广西师范大学学报（自然科学版） ›› 2020, Vol. 38 ›› Issue (6): 88-101.doi: 10.16088/j.issn.1001-6600.2020.06.011

• • 上一篇    下一篇

基于羧基载体LX-1000IDA的脂肪酶固定化研究

朱衡1,2,3,4, 张继福5, 张云1,2,3, 胡云峰1,2,3*   

  1. 1.中国科学院南海海洋研究所, 广东广州510301;
    2.中国科学院热带海洋生物资源与生态重点实验室(中国科学院南海海洋研究所), 广东广州510301;
    3.南方海洋科学与工程广东省实验室(广州), 广东广州511458;
    4.中国科学院大学, 北京100049;
    5.广东省中医院, 广东广州510120
  • 收稿日期:2019-09-23 发布日期:2020-11-30
  • 通讯作者: 胡云峰(1980—), 男, 山东日照人, 中国科学院南海海洋研究所研究员, 博导。 E-mail:yunfeng.hu@scsio.ac.cn
  • 基金资助:
    国家自然科学基金(21302199);南方海洋科学与工程广东省实验室(广州)人才团队引进重大专项(GML2019ZD0406);广东省自然科学基金(2018A030313151);广东省海洋渔业科技攻关与研发方向(A201701C12);中国科学院战略性先导科技专项(XDA11030404);中国科学院“科学”号高端用户项目(KEXUE2018G05)

Immobilization Method of Lipase Based on Carboxyl Carrier LX-1000IDA

ZHU Heng1,2,3,4, ZHANG Jifu5, ZHANG Yun1,2,3, HU Yunfeng1,2,3*   

  1. 1. South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou Guangdong 510301, China;
    2. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (South China Sea Institute of Oceanology, Chinese Academy of Sciences), Guangzhou Guangdong 510301, China;
    3. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou),Guangzhou Guangdong 511458, China;
    4. University of Chinese Academy of Sciences, Beijing 100049, China;
    5. Guangdong Provincial Hospital of Chinese Medicine, Guangzhou Guangdong 510120, China
  • Received:2019-09-23 Published:2020-11-30

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摘要： 本文利用碳二亚胺EDC作为羧基载体LX-1000IDA的活化剂,通过脂肪酶的表面氨基,将脂肪酶固定于羧基载体上,制成固定化海洋脂肪酶IDA-LIPASE。通过单因素实验、Plackett Burman试验和正交试验,对载体-酶液和交联剂的添加顺序、缓冲液浓度、温度、pH、载体量、时间和碳二亚胺EDC浓度等进行优化和筛选,得到的最佳固定化条件为:pH4.5、载体量0.2 g、时间6 h、温度25 ℃、EDC质量分数0.6%,制备得到酶活力为114 U/g的固定化酶。对该条件进行了10倍扩大固定化,制备得到的固定化脂肪酶IDA-LIPASE酶活力为210 U/g。对制备的固定化酶酶学性质鉴定表明:固定化酶与游离酶相比,最适反应温度提高10 ℃,最适反应pH向碱性方向移动0.5,重复使用7次还保留40%左右的酶活,具有优异的热稳定性;固定化酶在4 ℃保存30 d后保留84.6%酶活。该固定化酶IDA-LIPASE相比于游离酶更能适应工业生产环境,具备实际生产应用的潜力和价值。

关键词: 海洋脂肪酶, 固定化, 羧基载体, 1-(3-二甲氨基丙基)-3-乙基碳二亚胺, 酶学性质

Abstract: This paper used carbon diamine EDC as the activator of the carboxyl carrier LX-1000IDA, and the marine lipase was fixed on the carboxyl carrier through the surface amino resin of lipase to generate the fixed marine lipase IDA-LIPASE. To achieve the best immobilization effect, conditions such as the order of addition of carriers, enzymes and crosslinking agents, buffer concentration, temperature, pH, carrier quantity, time and EDC concentration were optimized and screened. After using single-factor experiment, Plackett Burman experiment, and orthogonal experiment, the optimized conditions of immobilization were determined as follows: pH 4.5, carrier amount 0.2 g, time 6h, temperature 25 ℃, 0.6% EDC, The immobilized enzyme was prepared with enzyme activity of 114 U/g. After 10 times of expanded immobilization, the activity of the immobilized IDA- LIPASE reached 210 U/g. After comparing the prepared immobilized lipase with free enzyme, it was found that: the optimal reaction temperature increased by 10 ℃, the optimal reaction pH shifted 0.5 to the alkaline direction, and the reusability was better. About 40% of the enzyme activity was still left after the 7th usage of immobilized catalysis, the immobilize lipase exhibited excellent thermal stability. In addition, the immobilized lipase remained 84.6% enzyme activity after preservation at 4 ℃ for 30 d and exhibited good storage stability. Compared with free enzyme, the immobilized enzyme IDA-LIPASE was more suitable for industrial production environment and possessed the potential and value of practical production.

