37.34 35.41 5.00
K1 232.45 144.11 139.35 150.38
K2 144.22 211.88 186.40 193.31
K3 131.24 151.91 182.15 164.22
R 101.21 67.77 47.04 42.93
Table 2 Results and analysis of orthogonal experiments for lipase immobilized condition optimization
影响因素 A(载体量) B(pH) C(温度) D(时间)
K1 U(1+2+3) U(1+4+7) U(1+6+8) U(1+5+9)
K2 U(4+5+6) U(2+5+8) U(2+4+9) U(2+6+7)
K3 U(7+8+9) U(3+6+9) U(3+5+7) U(3+4+8)
R A因素下Umax-Umin B因素下Umax-Umin C因素下Umax-Umin D因素下Umax-Umin
Table 3 Calculation process
Fig.3 Comparison of enzymatic properties between immobilized enzymes and free enzyme (a)Comparison of the optimal reaction temperature between free enzyme and immobilized enzymes LX-1000HA-PEGDGE-CRL,LX-1000EA-PEGDGE-CRL,LX-1000HA-GA-CRL (b)Comparison of the optimal reaction pH between free enzyme and immobilized enzymes LX-1000HA-PEGDGE-CRL,LX-1000EA-PEGDGE-CRL,LX-1000HA-GA-CRL (c)Comparison of thermal stability between free enzyme and immobilized enzymes LX-1000HA-PEGDGE-CRL,LX-1000EA-PEGDGE-CRL,LX-1000HA-GA-CRL (d)Comparison of pH stability between free enzyme and immobilized enzymes LX-1000HA-PEGDGE-CRL,LX-1000EA-PEGDGE-CRL,LX-1000HA-GA-CRL (e)Comparation of the operation stability of immobilized enzymes LX-1000HA-PEGDGE-CRL,LX-1000EA-PEGDGE-CRL and LX-1000HA-GA-CRL on the catalytic capacity (f)Comparation of the storage stability of the free enzyme and immobilized enzymes LX-1000HA-PEGDGE-CRL,LX-1000EA-PEGDGE-CRL and LX-1000HA-GA-CRL
[1]   陈秀琳 . 脂肪酶固定化的研究概况. 海峡药学, 2007,19(12):114-116.
[1]   Chen X L . Overview of lipase immobilization. Strait Pharmaceutical Journal, 2007,19(12):114-116.
[2]   Patrick A . Immobilisation and application of lipases in organic media. Chem Soc Rev, 2013,42(15):6406-6436.
[3]   Cygler M, Schrag J D . Structure and conformational flexibility of Candida rugosa lipase. Elsevier, 1999,1441(2-3):205-214.
[4]   Jaeger K E, Eggert T . Lipases for biotechnology. Elsevier Ltd, 2002,13(4):390-397.
[5]   魏雪, 孙丽超, 李淑英 , 等. 脂肪酶的固定化及其在食品领域的应用. 生物技术通报, 2016,32(11):59-64.
[5]   Wei X, Sun L C, Li S Y , et al. Immobilization of lipase and its application in food industry. Biotechnology Bulletin, 2016,32(11):59-64.
[6]   Guldhe A, Singh B, Mutanda T , et al. Advances in synthesis of biodiesel via enzyme catalysis: Novel and sustainable approaches. Elsevier Ltd, 2015,41(jan.):1447-1464.
[7]   Gandhi N N . Applications of lipase. Springer-Verlag, 1997,74(6):621-634.
[8]   Lima A C P. Cammarota M C. Gutarra M L E . Obtaining filamentous fungi and lipases from sewage treatment plant residue for fat degradation in anaerobic reactors. PEERJ , 2018,6(e5368):1-18.
[9]   陈枫, 韦仕航 . 微生物脂肪酶在动物饲料中的应用研究. 当代畜牧, 2014,17:84-85.
[9]   Chen F, Wei S H . Application of microbial lipase in animal feed. Contemporary Animal Husbandry, 2014,17:84-85.
[10]   黄霜霜 . 脂肪酶拆分手性药物布洛芬的工艺研究. 武汉:华中科技大学, 2015.
[10]   Huang S S . Study on lipase-catalyzed resolution of chiral drug ibuprofen. Wuhan: Huazhong University of Science and Technology, 2015.
[11]   Carlsson N, Gustafsson H, Tho?rn C , et al. Enzymes immobilized in mesoporous silica: A physical-chemical perspective. Advances in Colloid and Interface Science, 2014,205(2014):339-360.
[12]   Nadar S S, Rathod V K . Encapsulation of lipase within metal-organic framework (MOF) with enhanced activity intensified under ultrasound. Enzyme and Microbial Technology, 2018,108:11-20.