Key words: marine lipase, immobilization, carboxyl carrier, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, enzymatic property

中图分类号: 

  • Q814.2
[1] BASHEER S M, CHELLAPPAN S, BEENA P S, et al. Lipase from marine Aspergillus awamori BTMFW032: Production, partial purification and application in oil effluent treatment[J]. New Biotechnology, 2011, 28(6):627-638. DOI:10.1016/j.nbt.2011.04.007.
[2] JOSEPH D, CHAKRABORTY K. Production and biotechnological application of extracellular alkalophilic lipase from marine macroalga-associated shewanella algae to produce enriched C20-22 n-3 polyunsaturated fatty acid concentrate[J]. Applied Biochemistry Biotechnology, 2017, 185(1): 55-71. DOI: 10.1007/s12010-017-2636-8.
[3] DUMORNÉ K, SEVERE R. Marine enzymes and their industrial and biotechnological applications[J]. Minerva Biotecnologica, 2018, 30(4): 113-119. DOI: 10.23736/S1120-4826.18.02442-4.
[4] HASAN F, SHAH A A, JAVED S, et al. Enzymes used in detergents: Lipases[J]. African Journal of Biotechnology, 2010, 9(31): 4836-4844. DOI: 10.5897/AJBx09.026.
[5] KE M M, RAMESH B, HANG Y, et al. Engineering and characterization of a novel low temperature active and thermo stable esterase from marine Enterobacter cloacae[J].Internationd Journal of Biological Macromolecules, 2018, 118: 304-310. DOI: 10.1016/j.ijbiomac.2018.05.193.
[6] 卢昕. 生物传感器:国内研究现状及发展动向[J].广西师范大学学报(自然科学版), 1995,13(3): 61-65. DOI:10.16088/j.issn.1001-6600.1995.03.014.
[7] DAIHA K G, ANGELI R, de OLIVEIRA S D, et al. Are lipases still important biocatalysts? A study of scientific publications and patents for technological forecasting.[J]. PLoS ONE, 2015, 10(6): e0131624. DOI: 10.1371/journal.pone.0131624.
[8] NAVVABI A, RAZZAGHI M, FERNANDES P, et al. Novel lipases discovery specifically from marine organisms for industrial production and practical applications[J]. Process Biochemistry, 2018, 70(6): 61-70. DOI: 10.1016/j.procbio.2018.04.018.
[9] CARVALHO A L, FONSECA T S, MATTOS M C, et al. Recent advances in lipase-mediated preparation of pharmaceuticals and their intermediates[J]. International Journal of Moleculor Science, 2015, 16(12): 29682-29716. DOI:10.3390/ijms161226191.
[10] DUARTE A W F, DOS SANTOS J A, VIANNA M V, et al. Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments[J]. Critical Reviews in Biotechnology, 2018, 38(4): 600-619. DOI: 10.1080/07388551.2018.1379468.
[11] DALMASO G Z L D, FERREIRA D, VERMELHO A B. Marine extremophiles: a source of hydrolases for biotechnological applications[J]. Marine Drugs, 2015, 13(4): 1925-1965. DOI: 10.3390/md13041925.
[12] CHAROENPANICH J, SUKTANARAG S, TOOBBUCHA N. Production of a thermostable lipase by Aeromonas sp EBB-1 isolated from marine sludge in Angsila, Thailand[J]. Science Asia, 2011, 37(2): 105-114. DOI: 10.2306/scienceasia1513-1874.2011.37.105.
[13] RAMANI K, SARANYA P, JAIN S C, et al. Lipase from marine strain using cooked sunflower oil waste: production optimization and application for hydrolysis and thermodynamic studies[J]. Bioprocess and Biosystems Engineering, 2013, 36(3): 301-315. DOI: 10.1007/s00449-012-0785-2.
[14] LU P, GAO X W, DONG H, et al. Identification of a novel esterase from marine environmental genomic DNA libraries and its application in production of free all-trans-astaxanthin[J]. Journal of Agricultural and Food Chemistry, 2018, 66(11): 2812-2821. DOI: 10.1021/acs.jafc.7b06062.
[15] BENDIKIENE· V, KIRILIAUSKAITE· V, JUODKA B. Production of environmentally friendly biodiesel by enzymatic oil transesterification[J]. Journal of Environmental Engineering and Landscape Management, 2011, 19(2): 123-129. DOI: 10.3846/16486897.2011.579448.
[16] 钟成华, 张文东, 刘鹏, 等. 包埋固定化复合菌低温下处理养猪废水研究[J]. 广西师范大学学报(自然科学版), 2011, 29(3): 52-56. DOI:10.16088/j.issn.1001-6600.2011.03.008.
[17] BILAL M, ZHAO Y P, NOREEN S, et al. Modifying bio-catalytic properties of enzymes for efficient biocatalysis: a review from immobilization strategies viewpoint[J].Biocatalysis and Biotransformation, 2019, 37(3): 159-182. DOI: 10.1080/10242422.2018.1564744.
[18] 钱明华, 张继福, 张云, 等. 大孔树脂吸附-交联法固定脂肪酶[J]. 华南农业大学学报, 2019, 40(2): 103-110. DOI:10.7671/j.issn.1001-411X.20180431.
[19] 林海蛟, 王云鹏, 张云, 等. 无机载体吸附-交联固定化海洋脂肪酶技术研究[J]. 江西农业大学学报, 2019, 41(1): 186-196. DOI: 10.13836/j.jjau.2019023.
[20] 徐珊. 优化脂肪酶固定化工艺的研究[D]. 广州: 暨南大学, 2018.
[21] 杨秀芳, 陈梅, 马养民. 包埋-交联结合法固定β-呋喃果糖苷酶的研究[J]. 中国酿造, 2010(10): 56-59. DOI: 10.3969/j.issn.0254-5071.2010.10.018.
[22] 徐珊, 李任强, 张继福, 等. 使用国产环氧树脂LXEP-120固定化脂肪酶研究[J]. 广西师范大学学报(自然科学版), 2018, 36(4): 108-118. DOI: 10.16088/j.issn.1001-6600.2018.04.014.
[23] 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[J]. 中国生物工程杂志, 2019, 39(7): 71-78. DOI: 10.13523/j.cb.20190710.
[24] 吴晓燕, 刘茜, 焦庆才, 等. 固定化米曲霉氨基酰化酶拆分DL-茶氨酸[J]. 广西师范大学学报(自然科学版), 2008, 26(4): 107-111. DOI: 10.16088/j.issn.1001-6600.2008.04.008.
[25] HONG J, GONG P J, YU J H, et al. Conjugation of α-chymotrypsin on a polymeric hydrophilic nanolayer covering magnetic nanoparticles[J]. Journal of Molecular Catalysis B: Enzymatic, 2006, 42(3/4): 99-105. DOI: 10.1016/j.molcatb.2006.07.008.
[26] HONG J, XU D M, GONG P J, et al. Conjugation of enzyme on superparamagnetic nanogels covered with carboxyl groups[J]. Journal of Chromatography B, 2007, 850(1/2): 499-506. DOI: 10.1016/j.jchromb.2006.12.035.
[27] ZHU Y T, REN X Y, LIU Y M, et al. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays[J]. Materials Science and Engineering: C, 2014, 38(2): 278-285. DOI: 10.1016/j.msec.2014.02.011.
[28] ZHU J, ZHANG J, LAI Q M, et al. Covalent immobilization of lipase onto citric acid-esterified loofah sponge[J]. Bioresources, 2013, 8(3): 3289-3298. DOI: 10.15376/biores.8.3.3289-3298.
[29] NETA N D A S, SANTOS J C S, SANCHO S D O, et al. Enzymatic synthesis of sugar esters and their potential as surface-active stabilizers of coconut milk emulsions[J]. Food Hydrocolloids, 2012, 27(2): 324-331. DOI: 10.1016/j.foodhyd.2011.10.009.
[30] 陈振锋, 王修建, 梁宏, 等. 含硫香料:硫代香叶醇的合成研究[J]. 广西师范大学学报(自然科学版), 1998, 16(3): 57-60. DOI:10.16088/j.issn.1001-6600.1998.03.011.
[31] 马骁, 侯彬彬, 赵玮钦. 改性硅藻土固定化脂肪酶催化蓖麻油制备生物柴油[J]. 山东化工, 2015, 44(24): 4-6, 10. DOI: 10.19319/j.cnki.issn.1008-021x.2015.24.004.
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