[13]   Hilal N, Nigmatullin R, Alpatova A . Immobilization of cross-linked lipase aggregates within microporous polymeric membranes. Journal of Membrane Science, 2004,238(1):131-141.
[14]   Zhang S, Shang W T, Yang X X , et al. Immobilization of lipase using alginate hydrogel beads and enzymatic evaluation in hydrolysis of p-nitrophenol butyrate. Bulletin of the Korean Chemical Society, 2013,34(9):2741-2746.
doi: 10.5012/bkcs.2013.34.9.2741
[15]   Feng W, Ji P J . Enzymes immobilized on carbon nanotubes. Biotechnology Advances, 2011,29(6):889-895.
doi: 10.1016/j.biotechadv.2011.07.007
[16]   Xia J J, Zou B, Zhu G B , et al. Quick separation and enzymatic performance improvement of lipase by ionic liquid-modified Fe3O4 carrier immobilization. Bioprocess and Biosystems Engineering, 2018, 2018,41(5):739-748.
[17]   Zou B, Chu Y H, Xia J J , et al. Immobilization of lipase by ionic liquid-modified mesoporous SiO2 adsorption and calcium alginate-embedding method. Appl Biochem Biotechnol , 2018,185(3):606-618.
[18]   Dicosimo R, Mcauliffe J, Poulose A J , et al. Industrial use of immobilized enzymes. Chem Soc Rev, 2013,42(15):6437-6474.
[19]   Tran D N, Balkus K J . Perspective of recent progress in immobilization of enzymes. ACS Catalysis, 2011,1(8):956-968.
[20]   Pulat M, Akalin G O . Preparation and characterization of gelatin hydrogel support for immobilization of Candida rugosa lipase. Artificial Cells, Nanomedicine, and Biotechnology, 2013,41(3):145-151.
[21]   Aissaoui N, Landoulsi J, Bergaoui L , et al. Catalytic activity and thermostability of enzymes immobilized on silanized surface: Influence of the crosslinking agent. Enzyme and Microbial Technology, 2013,52(6):336-343.
[22]   周清华 . 组合固定化法增强交联脂肪酶聚集体稳定性的研究. 芜湖:安徽工程大学, 2018.
[22]   Zhou Q H . Enhanced the stability of cross-linked lipase aggregates by combined immobilization. Wuhu: Anhui Polytechnic University, 2018.
[23]   卢大胜, 雍克岚, 陈旭 , 等. 新型交联试剂三羟甲基磷固定α-葡萄糖苷酶. 食品科学, 2005,26(6):61-64.
[23]   Lu D S, Yong K L, Chen X , et al. Immobilization of α-glucosidase with a novel coupling reagent-tris(hydroxymethyl)phosphine. Food Science, 2005,26(6):61-64.
[24]   Csiffáry G, Füt? P, Adányi N , et al. Ascorbate oxidase-based amperometric biosensor for L-ascorbic acid determination in beverages. Food Technology and Biotechnology, 2016,54(1):31-35.
[25]   Boka B, Adanyi N, Szamos J . Putrescine biosensor based on putrescine oxidase from Kocuria rosea. Enzyme and Microbial Technology, 2012,51(5):258-262.
[26]   杨建军, 马晓迅, 王贵军 . 大孔树脂吸附固定化脂肪酶机理研究. 西北大学学报(自然科学版), 2009,39(2):241-245.
[26]   Yang J J, Ma X X, Wang G J . A study on mechanism of lipase immobilized with macroporous resins. Journal of Northwestern University (Natural Science Edition), 2009,39(2):241-245.
[27]   Cirillo G, Nicoletta F P, Curcio M , et al. Enzyme immobilization on smart polymers: Catalysis on demand. Elsevier B V, 2014,83:62-69.
[28]   冯超, 蒋丽娟, 黎继烈 , 等. 固定化脂肪酶研究进展. 食品工业科技, 2011,32(2):373-375,378.
[28]   Feng C, Jiang L J, Li J L , et al. Research progress of immobilized lipase. Food Industry Technology, 2011,32(2):373-375,378.
[29]   孟策, 戴小敏, 刘袖洞 . 固定化脂肪酶的载体材料. 辽宁化工, 2018,47(12):1244-1249.
[29]   Meng C, Dai X M, Liu X D . Carrier materials for lipase immobilization. Liaoning Chemical Industry, 2018,47(12):1244-1249.
[30]   李阳, 韦伟, 曹茜 , 等. 脂肪酶固定化新材料. 中国粮油学报, 2014,29(7):122-128.
[30]   Li Y, Wei W, Cao Q , et al. New materials for immobilized lipase. Journal of the Chinese Cereals and Oils Association, 2014,29(7):122-128.
[31]   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. 皮革科学与工程, 2011,21(1):22-27.
[31]   Hou A J, Xu B B, Liang L , et al. A modified colorimetric assay of lipase activity using emulsified oliveoil as the substrate. Leather Science and Engineering, 2011,21(1):22-27.
[32]   Wang S G, Zhang W D, Li Z , et al. Lipase immobilized on the hydrophobic polytetrafluoroethene membrane with nonwoven fabric and its application in intensifying synthesis of butyl oleate. Applied Biochemistry and Biotechnology, 2010,162(7):2015-2026.
doi: 10.1007/s12010-010-8977-1
[33]   Saisuburamaniyan N, Krithika L, Dileena K P , et al. Lipase assay in soils by copper soap colorimetry. Analytical Biochemistry, 2004,330(1):3-70.
[34]   Wang S G, Zheng D B, Yin L Y , et al. Preparation, activity and structure of cross-linked enzyme aggregates (CLEAs) with nanoparticle. Enzyme and Microbial Technology, 2017,107:22-31.
[35]   Talukder M M R., Zaman M M, Hayashi Y , et al. Ultrasonication enhanced hydrolytic activity of lipase in water/isooctane two-phase systems. Biocatalysis and Biotransformation, 2006,24(3):194-289.
[36]   Zhang W D, Qing W H, Ren Z Q , et al. Lipase immobilized catalytically active membrane for synthesis of lauryl stearate in a pervaporation membrane reactor. Bioresource Technology, 2014,172:16-21.
doi: 10.1016/j.biortech.2014.08.019
[37]   Zheng G, Shu B, Yan S . Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres. Enzyme and Microbial Technology, 2003,32(7), 776-782.
doi: 10.1016/S0141-0229(03)00051-6
[38]   Liu X Q, Guan Y P, Shen R , et al. Immobilization of lipase onto micron-size magnetic beads. Chromatography B, 2005,822(1-2):91-97.
[39]   徐珊, 李任强, 张继福 , 等. 孵育对环氧树脂固定化脂肪酶的稳定性研究. 华南农业大学学报, 2019,40(3):61-66.
[39]   Xu S, Li R Q, Zhang J F , et al. Effect of incubation on the stabilization of lipase immobilized by epoxy resin. Journal of South China Agricultural University, 2019,40(3):61-66.
[40]   朱衡, 林海蛟, 张继福 , 等. 氨基载体共价结合固定化海洋假丝酵母脂肪酶. 中国生物工程杂志, 2019,39(7):71-78.
[40]   Zhu H, Lin H J, Zhang J F , et al. Covalent immobilization of marine Candida rugosa lipase using amino carrier. China Biotechnology, 2019,39(7):71-78.
[41]   朱衡, 张继福, 张云 , 等. 聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶. 中国生物工程杂志, 2020,40(Z1):124-132.
[41]   Zhu H, Zhang J F, Zhang Y , et al. Immobilization of lipase through cross-linking of polyethylene glycol diglycidyl ether with amino carrier LX-1000EA. China Biotechnology, 2020,40(Z1):124-132.
[42]   Reshmi R, Sanjay G, Sugunan S . Immobilization of alpha-amylase on zirconia: A heterogeneous biocatalyst for starch hydrolysis. Catal Commun, 2007,8(3):393-399.
[43]   徐然, 韩生华, 王立刚 , 等. 氨基硅胶固定脂肪酶的制备及其酶学性质研究. 山西大同大学学报, 2019,35(5):1-5.
[43]   Xu R, Han S H, Wang L G , et al. Preparation and enzymatic properties of lipase immobilized on amino silica gel. Journal of Shanxi Datong University, 2019,35(5):1-5.
[44]   张权, 盛军, 刘均忠 , 等. 壳聚糖固定化海洋微生物YS2071脂肪酶及其酶学性质. 渔业科学进展, 2015,36(6):100-106.
[44]   Zhang Q, Sheng J, Liu J Z , et al. Chitosan immobilization of lipase from marine microorganism YS2071 and its enzyme characteristics. Progress in Fishery Sciences, 2015,36(6):100-106.
[45]   周蕊, 邢炎华, 王燕 . 以壳聚糖-戊二醛为载体柔性固定假丝酵母脂肪酶Candida rugosa lipase. 当代化工, 2019,48(8):1686-1689.
[45]   Zhou R, Xing Y H, Wang Y . Immobilization of Candida rugosa lipase by using chitosan-glutaraldehyde as support. Contemporary Chemical Industry, 2019,48(8):1686-1689.
[47]   Kim M, Park J M, Um H J , et al. Immobilization of cross-linked lipase aggregates onto magnetic beads for enzymatic degradation of polycaprolactone. Journal of Basic Microbiology, 2010,50(3):218-226.
[48]   Zou B, Hu Y, Jiang L , et al. Mesoporous material SBA-15 modified by amino acid ionic liquid to immobilize lipase via ionic bonding and cross-linking method. Ind Eng Chem Res, 2013,52(8):2844-2851.
[49]   Vitola G, Büning D, Schumacher J , et al. Development of a novel immobilization method by using microgels to keep enzyme in hydrated microenvironment in porous hydrophobic membranes. Macromolecular Bioscience, 2017,17(5):1-12.
[1] CHEN Kai-tong,ZHENG Wen-long,YANG Li-rong,XU Gang,WU Jian-ping. Immobilized L-threonine Aldolase by Amino Resin and Its Application[J]. China Biotechnology, 2021, 41(9): 55-63.
[2] ZHOU Hui-ying,ZHOU Cui-xia,ZHANG Ting,WANG Xue-yu,ZHANG Hui-tu,JI Yi-zhi,LU Fu-ping. Enhancing the Expression of the Substrate by the Extracellular Secreted Enzymes and Improving the Alkaline Protease Production in Bacillus licheniformis[J]. China Biotechnology, 2021, 41(2/3): 53-62.
[3] WEI Zi-xiang,ZHANG Liu-qun,LEI Lei,HAN Zheng-gang,YANG Jiang-ke. Improving the Activity and Thermal Stability of Thermomyces lanuginosus Lipase by Rational Design[J]. China Biotechnology, 2021, 41(2/3): 63-69.
[4] ZHU Heng,ZHANG Ji-fu,ZHANG Yun,SUN Ai-jun,HU Yun-feng. Immobilization of Lipase Through Cross-linking of Polyethylene Glycol Diglycidyl Ether with Amino Carrier LX-1000EA[J]. China Biotechnology, 2020, 40(1-2): 124-132.
[5] Heng ZHU,Hai-jiao LIN,Ji-fu ZHANG,Yun ZHANG,Ai-jun SUN,Yun-feng HU. Covalent Immobilization of Marine Candida Rugosa Lipase Using Amino Carrier[J]. China Biotechnology, 2019, 39(7): 71-78.
[6] Hai-jiao LIN,Ji-fu ZHANG,Yun ZHANG,Ai-jun SUN,Yun-feng HU. The Effective of Additives on the Immobilization of Lipase by Microporous Absorbent Resin[J]. China Biotechnology, 2019, 39(4): 38-51.
[7] ZHANG Ying,WANG Ying,YANG Li-rong,WU Jian-ping. DA-F127 Hydrogel Embedded Immobilized the Nitrile Hydratase-Containing Cells[J]. China Biotechnology, 2019, 39(11): 70-77.
[8] Yan HUANG,Yi-rong SUN,Jing WU,Ling-qia SU. Optimization of High Density Fermentation of Recombinant Humicola insolens Cutinase[J]. China Biotechnology, 2019, 39(1): 63-70.
[9] Qian-qian GUO,Deng-ke GAO,Xiao-tao CHENG,Fu-ping LU,Tanokura Masaru,Hui-min QIN. Heterologous Expression, Purification and Enzymatic Characterization of Cholesterol Oxidase PsCO4[J]. China Biotechnology, 2018, 38(6): 34-42.
[10] Kai DU,Zhuo-ling ZHANG,Ting-hua LI,Wei RAO. The Research Progress of Antibody Immobilization[J]. China Biotechnology, 2018, 38(4): 78-89.
[11] Hong-qiu SHI,Dai-ming ZHA,Bing-huo ZHANG,Han-quan LI. Research Advances in Whole-cell Lipases[J]. China Biotechnology, 2018, 38(11): 51-58.
[12] LI Ji-bin, CHEN Zhi, CHEN Hua-you. Research Progress on Cloning, Expression,Immobilization and Molecular Modification of Nitrilase[J]. China Biotechnology, 2017, 37(9): 141-147.
[13] YANG Jian-wei, XUE Zheng-lian, ZHU Hao, YANG Meng, WANG Zhou. Study on the Mutagenic Effect of Phospholipase A1 Recombinant Plasmid by ARTP[J]. China Biotechnology, 2017, 37(6): 78-85.
[14] LI Shuai, SHAN Hong-yu, DONG Xiao-yu, GUO Chang-hong, GUO Dong-lin. The Role of Phosphoinositide Phospholipase C in Expression Regulation of DREB2[J]. China Biotechnology, 2016, 36(4): 110-115.
[15] CAO Ying-ying, DENG Dun, ZHANG Yun, SUN Ai-jun, XIA Fang-liang, HU Yun-feng. Cloning, Expression and Characterization of a Novel Psychrophile Lipase from the Deep Sea of the South China Sea[J]. China Biotechnology, 2016, 36(3): 43-52.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会